key: cord-018771-xqlb74px authors: turinsky, sebastian; steuernagel, claus title: bluttransfusion date: 2013-04-04 journal: praxis der intensivmedizin doi: 10.1007/978-3-642-34433-6_4 sha: doc_id: 18771 cord_uid: xqlb74px bei einem 58-jährigen patienten mit einem hb-wert von 6,5 g/dl, der nach operativer ausschaltung eines bauchaortenaneurysmas auf der intensivstation unerwartet nachblutet, soll eine bluttransfusion durchgeführt werden. wenige minuten nach der anforderung aus der blutbank treffen die bestellten erythrozytenkonzentrate ein – es sind erythrozytenkonzentrate der blutgruppe a. sie sind eindeutig auf den namen des patienten ausgezeichnet, die identität des patienten wird erneut überprüft. der durchgeführte bedside-test zeigt aber die blutgruppe b! was muss der intensivarzt nun tun? bei einem 58-jährigen patienten mit einem hb-wert von 6,5 g/dl, der nach operativer ausschaltung eines bauchaortenaneurysmas auf der intensivstation unerwartet nachblutet, soll eine bluttransfusion durchgeführt werden. wenige minuten nach der anforderung aus der blutbank treffen die bestellten erythrozytenkonzentrate ein -es sind erythrozytenkonzentrate der blutgruppe a. sie sind eindeutig auf den namen des patienten ausgezeichnet, die identität des patienten wird erneut überprüft. der durchgeführte bedside-test zeigt aber die blutgruppe b! was muss der intensivarzt nun tun? die gabe von blut wurde erstmals erfolgreich im 17. jahrhundert durchgeführt und zählt damit zu den ältesten medizinischen therapiemaßnahmen. bedingt durch ihren häufig lebensrettenden effekt wurden bluttransfusionen über einen langen zeitraum sehr großzügig gehandhabt. erst durch das auftreten verschiedener unerwünschter reaktionen und nebenwirkungen rückte die transfusion zunehmend in den fokus wissenschaftlicher studien und wurde in den letzten jahren wie kaum ein anderes thema der intensivmedizin kritisch diskutiert. hervorzuheben ist v. a. die untersuchung von hebert et al. zur anämie bei intensivpatienten, in der gezeigt werden konnte, dass patienten durch eine restriktive transfusionstherapie (transfusion ab einem hämoglobinwert <7 g/dl, zielwert 9 g/dl) gegenüber patienten, bei denen eine großzügigere transfusionsstrategie angewendet wurde (transfusion ab einem hämoglobinwert <10 g/dl, zielwert 12 g/dl), keinen nachteil haben. auch die zunehmende verknappung an blutprodukten wegen eines mangels an potenziellen spendern und einer stagnation der spendebereitschaft sowie steigende herstellungspreise spielen eine weitere wesentliche rolle bei der indikationsstellung zur bluttransfusion. vor diesem hintergrund stellen die 2008 erschienenen querschnittsleitlinien sowie die 2010 erschienenen richtlinien zur gewinnung von blut und blutbestandteilen und zur anwendung von blutprodukten (hämotherapie) der bundesärztekammer eine wesentliche unterstützung für den transfundierenden intensivmediziner dar. erythrozytenkonzentrate erythrozytenkonzentrate (ek) werden aus frischem vollblut einer einzelspende gewonnen. durch zentrifugation und filtration des vollbluts werden die erythrozyten abgetrennt. es bleiben leukozyten und thrombozyten (sog. »buffy coat«) sowie zellfreies plasma übrig. land nur nach entfernung der leukozyten als sog. leukozytendepletierte ek zugelassen, um eine übertragung zellständiger viren und eine immunisierung gegen leukozytenantigene zu verhindern. 500 ml vollblut ergeben ca. 220-330 ml ek mit einem hämatokrit zwischen 50 und 75%, entsprechend einem hämoglobingehalt zwischen 17 und 25 g/dl, und 250 ml frischplasma. durch zugabe von stabilisatoren und additiver lösung werden der energiehaushalt und die stabilität der ek verbessert, sodass sie unter optimalen umständen (erschütterungsfreier kühlschrank mit kontinuierlicher temperaturüberwachung +4°c ± 2°c) bis zu 49 tage gelagert werden können. ein »einfaches« leukozytendepletiertes ek kostet ca. 85 €, bei bestimmten blutgruppe wie 0 rh-negativ kommt noch ein zuschlag von 10-20 € hinzu. neben den am meisten verwendeten leukozytendepletierten ek stehen folgende besondere zubereitungen zur verfügung: gewaschene erythrozytenkonzentrate diese können dann transfundiert werden, wenn trotz leukozytendepletion eine unverträglichkeitsreaktion beim empfänger aufgetreten ist. ein leukozytendepletiertes ek wird unmittelbar vor der transfusion gewaschen und muss dann innerhalb von 6 h ohne weitere lagerung transfundiert werden. seit der generellen einführung von leukozytendepletierten ek gibt es nur noch wenige indikationen für gewaschene ek. im falle einer massiven, nicht zu stillenden blutung ist eine hämoglobinkonzentration im bereich von 10 g/dl (hämatokrit 30%) anzustreben, da erythrozyten den thrombozytenaktivator adp freisetzen, die thrombozyten in die nähe der gefäßwand abdrängen und dadurch einen günstigen effekt auf die primäre hämostase haben. bei vorliegen einer chronischen anämie, z. b. im rahmen eines tumorleidens oder einer nierenerkrankung, kommt es langfristig zu adaptationsvorgängen, die zu einer sicherung der o 2 -versorgung führen. dennoch kann die anämie den verlauf einer erkrankung negativ beeinflussen und führt zu keiner besseren toleranz bei weiteren hämoglobinabfällen. patienten mit einer chronischen anämie sollten daher bei einer hämoglobinkonzentration unter 7-8 g/dl (hämatokrit <21-24%) transfundiert werden (evidenzgrad 1 c), sofern andere maßnahmen wie eisensubstitution oder gabe von erythropoetin versagen. sowohl der zunehmende mangel an blutprodukten als auch diverse andere umstände wie die ablehnung durch den patienten oder eine verzögerte verfügbarkeit von blutprodukten im notfall machen es erforderlich, alternativen zur transfusion von fremdblut zu kennen. so kann in akut lebensbedrohlichen situationen, z. b. bei schwerer blutungsanämie, durch beatmung mit einer fio 2 = 1,0 eine zunahme des physikalisch im blut gelösten sauerstoffs erreicht werden, die etwa dem effekt der transfusion von 1-2 ek entspricht. bei der anwendung von intravenösen eisenpräparaten wie eisencarboxymaltose (z. b. ferinject) oder eisensucrose (z. b. venofer) handelt es sich um therapieoptionen, die schnell wirken, mit geringem risiko verbunden sind und nicht nur bei patienten mit nachgewiesenem eisenmangel zu einer anhebung des hämoglobinwerts und somit einer reduktion von transfusionen führen. bei operativen eingriffen mit erwartet größeren blutverlusten (richtwert >1 l) kann kurzfristig eine maschinelle autotransfusion (mat, engl. »cell salvage«) durchgeführt werden. zahlreiche untersuchungen konnten zeigen, dass die mat insbesondere im bereich der herz-, gefäß-und orthopädischen chirurgie eine sichere und wirksame alternative zur allogenen transfusion darstellt. möglicherweise ist es zukünftig möglich, durch anwendung spezieller filter die mat auch bei onkologischen oder septischen eingriffen einzusetzen. durch die anschließende schockgefrierung innerhalb von 6 h auf mindestens -30°c bleibt die aktivität der labilen gerinnungsfaktoren erhalten, und das gfp wird -je nach lagerungstemperatur -für 1-2 jahre haltbar gemacht. die plasmaspiegel der gerinnungsfaktoren und inhibitoren der hämostase variieren sehr stark, besonders für faktor viii und fibrinogen. die aktivität der gerinnungsfaktoren v, viii, ix und xi ist beim apherese-gfp deutlich höher als beim vollblut-gfp, die durchschnittliche aktivität liegt bei 100 ie pro 100 ml, also 1 ie pro 1 ml gfp. nach viermonatiger lagerung muss eine serologische untersuchung des gfp-spenders auf spezifische virusmarker (hiv, hbv, hcv) erfolgen, in dieser zeit befindet sich das gfp in »quarantänelagerung«. erst wenn die serologischen untersuchungen negativ verlaufen sind, kann das gfp verwendet werden. einmal aufgetaute gfp müssen innerhalb von 6 h transfundiert werden. 250 ml gfp kosten etwa 50 €. was? neben seiner funktion als substrat für die fibrinbildung ist fibrinogen das verbindungsmolekül für 6 praxistipp 1 ml gfp enthält durchschnittlich 1 ie jedes gerinnungsfaktors. transfundiert man einem patienten 1 ml gfp pro kg körpergewicht, dann erhöht dies 5 den spiegel der gerinnungsfaktoren und inhibitoren oder den quickwert um etwa 1 ie/dl oder 1% und 5 den fibrinogenwert um 2-3 mg/dl. allergische transfusionsreaktionen sind folge von antikörpern des empfängers gegen plasmaproteine des spenders und treten mit einer wahrscheinlichkeit von etwa 0,5% auf. die klinische symptomatik ist mit urtikaria oder hautrötung meist milde und nimmt nur selten schwere formen wie bronchospasmus oder anaphylaktischen schock an. eine akute intravasale hämolyse und eine bakterielle kontamination sollten ausgeschlossen und eine symptomatische therapie wie bei anderen allergischen reaktionen durchgeführt werden. bei schweren anaphylaktischen transfusionsreaktionen, wie sie häufiger bei patienten mit iga-mangel und der ausbildung von anti-iga-antikörpern auftreten können, besteht die indikation zur transfusion gewaschener blutprodukte. eine bakterielle kontamination von blutprodukten ist sehr selten, und ein großteil der kontaminierten präparate führt nicht zu einer symptomatischen infektion. die klinische manifestation gleicht denen der nichthämolytischen, fieberhaften und der hämolytischen transfusionsreaktion, die daher im rahmen der diagnostik ausgeschlossen werden müssen. es sollte eine mikrobiologische diagnostik inkl. blutkulturen sowohl des empfängers als auch des blutprodukts durchgeführt werden. zudem sollte eine antibiotische therapie eingeleitet werden. eine transfusionsassoziierte virale infektion, bedingt durch eine mit den gängigen testverfahren nicht nachweisbare virämie des spenders, ist ebenfalls sehr selten (hepatitis-b-virus 1:500.000 bis 1:1.000.000, hiv-/ hepatitis-c-virus <1:1.000.000, cmv einzelfälle). eine virologische abklärung sollte angestrebt und ggf. eine antivirale therapie eingeleitet werden. die ursache eines »transfusion-related acute lung injury« (trali) sind leukozytenreaktive antikörper meist im spenderplasma, die über eine aktivierung der empfängerleukozyten zu einer störung der pulmonalen mikrozirkulation führen, die sich zunächst als lungenödem und im verlauf als ards darstellt. seltener kann diese reaktion auch durch leukozytenreaktive antikörper im empfängerplasma hervorgerufen werden oder ist gänzlich nichtimmunogen bedingt. aufgrund der klinischen symptomatik von rasch progredienter dyspnoe mit hypoxämie, die während oder bis zu 6 h nach der transfusion auftreten kann, werden bis zu 70% der patienten beatmungspflichtig. therapeutisch stehen die sicherung einer adäquaten oxygenierung sowie eine stabilisierung der kreislauffunktion im vordergrund. über die letalität des trali gibt es in der literatur abweichende aussagen -sie schwankt zwischen 10 und 25%. dennoch ist sie die häufigste todesursache nach der transfusion von blutprodukten. offenbar kam es bei der initialen blutabnahme zu einer verwechslung des patienten bzw. des patientenbluts. auch ein zweiter bedside-test zur kontrolle zeigt erneut die blutgruppe b. da die blutung klinisch fortschreitet und der patient in den op muss, entschließt sich der intensivarzt zur transfusion von erythrozytenkonzentraten der blutgruppe 0 rhesus-negativ und gfp der blutgruppe ab. vor der transfusion wird nochmals blut zur bestimmung der blutgruppe und zum kreuzen von erythrozytenkonzentraten abgenommen. querschnitts-leitlinien (bäk) zur therapie mit blutkomponenten und plasmaderivaten richtlinien zur gewinnung von blut und blutbestandteilen und zur anwendung von blutprodukten (hämotherapie) dtsch ärztebl a multicenter, randomized, controlled clinical trial of transfusion requirements in critical care the 2010 annual shot report strategies to reduce the use of blood products: a us perspective transfusion strategies for acute upper gastrointestinal bleeding hämotherapie der blutgerinnung im umgekehrten fall, bei der übertragung von rhesus-positivem blut auf einen rhesus-negativen patienten, muss 2-4 monate nach der transfusion eine serologische untersuchung zur feststellung von antikörpern gegen den rhesusfaktor erfolgen, da es bei der erneuten gabe von rhesus-positivem blut zu einer unverträglichkeitsreaktion kommen könnte. bei rhesus-negativen mädchen und frauen im gebärfähigen alter sollte die transfusion von rhesus-positiven erythrozytenkonzentraten unbedingt vermieden werden, da es durch die antikörperbildung bei einer schwangerschaft mit einem rhesus-positiven kind ebenfalls zu einer rhesusunverträglichkeitsreaktion kommen kann. ist im rahmen einer lebensbedrohlichen situation eine transfusion mit rhesus-positivem blut unvermeidlich, so kann die bildung von antikörpern durch eine immunisierung gegen das rhesusantigen verhindert werden (gabe von anti-d-immunglobulin, z. b. rhophylac).auch thrombozytenkonzentrate sollen ab0kompatibel, bevorzugt ab0-gleich, transfundieren werden, da in einzelfällen akute hämolytische transfusionsreaktionen auftreten können. ferner sollte auch bei thrombozyten der rhesus-faktor-d berücksichtigt werden, da tk geringe mengen an erythrozyten enthalten.die transfusion von gefrorenem frischplasma soll ab0-gleich oder ab0-kompatibel erfolgen (. tab. 4.6). als universell verträgliches plasmapräparat gilt plasma der blutgruppe ab. aufgrund der begrenzten verfügbarkeit von ab-plasma in mitteleuropa ist der generelle einsatz dieser blutgruppe jedoch nicht praktikabel. bei einem lebensbedrohlich stark blutenden patienten kann im notfall mit der transfusion folgender blutkonserven sofort begonnen werden: key: cord-103509-hynnba03 authors: wong, ten-tsao; liou, gunn-guang; kan, ming-chung title: a self-assembled protein nanoparticle serving as a one-shot vaccine carrier date: 2020-09-18 journal: biorxiv doi: 10.1101/2020.09.16.299149 sha: doc_id: 103509 cord_uid: hynnba03 in this paper, we are exploring the role of an amphipathic helical peptide in mediating the self-assembly of a fusion protein into a protein nanoparticle and the application of the nanoparticle as a one-shot vaccine carrier. out of several candidates, an amphipathic helical peptide derived from m2 protein of type a influenza virus is found to stimulate high antigenicity when fused to a fluorescent protein genetically. this fusion protein was found to form protein nanoparticle spontaneously when expressed and purified protein stimulates long-lasting antibody responses in single immunization. through modeling peptide structure and nanoparticle assembly, we have improved this vaccine carrier in complex stability. the revised vaccine carrier is able to stimulate constant antibody titer to a heterologous antigen for at least six months in single immunization. the immune response against a heterologous antigen can be boosted further by additional immunization in spite of high immune responses to carrier protein. subunit vaccine is a safe alternative to traditional inactivated or attenuated vaccines, but its efficacy is often hindered by the low antigenicity of recombinant protein. different approaches are utilized to resolve this issue, among them, virus like particle (vlp) and self-assembled protein nanoparticle (sapn) are considered the best platforms for subunit vaccine development(1). vlp is assembled from a recombinant capsid protein alone without the genomic nucleic acid and bound nucleocapsid protein so it is non-infectious (2) . the size of vlp is ranged between 20-200 nm that facilitates both draining efficiently to lymph node and also uptake by antigen presenting cells like dendritic cell and macrophage (3) . the other benefit of vlp based vaccine is the induction of b cell receptor clustering when presenting repetitive antigen to b cell, a function that can activate antibody class-switch and somatic hypermutation in a t cell dependent mechanism (4) . not only the virus like particle can be used for vaccine directly, heterologous antigen can be presented in the particle surface through genetic fusion (5) . the universal flu vaccine candidate, ectopic m2 domain (m2e), was genetically fused with hepatitis b core antigen(hbc) and assembled into a nanoparticle that provides full protection to homologous flu strain (6) . but the application of hbc based vlp in human vaccine development is restricted by the pre-existing anti-hbc antibody present in the 450 million chronic hbv carriers and the population exposed to hbv infection (7) . an artificially designed sapn may avoid the effect of the existing antibody. a sapn assembled from protein constituted by two coiled-coil domains that form trimer and pentamer respectively can be assembled into nanoparticles with specific sizes (8, 9) . this sapn stimulates strong immune responses to target protein fused to the terminal of constituting monomer after 3 immunizations even without adjuvant, but this immunity waned gradually (10) . the green fluorescent protein is a member of fluorescent protein family that are structurally conserved and emit fluorescent light from a chromophore when excited by photons of shorter wavelength (11) . the shared features of fluorescent proteins including a sturdy barrel shaped structure constituted by 11 β-sheets and an enclosed chromophore that emits fluorescent light when excited (12) . the function of the barrel shell is to provide a well organized chemical environment to ensure the maturation of chromophore and protects it from hostile elements (13) . so it is conceivable that the protein sequences among fluorescent protein family members in the barrel shell are highly variable and fluorescent proteins possess desirable biophysical properties can be selected using directed evolution (11, (14) (15) (16) (17) . the applications of fluorescent protein have been expanded into multiple areas beyond live imaging which includes serving as biological sensors (18, 19) , or detectors for protein-protein interaction or protein folding (20, 21) . amphipathic α-helical peptide (ahp) forms hydrophilic and hydrophobic faces when folded and is often identified in proteins related to phospholipid membrane interaction. the n-terminal amphipathic helical peptide is required for membrane anchorage of hepatitis c virus ns3 protein and the protease function of the ns3/ns4a complex (22, 23) . several anti-microbial peptides also possess amphipathic properties and function by forming membrane pores or causing membrane disruption (24) . the amphipathic α-helical of type a influenza virus m2 protein is required for m2 protein anchorage and induces membrane curvature required for virus budding (25, 26) . life protection from diseases through vaccination is considered a holy grail only achievable by some live attenuated viral vaccines with multiple doses up to date (27) . here in this report, we are describing the identification of a protein nanoparticle based on an amphipathic α-helical peptide (ahp) from m2 protein of type a influenza strain h5n1 and a green fluorescent protein. we have predicted the protein nanoparticle structure according to transmissive electronic microscope images using protein modeling and generated ahp mutants that provide higher stability and antigenicity to the protein nanoparticle. this modified protein nanoparticle is able to simulate long constant antibody response to an inserted heterologous antigen in single immunization without adjuvant. and this antibody response can be further boosted by additional immunization. the identification and application of this nanoparticle as a vaccine carrier was explored in this study. identification of an ahp-gfp protein complex with high antigenicity and stability. as described in our patent application filed in 2015, we have tested the immunogenicity of fusion proteins composed of an ahp and a gfp (28) . the results showed an increase of anti-gfp igg titer ranged between 2~3 log under a two immunizations regime ( figure s1 ). one of the peptides, ah3, that derived from m2 protein of type a influenza strain h5n1 gives extended stability to the gfp fusion protein when compared to another peptide, ah1 ( figure s2 ) as well as other peptides in our study (data not shown). since a stable protein is essential for vaccine carrier and may profits worldwide vaccination effort, we were interested in the mechanism of ah3-gfp stability and antigenicity. to study the potential mechanisms that contribute to the above mentioned properties of ah3-gfp fusion protein, we first checked the composition of ah3-gfp protein post expression and purification. one clue that led us to study the composition of ah3-gfp fusion protein is the difficulties encountered during protein purification. unlike other fusion proteins studied, both ah3-gfp and ah5-gfp fusion proteins are mostly expressed as insoluble inclusion body and the remaining soluble protein did not bind to ni-nta resin under normal condition of 300mm nacl. the ah3-gfp and ah5-gfp fusion proteins only started to bind to ni-nta resin after lowering the nacl concentration from 300mm to 50mm, an indication that hydrophobic interaction may induce a protein complex with n-terminal his tag hindered from binding to ni-nta ligand. also, the resistance of ah3-gfp fusion protein to hydrolysis suggested the linker between ah3 peptide and gfp is kept in a water tight complex. from these two clues, it was hypothesized that ah3-gfp fusion protein form stable protein complex through hydrophobic interaction mediated by n-terminal ah3 peptide. to test the hypothesis that ah3-gfp or ah5-gfp fusion protein forms a protein complex, we first used the protein concentration tube with different molecular weight cut off (mwco) to determine the protein complex sizes. as shown in figure 1a , when gfp protein with a molecular weight of 27kda is able to pass through membranes with mwco of 100kda, 300kda and 1000kda freely, ah3-gfp fusion protein purified from bacterial lysate was prevented from passing through the membrane with an mwco of 1000kda. with a molecular weight of 33kda, the purified ah3-gfp protein need to form a complex with more than 30 monomers to be excluded from passing a membrane with a 1000kda mwco. to explore further the geometric composition of the ah3-gfp protein complex, we examined the fusion protein under transmissive electronic microscope (tem). the tem results showed the ah3-gfp fusion protein forms a cylinder-like structure with length up to ~60 nm and a diameter around 10 nm (fig. 1b) . the difference in length suggests that the particle may be assembled along the long axis. when scanning along the long axis of the ah3-gfp particle, there is a repetitive pattern of two-one-two-one of white dots with two less visible dots on each side of the single dot. the predicted structure according to tem images is shown in fig.1d . we also examined protein geometric composition of ah5-gfp under tem, but there is no clear evidence of forming higher order protein complex, suggesting ah5-gfp protein complex is not as stable as ah3-gfp to withstand the conditions during negative staining. to find the correlation between protein complex formation and antigenicity, we immunized mice with purified ah3-gfp fusion protein and the recombinant gfp protein that pass through the membrane freely. proteins were prepared from lps synthesis defective e. coli strain, clearcoli bl21(de3), to avoid the interference of lps contamination, a known tlr4 ligand. the mice were immunized with purified proteins by single intramuscular injection and sera were collected at day 7, 14, 30 and 182 to evaluate anti-gfp igg titer by elisa. deoxycholate was added to test if deoxycholate in the concentration of 0.2% affects ah3-gfp antigenicity and related experiment was terminated at 30 days post immunization when it showed no effect on antigenicity of either gfp or ah3-gfp. these results suggest gfp alone is a poor antigen and only gained high antigenicity after fused with ah3 peptide (fig. 1c) . to understand the potential molecular mechanism leading to the assembly of ah3-gfp nanoparticle, we tried to build a protein model based on three observations: first, the particle was assembled through hydrophobic interaction and second, the particle assembled along the long axis and the third: ah3-gfp protein particle has a repetitive three-two pattern when observed under tem. we first assembled the two ah3 peptide as anti-parallel helices with hydrophobic sidechains of f4, f5, i8, l12 and l16 mediating intermolecular contacts. the assembled ah3 dimer created a hydrophobic core between two helices and the helix dimer is surrounded by hydrophilic sidechains from multiple lysine and arginine except one exposed hydrophobic patch, as predicted using deepview and marked by white mesh (fig. 2a) . this hydrophobic patch can be seen only to cover one face of the dimer (fig. 2b ). when the water accessible surface of ah3 dimer was calculated, two cavities could be seen located within the hydrophobic patch that provides contact points for two arg9 sidechains extruding from the opposite face of ah3 dimer. a second ah3 dimer can make close contact with the first dimer after turning counter clockwise looking down the hydrophobic patch for 36 o and forms a tetramer (fig. 2c) . the intermolecular energy between two dimers from this model was calculated to has a δg of -101 kcal/mol (fig. 2c) . after adding gfp protein structures onto the ah3 tetramer model, the ah3-gfp fusion protein tetramer will form a cross-shaped assembling unit and the stacking of every ah3-gfp tetramer on top of another tetramer will extend the particle length by 2.8 nm and turning the cross by 72 o . since the gfp protein barrel diameter is ranged between 2.7~3.5 nm, the out extending gfp from ah3-gfp tetrameric cross can spatially fit with the model (fig. 2d ). under this model, the protein nanoparticle will be extended continuously with a hydrophobic patch presenting on one end of the assembled particle constitutively and serving as a point for polymerization. after proving that the ah3-gfp protein complex possesses high antigenicity, we decided to explore the application of ah3-gfp protein nanoparticle as a vaccine carrier. for the hepatitis b core antigen, the amino acid 144 served as an insertion site for heterologous antigen fusion (29) . gfp protein has a thermal stable structure that constituted by 11 β-strands and 3 α-helices and some of the loops between strands have been explored as insertion sites for heterologous protein for various purposes (18, 19, 30) . among those candidates, loop173 linking strand 8 and strand 9 was chosen because it has a high capacity for foreign peptide insertion (fig 3a) (30) . the original ah3-gfp recombinant protein is constructed in pet28a vector with ah3 coding region inserted c-terminal to his-tag and thrombin cleavage site followed immediately by gfp cloned from pegfp-c2. this expression vector was low in soluble protein productivity and unable to express soluble recombinant protein when the peptide is inserted between d173 and g174. to resolve the expression and folding issues, we designed a new expression vector. first, we cloned ah3 peptide into the very n-terminal following methionine in the pet27 vector, and then to its c-terminal we inserted a synthesized sfgfp gene (31) with a created antigen insertion site following 175s of sfgfp. the antigen insertion site also included an 8xhis tag for recombinant protein purification. to verify vaccine carrier function, we inserted two copies of broad spectrum flu vaccine candidate, human m2 ectopic peptide (hm2e) separated by a 6 amino acid linker (fig. 3b) . the newly constructed vector was proven to be efficient for expressing soluble ah3-sfgfp-2hm2e fusion protein as a protein complex (data not shown). the ah3-sfgfp-2xhm2e protein complex under tem is not as stable as ah3-gfp unless first cross-linking the protein preparation with a heterobifunctional protein crosslinker, sulfo-smcc (fig. 3c ). following the previous established ah3-gfp protein model, we were seeking strategies to create a more stable ah3-sfgfp protein complex. first, we found the mutation of isoleucine 8 to leucine increase the intermolecular interaction(δg) from -16 kcal/mol to -37 kcal/mol (fig.3d) . second, we mutated lysine 13 to glutamic acid and generated additional electrostatic interactions between side chains of glu13 with arg10 and arg11 (fig. 3e ). to verify whether the protein modeling results are correct, including the presence of hydrophobic patch on ah3 peptide complex and a higher stability of ah3 based protein complex with i8l and/or k13e mutations. we first generated mutations in ah3 peptide in the context of ah3-sfgfp-2xhm2e construct (fig. 4a ). our hypothesis is that the hydrophobic patch of the ah3-gfp complex will bind bacterial membrane and co-sediment with it during ultracentrifugation. and then the methodology was verified by first centrifuge bacterial lysate prepared from clearcoli culture in a centrifuge tube preloaded with 15%(w/v), 45%(w/v) and 85%(w/v) sucrose solution in the volume of 7ml, 2ml, 1ml respectively ( fig. 4b left panel) . the distribution of bacterial membrane was marked by a lysochromic dye, sudan iii. the control sample contained sudan iii with lysis buffer alone (fig. 4b lane 1) . after ultracentrifugation, the bacterial membrane is sedimented to the junction of 15%/45% sucrose solution (fig.4b lane 2) . using the same protocol, ah3-gfp was found to co-sedimented with the bacterial membrane (fig. s3a) as well as the ah3-sfgfp-hm2e fusion protein but not a free gfp protein (fig s3b) these results are consistent with our protein structure modeling that arg11 serves as a main contact point for dimer stacking also it mediates the electrostatic interaction with mutated glu13 (fig. 3e ). as shown in the sucrose step gradient result, the presence of hydrophobic patch enables nonspecific interaction of the ah3-gfp protein complex with phospholipid membrane. which may restrict the free moving of protein nanoparticle and keep it from reaching draining lymph node for stimulating immunity (4) . to compare the antigenicity of protein complexes derived from either ah3-sfgfp-2xhm2e or lyrrle-sfgfp-2xhm2e, we immunized mice with a single injection of either recombinant proteins. post immunization, sera were collected at day 15, 50, 90 and 202 to evaluate anti-hm2e igg titer by elisa. the geometric mean titer of anti-hm2e igg reached the highest point for the ah3-sfgfp -2xhm2e group and then declined afterward. but of the lyrrle-sfgfp-2xhm2e group, the gmt reached highest point at day 50 and remained steady up to day 90 (fig. s4) . when the individual mouse serum result is observed separately, only one out of 5 mice from ah3-sfgfp-2xhm2e group has higher anti-hm2e igg titer at day 202 than day 15. but there are 4 out of 5 mice from the lyrrle-sfgfp-2xhm2e group shows a higher antibody titer in day 202 compared to day 15 (fig. 5a) . these results suggest that the two point mutations of ah3 in i8l and k13e enable the formation of a stable, high antigenic protein complex that stimulates long lasting immune responses in a single immunization. vaccine carrier like hbc based virus like particle (vlp) is often failed at boosting humoral immune responses after prime dose in a multiple doses protocol due to antigen competition. although gfp is a protein of low antigenicity, the fusion with ah3 strongly enhances its antigenicity as shown in figure 1c . to test if sfgfp backbone competes with inserted hm2e peptide for immune machinery, we immunized mice in a prime-boost protocol using the same protein preparations. the two consecutive injections were carried out 14 days apart and sera collected at day 14, 28 and 90 were subject to elisa assay using either hm2e peptide or sfgfp protein as coating antigen. the result shows the igg titer against hm2e elevated continuously after consecutive immunizations for both proteins as well as anti-sfgfp igg titer. the result suggests that although carrier protein ah3-sfgfp also has high antigenicity, it did not interfere with the immune response against the heterologous protein, hm2e (fig. 5b, 5c) vaccine as a tool to prevent infectious disease is the most cost effective strategy. especially for attenuated viral vaccines like vaccinia, mmr or oral polio vaccine, they produce long lasting even life time protective immune responses but these attenuated viral vaccines took decades for development. apparently, this strategy will not be able to timely develop a vaccine to ward off emerging global pandemic like covid-19. although the new vaccine technology like dna vaccine, mrna vaccine or adenovirus based vaccine that can quickly develop a subunit vaccine after the genomic information of pathogen become available, but the immune responses generated are often declined to base level within a year (32) (33) (34) (35) . this short lived immune response may expose vaccinated people to the risk of antibody dependent enhancement (ade) that is known to be devastating and leads to vaccine failure (11, 36) . in this study, we have created a self-assembled protein nanoparticle composed of ah3-fp and a more stable variant that stimulates long lasting antibody responses. the nature of this long lasting immune responses is not known but it may be mediated by long lasting plasma cell generated during ah3-fp immunization, the same mechanism that accounts for the lifelong protection of attenuated viral vaccines (27) . fluorescent protein family is a group of proteins with conserved barrel shaped structure with chromophore buried inside. since the function of this beta strand constituted barrel is to provide a suitable environment for chromophore maturation, the primary sequence of fluorescent protein barrel is prone to mutagenesis through either direct selection (11) or evolution (12) . also, fluorescent proteins of desired biophysical properties like thermal stability or folding efficiency can be obtained expert review of vaccines key: cord-273711-bxijla09 authors: zhao, zhixun; wu, guohua; zhu, xueliang; yan, xinmin; dou, yongxi; li, jian; zhu, haixia; zhang, qiang; cai, xuepeng title: rna interference targeting virion core protein orf095 inhibits goatpox virus replication in vero cells date: 2012-02-17 journal: virol j doi: 10.1186/1743-422x-9-48 sha: doc_id: 273711 cord_uid: bxijla09 background: goatpox is an economically important disease in goat and sheep-producing areas of the world. many vaccine strategies developed to control the disease are not yet completely successful. hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses. however, none of these studies has been attempted to study gtpv. in the interest of exploiting improved methods to control goat pox, it is participated that rnai may provide effective protection against gtpv. in this study we show the suppression of goatpox virus (gtpv) replication via knockdown of virion core protein using rna interference. results: four short interfering rna (sirna) sequences (sirna-61, sirna-70, sirna-165 and sirna-296) against a region of gtpv orf095 were selected. sense and antisense sirna-encoding sequences separated by a hairpin loop sequence were designed as short hairpin rna (shrna) expression cassettes under the control of a human u6 promoter. orf095 amplicon was generated using pcr, and then cloned into pegfp-n1 vector, named as p095/egfp. p095/egfp and each of the sirna expression cassettes (p61, p70, p165 and p296) were co-transfected into bhk-21 cells. fluorescence detection, flow cytometric analysis, retro transcription pcr (rt-pcr) and real time pcr were used to check the efficiency of rnai. the results showed that the orf095-specific sirna-70 effectively down-regulated the expression of orf095. when vero cells were transfected with shrna expression vectors (p61/gfp, p70/gfp, p165/gfp and p296/gfp) and then infected with gtpv, gtpv-orf095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (cpe) induced by gtpv. the results presented here indicated that dna-based sirna could effectively inhibit the replication of gtpv (approximately 463. 5-fold reduction of viral titers) on vero cells. conclusions: this study demonstrates that vector-based shrna methodology can effectively inhibit gtpv replication on vero cells. simultaneously, this work represents a strategy for controlling goatpox, potentially facilitating new experimental approaches in the analysis of both viral and cellular gene functions during of gtpv infection. gtpv is a member of the genus capripoxvirus of the family poxviridae [1] , which also includes the sheeppox virus (sppv) and the lumpy skin disease virus (lsdv) of cattle. both sheeppox and goatpox are endemic in africa, the middle east and many countries in asia, and the diseases caused by these viruses have a significant economic impact on the livestock industry in africa and asia [2] . gtpv genome is approximately 150 kbp double-stranded dna, which composes at least 147 putative genes, including conserved replicative and structural genes and genes likely involved in virulence and host range [3] . orf095 encodes the virion protein which constitutes a great part of the total protein content of the virion and is essential during the assembly and disassembly of virion. it is similar to myxoma virus (myxv) m093l (accession no.af170726) [4, 5] and vaccinia virus (vacv) a4l (accession no.m35027) that encodes a 39 kda acidic protein, a part of the viral core, and is synthesized at late stages after infection [6, 7] . rna-mediated interference (rnai) is a conserved gene-silencing mechanism, where by the doublestranded rna matching is used as a signal to trigger the sequence-specific degradation of homologous mrna [8] . rnai can be triggered by chemically synthesised and enzymatically produced 21-25 nt long rna duplexes in mammalian cells [9, 10] . since the effect of short interfering rnas (sirnas) is generally temporal in transfected animal cells, small rna expression vectors have been developed to induce long-lasting rna silencing in mammalian cells [11] [12] [13] [14] . rnai represents a new antiviral method and is being increasingly used to inhibit the replication of viral pathogens [15] such as foot-and-mouth disease [16, 17] , porcine reproductive and respiratory syndrome virus [18] , newcastle disease virus [19] , classical swine fever virus [20] and monkeypox virus [21] . hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses [11, [22] [23] [24] [25] [26] . this study provides not only an experimental basis for the development of a new anti-gtpv strategy, but also for a new approach to the study of gtpv infection and replication. goatpox virus strain a/goat/qinghai/av40/2006(a celladapted strain) was used in this study and maintained in african green monkey kidney cells (vero). baby hamster kidney cells (bhk-21) and the gtpv permissive cell line vero (lanzhou veterinary research institute, chin) were cultured in dulbecco's modified eagle's medium (dmem; sigma) supplemented with 10% heat-inactivated fetal bovine serum (fbs; hangzhou, china), 100 u/ml penicillin and 100 μg/ml streptomycin (sigma). cultures were incubated at 37°c with 5% co2. the cdna cassettes corresponding to the conserved gene of the gtpv genome was cloned into the pegfp-n1 vector ( figure 1a ). directed cloning pcr was used to amplify the orf095 gene, using the following primers (sense: 5'-gtcctcgagatggacttcat-gaaaaaatatactaa-3' and antisense: 5'-gcggatccttgctgttattatcatctagtttg-3') used for amplification contained the target sequences for xhoi (ctcgag) and bamhi (ggatcc) incorporated at the 5' of the viral complementary sequence. forward primer contained an atg sequence, before the sequence that codified for the protein, as a start initiation codon of protein translation. the reverse primer uncontained a tta sequence, which was used in the characterization of orf095 protein and egfp coexpression. pcr products were digested with xhoi and bamhi, and cloned into the pegfp-n1 expression vector (invitrogen, inc., shanghai, china). the final construct p095/egfp ( figure 1a ) was analyzed by restriction digestion and sequenced. plasmids used for sirnas. an inverted repeat is inserted at the 3'-end of the human u6 promoter. the forward sequence of the repeat is 21 or 63 nt long, corresponding to the region of interest of the orf095 gene. the forward and reverse motifs are separated by a 7-nt spacer, 5'-tcaagag-3'. the transcriptional termination signal of five ts is added at the 3'-end of the inverted repeat. the synthesized rna is predicted to fold back to form a hairpin dsrna, which would be finally processed into the putative sirnas. transfection were purified with the wizard purefection tm plasmid dna purification system (promega, usa) and quantified by biophotometer (eppendorf, germany). as the aagn18 uu sequence (n, any nucleotide) has been found to be preferred for sirna-mediated gene silencing under the control of the polu6 promoter [27] , we searched for this sequence in the orf of orf095 gene. four fragments (orf095-61, orf095-70, orf095-165 and orf095-296) in the coding region of orf095 gene were selected according to the web-based criteria (http://www.ambion.com). these selected sequences were then submitted to a blast searching against human genome sequence to check whether or not these potential targets have homologues in the human genome was not targeted. to construct hairpin sirna expression cassette, the following dna oligonucleotides were synthesized: gtpv-orf095-61, gtpv-orf095-70, gtpv-orf095-165 and gtpv-orf095-296 ( figure 1a ). the 21 nt target sequences served as a basis for the design of the two complementary 51-53mer sirna template oligonucleotides that were synthesized, annealed, and inserted into bamhi and bbsi sites of the sirna expression vectors pgpu6/neo and pgpu6/gfp/neo (genepharma co., ltd, shanghai, china), respectively. the recombinant plasmids were designated as p61, p61/gfp, p70, p70/gfp, p165, p165/ gfp, p296 and p296/gfp. the pc and pc/gfp negative controls (genepharma co., ltd, shanghai, china) were negative control plasmid, which encode hairpin sirna that does not have homologues in mice, human and carpripoxvirus genome databases. transfection of the sirna expression cassettes into bhk-21 cells bhk-21 cells were seeded in six-well plates and cultured at 37°c and 5% co2 overnight. when the cells showed 70-80% confluence, 2.5 μg of p61, p70, p165 or p296 each were cotransfected with an equal amount of p095/ egfp using fugene hd transfection reagent (roche, germany) according to the manufacturer's recommendations, respectively. simultaneously, 2.5 μg of p095/ egfp were cotransfected with 2.5 μg of pc. non-transfected bhk-21 cells were also used as a control. after an additional 24 h of incubation, cells were observed for the expression of green fluorescent protein in the transfected cells was monitoring fluorescent microscope (olympus, japan). cells were further subjected to fluorescence-activated cell sorting (facs). at 48 h posttransfection, the transfected cells and the controls were washed gently in phosphate-buffered saline (pbs), trypsinized and resuspended in pbs. egfp positive cells and egfp expression signal were evaluated by the facs calibur flow cytometry system (becton dickinson, usa). to confirm the efficacy of rnai, rt-pcr was used to amplify the target gene in the transfected cells. total rna was extracted from bhk-21 culture with trizol reagent (takara, dalian), and incubated for 1 h at 37°c with dnase rq1 (takara, dalian). to detect orf095 mrna expression in bhk-21 cells, rt-pcr was conducted using 1.8 μg of rna extracts with superscript one-step rt-pcr system (gibco, brl). retrotranscription β-actin as a control was also amplified using the primers 5'-cacccgcgagtacaaccttc-3' (sense) and 5'-cccatacccaccatcacacc-3' (antisense). pcr was run for 30 cycles with 95°c for 30 s, 56°c for 45 s and 72°c for 45 s. to verify primer specificity, a melting curve was analyzed, and rt-pcr products were further cloned into pmd18-t for sequencing. in order to use full-length orf095 gene as a quantitative rt-pcr standard, selected primer sequences were ctgtctacatgattaacccactcgttcttc (orf095 fp primer), and gaagtcgactac ccctctccctatcagggtcatc (orf095 rp primer). one additional primer was synthesized for quantification of the orf095 in real-time pcr: 5-fam-ccttgctcgcgaatttctcaccgatamra-3 (taqman probe). the target region of real time rt-pcr was 263-389 bp of orf095. for quantitative analysis of the orf095 gene, 100 ng total rna from p095/egfp-transfected cells was mixed with 1 μl orf095 primer, heated to 65°c for 5 min and chilled on ice for 2 min. to this primer template mix was added 5× buffer (4 μl), 10 mmol/μl dntp (1 μl), rnasin (1 μl), amv reverse transcriptase (1 μl, promega, usa) and ddh 2 o to a total volume of 20 μl. the reaction mixture was incubated at 42°c for 45 min, followed by inactivation of reverse transcriptase at 75°c for 15 min. real-time pcr was performed with the abi prism 7000 sequence detection system using 2 μl transcriptase products as template under the conditions of 95°c for 15 min, followed by 50 cycles of denaturation at 95°c for 30 s, annealing, and extension at 60°c for 30 s. the quantitative standard curve for determination of orf095 copy number was created by real-time pcr of standard plasmid p095/egfp serial 10-fold dilutions of a stock containing 10 8 copies/μl. the specificity of the real-time pcr was confirmed by sequencing of the product. four sirnas targeting orf095 were designed to inhibit gtpv orf095 gene expression in bhk-21 cells. we used a modified cmv promoter, a typical rna poliii promoter, to drive the transcription of the sirnas. to monitor the effects of the sirnas, eukaryotic expression plasmid p095/egfp was constructed, in which the orf095 gene were fused to the 5'-end of the egfp coding sequence, and cotransfected with their specific sirna expression plasmids. so the inhibitory effects of the orf095-specific sirnas on the orf095 expression could be indirectly evaluated by the expression of egfp in the transfected cells. to test whether the expressed sirnas inhibited gtpv production, we first assessed the growing capacity of gtpv in vero cells expressing sirnas. vero cells were seeded in six-well plates and cultured at 37°c and 5% co 2 overnight. when the cell reached 70-80% confluence, 2.5 μg of p61/gfp, p70/gfp, p165/gfp or p296/ gfp each were cotransfected with an equal amount of pc/gfp as described above, respectively. the nonspecific vector pc/gfp and non-transfected vero cells were also used as a control. 24 h posttransfection, the cells were infected with gtpv at a multiplicity of infection (moi) of about 0.01. briefly, after removing the culture medium, gtpv (200 μl) in infection medium (2.5 μg/ml trypsin), respectively, were added to each well. the cultures were then incubated at 37°c, 5% co 2 for 4 h, at which point the culture medium was replaced with fresh dmem containing 2% fetal bovine serum. to determine transfection efficiency, we monitored gfp fluorescence intensity of transfected cells using fluorescent microscope analysis. culture supernatants were collected for virus titration. six days post infection, supernatants was harvested from the infected cultures and virus titer (tcid50) was determined three times on vero cells. virus infectivity was determined by serial dilutions of the samples in 96-well plates and the virus titer was calculated as a tcid50 by the reed-muench method [28] . a viral suspension titrated at 10 -1 to 10 -8 tcid50 per 0.1 ml was used for viral challenge. vero cells (about 80% confluent) grown in 96-well plates were transiently transfected with 0.1 μg p61/gfp, p70/gfp, p165/gfp and p296/gfp, respectively, per well. after 6 h of transfected, the transfection medium was removed and the cells were washed twice with dmem medium. the transfected cells were then infected with 100 tcid50 of virus per 0.1 ml per well. after additional 1 h incubation, the inoculum was removed and the cells were washed twice with dmem medium. the cells were then maintained in dmem medium supplemented with 10% fetal bovine serum for 6 days. for detecting the therapeutic potential of sirna, in another parallel experiment, transfection was performed 1 h post-infection with the virus. gtpv replication in vero cells was evaluated by virus titer (tcid50). transient cellular transfection and analysis of the targeted gene and egfp expression in bhk-21 cells different sirnas suppressed the expression of fusion green fluorescent protein in bhk-21 cells is different. the sirnas targeting to the conserved region of gtpv genome were generated in vitro by human recombinant dicer enzyme, as described in figure 1b . to identify an effective inhibitory effect of sirnas, the cdna cassettes of these regions were inserted into the 5' end of enhanced green fluorescent protein (egfp) gene to construct reporter plasmids ( figure 2 ). the reporter plasmids were used to cotransfect bhk-21 cells with either the homologous sirnas or the heterologous sirnas. the results showed that the number of egfp-expressing cell was markedly reduced in the sample transfected with homologous sirnas than sample transfected with heterologous sirnas or non-transfected ( figure 3a ). facs demonstrated that the levels of inhibition mediated by the sirnas were similar among the different experiment groups and significantly higher than the control group (cotransfection with heterologous sirnas or without sirnas). the inhibitory effects of the sirnas on expression of egfp were quantitatively validated by flow cytometry 48 h posttransfection. the extent of egfp down regulation was assessed by the mean fluorescence of the positive cells (lr-values) and the rate of egfp positive cells figure 3b ). compared with the pc, the lr-values of the egfp positive cells were reduced in the cells transfected with orf095 sirna-specific expression plasmids p61, p70, p165 and p296, and were reduced by 90.9%, 96.7%, 66.0%and 69.32%, respectively. to further demonstrate the levels of inhibition, cells were collected 48 h post-transfection and rt-pcr analysis was performed. the level of target rna, as determined by rt-pcr, was also significantly reduced in cells transfected with homologous sirnas ( figure 4a) . to measure the level of gene suppression accurately, qpcr primers and taqman probes directing to orf095 were designed. we also designed probes and primers directed toβ-actin sequence (serve as internal reference). when normalized for loading differences using the βactin mrna, the orf095 message in the cells transfected with p61, p70, p165 and p296 were reduced by 69%, 97%, 22%and 57% (orf095 message copies ratios of cells transfected with shrna expression vectors/cells transfected with empty vector)( figure 4b ). there was no significant inhibition in cells transfected with the empty vector pegfp-n1 and nonspecific shrna expression vector pc. mrna levels of orf095 (average orf095 mrna levels in cells treated with p61, p70, p165, p296, pc and empty vector were 0.416, 0.036, 1.046, 0.580, 1.345, respectively) or β-actin suggested that the reductions in orf095 message did not result from poor transfection, nonspecific inhibition or toxicity, because the average mrna levels of β-actin for experimental cells were not significantly reduced compared to the control cells. in addition, the suppressive effect was found to be gene-specific, because the inhibitory effect of empty vector and nonspecific shrna expression vector pc were negligible. these results suggest that the sirna generated by in vitro transcription effectively and specifically inhibit the expression of gtpv orf095 conserved regions in bhk-21 cells. to investigate whether or not knockout of orf095 relieves cytopathic effect (cpe) induced by gtpv, vero cells were transfected by plasmids expressing orf095 protein-targeted shrnas (p61/gfp, p70/gfp, p165/gfp and p296/gfp), respectively. nonspecific shrna expression vector pc/gfp was transfected in parallel. 4 h post-transfection, the cultures were infected with gtpv and checked daily. six days later, we found that cells pre-transfected with p70/gfp exhibited less cpe, whereas other shrna-treated cells and the empty vector control demonstrated the same typical gtpv-induced cpe as cells infected only with virus, as shown in figure 5 . the tcid50 assay was performed to examine the effect of sirna on production of viable virus, and the results ( figure 6 ) showed that in control cells the titers reached 10 -5.12 tcid50/0.1 ml at 6 days post-infection. in contrast, the titers in the cells transfected with p61/ gfp, p70/gfp, p165/gfp or p296/gfp, were 10 -3.75 , 10 -2.48 , 10 -4.73 and 10 -4.66 /0.1 ml 6 days post-infection rnai is a process of sequence-specific, post-transcriptional gene silencing that is initiated by double stranded rna. introduction of sirna results in degradation of sirna specific transcripts thus reducing the expression of their protein product. in plants, it is a natural antiviral defense mechanism [29] . in mammalian cells, however, dsrnas longer than 30 nt activate an antiviral defense leading to the nonspecific degradation of rna transcripts and a general shutdown of host-cell protein translation [30] . the successful use of sirna in animal cells encouraged the development of sirna expression vector [31] and numerous studies have demonstrated that dna-based sirna is a promising approach for antiviral therapy in mammals. rnai represents a new antiviral method and is being increasingly used to inhibit the replication of viral pathogens [15] , such as hiv-1 [32] , hepatitis c [33] , influenza [34] , severe acute respiratory syndrome [35] and hepatitis e viruses [36] . this study has demonstrated the use of pgpu6/neo or pgpu6/gfp/neo vector-based rnai against gtpv, a major pathogen of goats and sheep. four different sirna targeting viral gene orf095, one key gene involved in gtpv replication, successfully reduced viral replication. the results showed that the orf095-specific sirnas p70 could effectively down-regulate the expression of orf095, while p61, p165 and p296 displayed weak activity. additionally, expression of the housekeeping gene β-actin was also analyzed by rt-pcr and quantitative real-time pcr, and no significant difference in the expression of β-actin was observed between the sirnas treatment groups and pc treatment groups. different sirna sequences display widely different efficacies with regard to suppression of gene expression, requiring screening of multiple sequences [37] . in this research, we have selected four target sequences for rna interference by the software applications, "sirna target finder and design tool" available at http://www.ambion. com/. as the orf095 gene is well conserved in gtpv and orf095 protein is a virion core protein and assembly protein in gtpv, we selected orf095 as a target gene. in order to generate shrna expression cassettes quickly and accurately, we employed a pcr-based strategy to clone sirna sequences. in this strategy, sirna sequences were designed as a single primer sequence of which 19 bp complementary to the human u6 promoter were added. the resulting pcr products are shrna expression cassettes including the human u6 promoter. shrnas that have been generated from this expression system are efficiently processed by dicer into sirnas. in addition, in this study, we selected pegfp-n1 vector that contains an egfp gene as report gene and can be transfected into mammalian cells using any standard transfection method. vero cells transfected with p61/gfp, p70/gfp, p165/ gfp, p296/gfp and pc/gfp were examined for cpe by virus titration. all results demonstrated that sirna-70 is the most effective one, and result showed that minimum concentration of the construct p70/gfp is 2.5 μg required to induce maximum inhibition. blasting orf095 sequence in genbank revealed that there were 8 capripoxvirus isolates containing identical sequence corresponding to orf095. in view of the sequences of the orf095 genes of gtpv strains from the same genotype, they all share high homology (95-100%). therefore, orf095 gene is a good target to suppress gtpv replication by rnai. in conclusion, this study demonstrates that vector-based shrna methodology can effectively inhibit gtpv replication on vero cells. further study will be required to determine whether such treatment protect against gtpv infection in vivo. still, this work represents a strategy for controlling goatpox, potentially facilitating new experimental approaches in the analysis of both viral and cellular gene functions during of gtpv infection. some observations on the occurrence of lumpy 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rna interference (rnai) targeting rna-dependent rna polymerase approaches for chemically synthesized sirna and vector-mediated rnai rna interference targeting virion core protein orf095 inhibits goatpox virus replication in vero cells we thank yadong zheng for thoughtful discussions and assistance during manuscript preparation. authors' contributions qz and xc designed research; zz performed research and wrote the paper; gw helped to construct partial plasmids and analyzed data; xz contributed new reagents/analytic tools; xy provided partial plasmids. hz helped to culture cells; jl helped to culture viruses; yx d had a co-ordination role in this work. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-334855-s0ci3r8w authors: andersen, petter i.; krpina, klara; ianevski, aleksandr; shtaida, nastassia; jo, eunji; yang, jaewon; koit, sandra; tenson, tanel; hukkanen, veijo; anthonsen, marit w.; bjoras, magnar; evander, magnus; windisch, marc p.; zusinaite, eva; kainov, denis e. title: novel antiviral activities of obatoclax, emetine, niclosamide, brequinar, and homoharringtonine date: 2019-10-18 journal: viruses doi: 10.3390/v11100964 sha: doc_id: 334855 cord_uid: s0ci3r8w viruses are the major causes of acute and chronic infectious diseases in the world. according to the world health organization, there is an urgent need for better control of viral diseases. repurposing existing antiviral agents from one viral disease to another could play a pivotal role in this process. here, we identified novel activities of obatoclax and emetine against herpes simplex virus type 2 (hsv-2), echovirus 1 (ev1), human metapneumovirus (hmpv) and rift valley fever virus (rvfv) in cell cultures. moreover, we demonstrated novel activities of emetine against influenza a virus (fluav), niclosamide against hsv-2, brequinar against human immunodeficiency virus 1 (hiv-1), and homoharringtonine against ev1. our findings may expand the spectrum of indications of these safe-in-man agents and reinforce the arsenal of available antiviral therapeutics pending the results of further in vitro and in vivo tests. every year, emerging and re-emerging viruses, such as ebola virus (ebov), marburg virus (marv), and rift valley fever virus (rvfv), surface from natural reservoirs and kill people [1, 2] . in addition, influenza a virus (fluav), human immunodeficiency (hiv-1), herpes simplex (hsv), and other viruses regularly infect human population and represent substantial public health and economic burden [3, 4] . the world health organization (who) and the united nations (un) have called for better control of viral diseases (https://www.who.int/blueprint/priority-diseases/en/; https://sustainabledevelopment.un.org/). developing novel virus-specific vaccines and antiviral drugs can be time-consuming and costly [5, 6] . in order to overcome these time and cost issues, academic institutions and pharmaceutical companies have focused on the repositioning of existing antivirals from one viral disease to another, considering that many viruses utilize the same host factors and pathways to replicate inside a cell [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . broad-spectrum antiviral agents (bsaas) are small-molecules that inhibit a wide range of human viruses. we have recently reviewed approved, investigational and experimental antiviral compounds and identified 108 bsaas, whose pharmacokinetics (pk) and toxicity had been studied in clinical trials [16] . we tested 40 of these bsaas against human metapneumovirus (hmpv), hepatitis c virus (hcv), cytomegalovirus (cmv), and hepatitis b virus (hbv). we demonstrated novel antiviral effects of azacytidine, itraconazole, lopinavir, nitazoxanide, and oritavancin against hmpv, as well as cidofovir, dibucaine, azithromycin, gefitinib, minocycline, oritavancin, and pirlindole against hcv [17] . we also tested 55 bsaas, including these 40, against fluav, rvfv, echovirus 1 (ev1), zikv, chikv, rrv, hiv-1 and hsv-1. we identified novel activities for dalbavancin against ev1, ezetimibe against hiv-1 and zikv, and azacytidine, cyclosporine, minocycline, oritavancin and ritonavir against rvfv [18] . here, we evaluated the efficacy of 43 bsaas, which do not overlap with 55 agents we tested before. we identified novel in vitro activities of obatoclax and emetine against hsv-2, ev1, hmpv and rvfv. moreover, we demonstrated novel antiviral effects of emetine against fluav, niclosamide against hsv-2, brequinar against hiv-1, and homoharringtonine against ev1 in vitro. to identify potential bsaas, we have reviewed approved and investigational safe-in-man antiviral agents using drug bank and clinical trials websites, respectively. in addition, we reviewed investigational and approved safe-in-man antibacterial, antifungal, antiprotozoal, antiemetic, etc. agents, for which antiviral activities have been reported in pubmed. by excluding vaccines and interferons, we identified 108 molecules that inhibit the replication of several viruses in man [16] . most recently, novel antiviral activities have been reported for some of these agents [17] . forty-three compounds were used in this study, and their suppliers and catalogue numbers are summarized in table s1 . to obtain 10 mm stock solutions compounds were dissolved in dimethyl sulfoxide (dmso, sigma-aldrich, steinheim, germany) or milli-q water. the solutions were stored at −80 • c until use. madin-darby canine kidney (mdck, american type culture collection (atcc)) and african green monkey kidney epithelial (vero-e6, atcc) cells were grown in dulbecco's modified eagle's medium (dmem; gibco, paisley, scotland) supplemented with 100 u/ml penicillin and 100 µg/ml streptomycin mixture (pen/strep; lonza, cologne, germany), 2 mm l-glutamine, and 10% heat-inactivated fetal bovine serum (fbs; lonza, cologne, germany). human telomerase reverse transcriptase-immortalized retinal pigment epithelial (rpe, atcc) cells were grown in dmem-f12 medium supplemented with pen/strep, 2 mm l-glutamine, 10% fbs, and 0.25% sodium bicarbonate (sigma-aldrich, st. louis, usa). ach-2 cells, which possess a single integrated copy of the provirus hiv-1 strain lai (nih aids reagent program), were grown in rpmi-1640 medium supplemented with 10% fbs and pen/strep. tzm-bl cells were grown in dmem supplemented with 10% fbs and pen/strep. human lung adenocarcinoma epithelial a549 cells were cultured in dmem medium containing 10% fbs and pen/strep. a549-npro cells (kindly provided by prof. steve goodbourn, university of london), which stably express bvdv npro protein, which inhibits ifn production, were cultured in dmem containing 10% fbs, pen/strep, and 10 µg/ml puromycin. all cell lines were grown in a humidified incubator at 37 • c in the presence of 5% co2. hsv-2 strain g was from the atcc. ev1 (farouk strain; atcc) was from prof. marjomäki (university of of jyväskylä) [19] . rvfv encoding the far-red fluorescent protein instead of non-structural (ns) protein (rvfv-rfp) was from profs. hartmut hengel and friedemann weber (university medical center freiburg) [20] . hmpv nl/1/00 strain, encoding green fluorescent protein (hmpv-gfp), was from vironovative and erasmus mc [21] . the gfp-expressing influenza a/pr/8-ns116-gfp strain (fluav-gfp) was generated by drs. andrej egorov (vienna) [22] . all the experiments with viruses were performed in compliance with the guidelines of the national authorities using appropriate biosafety laboratories under appropriate ethical and safety approvals. fluav-gfp was amplified in a monolayer of mdck cells in dmem containing pen/strep, 0.2% bovine serum albumin, 2 mm l-glutamine, and 1 µg/ml l-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin (tpck)-trypsin (sigma-aldrich, st. louis, usa). hmpv-gfp, rvfv-rfp and the wild-type hsv-2 strain were amplified in a monolayer of vero-e6 cells in the dmem medium containing pen/strep, 0.2% bovine serum albumin, 2 mm l-glutamine, and 1 µg/ml tpck-trypsin. ev1 was amplified in a monolayer of a549 cells in the dmem media containing pen/strep, 0.2% bovine serum albumin, and 2 mm l-glutamine. for the production of hiv-1, 6 × 10 6 ach-2 cells were seeded in 10 ml medium. virus production was induced by the addition of 100 nm phorbol-12-myristate-13-acetate. the cells were incubated for 48 h. the hiv-1-containing medium was collected. the hiv-1 concentration was estimated by measuring the concentration of hiv-1 p24 in the medium using anti-p24-elisa, which was developed in-house. recombinant purified p24 protein was used as reference. the virus stocks were stored at −80 • c. approximately 4 × 10 4 rpe cells were seeded per well in 96-well plates. the cells were grown for 24 h in dmem-f12 medium supplemented with 10% fbs, and pen/strep. the medium was replaced with dmem-f12 medium containing 0.2% bovine serum albumin, 2 mm l-glutamine, and 1 µg/ml tpsk-trypsin. the compounds were added to the cells in 3-fold dilutions at seven different concentrations starting from 10 or 30 µm. saliphenylhalamide, abt-263 and dmso were added to the control wells. saliphenylhalamide is an inhibitor of cellular vacuolar atpase, which protects cells from virus-mediated death, whereas abt-263 is an inhibitor of anti-apoptotic bcl-2 proteins, which facilitates death of cells with viral nucleic acids [23] [24] [25] [26] [27] [28] . rpe cells were infected with hsv-2, fluav-gfp, hmpv-gfp or rvfv-rfp viruses at multiplicity of infections (moi) of 0.1, 0.5, 0.1 and 1, respectively. hsv-2-infected rpe cells were imaged after 72 h in the phase-contrast mode. rvfv-mediated rfp expression and fluav-mediated gfp expression were visualized after 24 h, whereas hmpv-mediated gfp expression was recorded after 96 h using fluorescent microscopy (zeiss observer z1, zaventem, belgium). image j software (v.ij 1.46r, nih) was used to determine fluorescent intensities. rpe cells were treated with bsaas or control compounds as described above and infected with hsv-2, ev1, fluav-gfp, hmpv-gfp or rvfv-rfp viruses at multiplicity of infections (moi) of 0.1, 0.1, 0.5, 0.1 and 1, respectively. after 48 h of infection, the medium was removed from the cells. the viability of mock-and virus-infected cells were measured using cell titer glow assay (ctg; promega, madison, usa). the luminescence/fluorescence were read with a pherastar fs plate reader (bmg labtech, ortenberg, germany). for testing compound toxicity and efficacy against hiv-1, approximately 4 × 10 4 tzm-bl cells were seeded in each well of a 96-well plate. tzm-bl cells express firefly luciferase under control of hiv-1 long terminal repeat (ltr) promoter allowing quantitation of the viral infection (tat-protein expression by integrated hiv-1 provirus) using firefly luciferase assay. the cells were grown for 24 h in cell growth medium. compounds were added to the cells in three-fold dilutions at seven different concentrations starting from 30 µm. no compounds were added to the control wells. the cells were infected with hiv-1 (corresponding to 300 ng/ml of hiv-1 p24) or mock. at 48 hpi, the media was removed from the cells, the cells were lysed, and firefly luciferase activity was measured using the luciferase assay system (promega, madison, wi, usa) and pherastar fs plate reader. in a parallel experiment, cell tox green reagent (ctxg; promega, madison, wi, usa) was added to the cells and fluorescence was measured with a plate reader. the half-maximal cytotoxic concentration (cc 50 ) for each compound was calculated based on viability/death curves obtained on mock-infected cells after non-linear regression analysis with a variable slope using graphpad prism software version 7.0a. the half-maximal effective concentrations (ec 50 ) were calculated based on the analysis of reporter protein expression or the viability/death of infected cells by fitting drug dose-response curves using four-parameter (4pl) logistic function f (x): where f (x) is a response value at dose x, a min and a max are the upper and lower asymptotes (minimal and maximal drug effects), m is the dose that produces the half-maximal effect (ec 50 or cc 50 ), and λ is the steepness (slope) of the curve. a relative effectiveness of the drug was defined as selectivity index (si = cc 50 /ec 50 ). the threshold of si used to differentiate between active and inactive compounds was 3. rpe cells were treated with combinations of increasing concentrations of obatoclax and emetine. the cells were infected with fluav-gfp at moi 0.5. after 24 h, gfp fluorescence was recorded using fluorescent microscopy. in a parallel experiment, the viability of infected cells was measured using the ctg assay. to test whether the drug combinations act synergistically, the observed responses were compared with expected combination responses. the expected response of the emetine-obatoclax drug combination on the viability of fluav-and mock-infected rpe cells was calculated using bliss reference model [29] . for testing the production of hsv-2 and ev1 viruses in compound-treated and non-treated rpe cells, the media from the cells were serially (10-fold) diluted, starting from 10 −3 to 10 −8 in serum-free growth media containing 0.2% bovine serum albumin, and applied to a monolayer of a549-npro cells in 12-well plates. after one hour, cells were overlaid with growth medium containing 1% carboxymethyl cellulose and 1% fbs and incubated for 72 h. the cells were fixed and stained with crystal violet dye. the plaques were calculated in each well. the titers were expressed as plaque-forming units per ml (pfu/ml). forty-three safe-in-man bsaas used in this study reached different stages of drug development process (figure 1 , tables s2 and s3). altogether, these bsaas inhibit the replication of 52 viruses belonging to (−) single-stranded (ss)rna, (+)ssrna, ssrna-reverse transcriptase (rt), ssdna, double-stranded (ds)dna, or dsdna-rt virus groups. we tested 43 bsaas against wild-type hsv-2 in rpe cells. seven different concentrations of the compounds were added to virus-or mock-infected cells. cell viability was monitored by microscopy and the ctg assay. after the initial screening, we identified four compounds (obatoclax, emetine, niclosamide and ganciclovir) that at none-cytotoxic concentrations rescued cells from virus-mediated death (figure 2a) . to determine the efficiency and toxicity of these hsv-2 inhibitors, we measured the viability of mock-and virus-infected cells after 72 h using the ctg assay ( figure 2b ). the sis for obatoclax, emetine, niclosamide and ganciclovir were 12, 37, 3 and >750, respectively (table s4) . we titrated hsv-2 produced from drug-treated and non-treated cells in a540-npro cells ( figure 2c ). the experiment revealed that 0.12 µm obatoclax, 0.04 µm emetine, 0.37 µm niclosamide and 0.37 µm ganciclovir lowered the production of hsv-2 in rpe cells. thus, we identified novel anti-hsv-2 activities of obatoclax, emetine and niclosamide and confirmed the known anti-hsv-2 activity of ganciclovir. similarly, we examined 43 bsaas against wild-type ev1 in rpe cells. we monitored the viability of mock-and virus-infected cells by microscopy and the ctg assay. after the initial screening, we identified three compounds (obatoclax, emetine, and homoharringtonine), which rescued cells from virus-mediated death at none-cytotoxic concentrations. to determine the efficiency and toxicity of novel ev1 inhibitors, we measured the viability of mock-and virus-infected cells after 48 h using the ctg assay ( figure 3a ). the sis for obatoclax, emetine, and homoharringtonine were 25, >300, and >300, respectively (table s4) . we titrated ev1 produced from drug-treated and non-treated cells in a549-npro cells ( figure 3b ). the experiment revealed that all three compounds lowered the production of ev1, confirming novel antiviral activities of obatoclax, emetine, and homoharringtonine. we also examined the toxicity and antiviral activity of 43 bsaas against hiv-1-mediated firefly luciferase expression. the firefly luciferase open reading frame is integrated into the genome of tzm-bl cells under the hiv-1 ltr promoter. our primary screen identified two compounds (brequinar and suramin) that suppressed hiv-1-mediated firefly luciferase expression without detectable cytotoxicity. we performed a validation experiment with anti-hiv-1 compounds and calculated selectivity. the si for brequinar was >750, whereas the si for suramin was >375 (figure 4 ; table s4 ). thus, we identified novel activity of brequinar and confirmed known activity of suramin. in addition, we examined the toxicity and antiviral activity of 43 bsaas against rfp-expressing rvfv in rpe cells. both fluorescent microscopy and the ctg assay showed that obatoclax and emetine inhibited rvfv-mediated rfp expression at non-cytotoxic concentrations ( figure 5a-c) . the si for obatoclax was >100, and the si for emetine was >75 (table s4 ). next, we tested 43 bsaas against gfp-expressing hmpv. hmpv-mediated gfp expression and cell viability were measured after 72 h. after the initial screening, we identified two compounds, obatoclax and emetine, which lowered gfp expression without detectable cytotoxicity. replicate experiments confirmed these hits ( figure 6a-c) . the si for obatoclax was six and for emetine was 10 (table s4 ). next, we tested 43 bsaas against gfp-expressing fluav in rpe cells. after the initial screening, we identified two compounds, obatoclax and emetine, which lowered gfp expression without detectable cytotoxicity. we repeated the experiment with these compounds and confirmed initial hits ( figure 7a -c). the si for obatoclax was 31 and for emetine was >300 (table s4) . thus, we identified novel anti-fluav activity of emetine and confirmed known activity of obatoclax. to test whether the obatoclax-emetine combinations act synergistically against fluav-mediated gfp expression and rescue infected cells from death, the observed responses were compared with expected combination responses. the deviations in observed and expected responses showed no synergistic effect ( figure 7d ,e; figure s1 ). this result indicates that obatoclax and emetine target distinct cellular pathways essential for virus infection. here, we tested 43 safe-in-man bsaas against (−)ssrna, (+)ssrna, rt-ssrna and dsdna viruses and identified novel activities for five agents (table 1, figure s2 ). we identified novel activities of niclosamide against hsv-2, brequinar against hiv-1, homoharringtonine against ev1, obatoclax against hsv-2, ev1, hmpv and rvfv, and emetine against hsv-2, ev1, hmpv, rvf and fluav. we also confirmed antiviral activities of ganciclovir against hsv-2, suramin against hiv-1, and obatoclax against fluav [24, 30, 31] . our results pointed out that an evasion mechanism observed in one virus could be relevant for other viruses and that existing bsaas could be re-positioned to other viral infections. obatoclax was originally developed as an anticancer agent. several phase ii clinical trials were completed that investigated the use of obatoclax in the treatment of leukemia, lymphoma, myelofibrosis, and mastocytosis. in addition, its antiviral activity was reported against fluav, zikv, wnv, yfv, sinv, junv, lasv, and lcmv in vitro [24, 26, 32, 33] . it was shown that obatoclax inhibited viral endocytic uptake by targeting cellular induced myeloid leukemia cell differentiation protein mcl-1 [24] . given that obatoclax also inhibits rvfv, ev1, hmpv and hsv-2, it could be pursued as a potential bsaa candidate. emetine is an anti-protozoal drug. it is also used to induce vomiting. in addition, it possesses antiviral effects against zikv, ebov, rabv, cmv, hcov-oc43 and hiv-1 [34] [35] [36] [37] [38] . it was proposed that emetine can directly inhibit viral polymerases, though it may have some other targets as well [39] . given that emetine also inhibits fluav, rvfv, ev1, hmpv and hsv-2, it may represent a promising bsaa candidate. niclosamide is an orally bioavailable anthelmintic drug and potential antineoplastic agent. in addition, it inhibits the broadest range of viruses, including hsv-2, in vitro and, in some cases, in vivo [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] . it was shown that niclosamide induces endosomal neutralization and prevents virus entry into host cells. this supports the further development of niclosamide as a bsaa. homoharringtonine is an anticancer drug which is indicated for treatment of chronic myeloid leukemia. it also possesses antiviral activities against hbv, mers-cov, hsv-1 and vzv [50] [51] [52] [53] . homoharringtonine binds to the 80s ribosome and inhibits viral protein synthesis by interfering with chain elongation [51] . given that homoharringtonine also inhibits ev1, it may represent a promising bsaa candidate. brequinar is an investigational anticancer agent (phase i/ii). brequinar attenuates the replication of denv, wnv, yfv, lasv, junv, lcmv, vsv, hiv-1, and powv (nct03760666) [32, 54] . it inhibits dihydroorotate dehydrogenase, thereby blocking de novo pyrimidine biosynthesis, which is essential for the transcription and replication of viral rna. given that brequinar also inhibits hiv-1, it may represent a promising bsaa candidate. the human non-malignant rpe cell line represents an excellent model system for studying the infection of different viruses [17, 18, 24, 26] . in addition, different viral strains expressing reporter proteins, such as rvfv-rfp, hmpv-gfp and fluav-gfp, are excellent tools for drug screening [17, 18, 24, 27] . however, the number of novel and confirmed antiviral activities of bsas could be higher if we had used other cell lines and viral strains, different virus loads, different measurement endpoints, a different time of compound addition, as well as a higher purity and concentration range of 43 bsaas. moreover, antiviral properties of bsaas detected in cell-line-based assays might not be reproduced in vivo because systemic mechanisms may compensate the blocked target effect. thus, follow-up studies are needed to validate our initial hits. altogether, we expanded the spectrum of antiviral actions of niclosamide, brequinar, homoharringtonine, obatoclax and emetine in vitro. importantly, pk and safety studies have been performed on these compounds in laboratory animals and humans. this information could be used to initiate efficacy studies in vivo, saving time and resources. the most effective and tolerable bsaas or their combinations will have a global impact, improving the preparedness and protection of the general population from emerging and re-emerging viral threats and the rapid management of drug-resistant strains, as well as being used for first-line treatment or for prophylaxis of viral co-infections. bsaas could have a pivotal role in the battle against emerging and re-emerging viral diseases. the development of novel bsaas could save time and resources which are required for the development of their alternatives-virus-specific drugs and vaccines. in future, bsaas could have a global impact by decreasing morbidity and mortality from viral and other diseases, maximizing the number of healthy life years, improving the quality of life of infected patients, and decreasing the costs of patient care. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/11/10/964/s1. table s1 : compounds, their suppliers and catalogue numbers; table s2 : developmental status of broad-spectrum antivirals used in the study; table s3 : human viruses and associated diseases; figure s1 : the expected response of the emetine-obatoclax drug combination on the viability of fluav-and mock-infected rpe cells, as measured with the ctg assay using the bliss reference model; figure s2 available online: wwwwhoint/medicines/ebola-treatment/who-list-of-top-emerging-diseases/en emerging virus diseases: can we ever expect the unexpected? emerg. microbes infect global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study global, regional, and national disability-adjusted life-years (dalys) for 359 diseases and injuries and healthy life expectancy (hale) for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study approved antiviral drugs over the past 50 years infectious disease. combating emerging viral threats srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target experimental drugs poised for use in ebola outbreak an off-target effect of bx795 blocks herpes simplex virus type 1 infection of the eye repurposing of kinase inhibitors as broad-spectrum antiviral drugs the future of antivirals: broad-spectrum inhibitors approaches for identification of hiv-1 entry inhibitors targeting gp41 pocket drug repurposing: progress, challenges and recommendations editorial: drug repositioning: current advances and future perspectives review of drug repositioning approaches and resources expanding the activity spectrum of antiviral agents common nodes of virus-host interaction revealed through an integrated network analysis. front novel activities of safe-in-human broad-spectrum antiviral agents internalization of echovirus 1 in caveolae t7 rna polymerase-dependent and -independent systems for cdna-based rescue of rift valley fever virus an improved plaque reduction virus neutralization assay for human metapneumovirus rescue of influenza virus expressing gfp from the ns1 reading frame antiviral properties of chemical inhibitors of cellular anti-apoptotic bcl-2 proteins saliphenylhalamide, and gemcitabine inhibit influenza a virus infection anticancer compound abt-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism the proton translocation domain of cellular vacuolar atpase provides a target for the treatment of influenza a virus infections inhibition by cellular vacuolar atpase impairs human papillomavirus uncoating and infection synergyfinder: a web application for analyzing drug combination dose-response matrix data the anti-parasitic drug suramin potently inhibits formation of seminal amyloid fibrils and their interaction with hiv-1 ]methyl)guanine: a selective inhibitor of herpes group virus replication the reframe library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors obatoclax inhibits alphavirus membrane fusion by neutralizing the acidic environment of endocytic compartments high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry efficacy and mechanism of action of low dose emetine against human cytomegalovirus natural plant alkaloid (emetine) inhibits hiv-1 replication by interfering with reverse transcriptase activity emetine inhibits replication of rna and dna viruses without generating drug-resistant virus variants identification of broad-spectrum antiviral compounds by targeting viral entry arbidol and other low-molecular-weight drugs that inhibit lassa and ebola viruses the antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mtor niclosamide rescues microcephaly in a humanized in vivo model of zika infection using human induced neural stem cells niclosamide inhibits lytic replication of epstein-barr virus by disrupting mtor activation antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission identification of three antiviral inhibitors against japanese encephalitis virus from library of pharmacologically active compounds 1280 niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects thiazolides as novel antiviral agents. 2. inhibition of hepatitis c virus replication inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide anti-varicella-zoster virus activity of cephalotaxine esters in vitro the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs effect of cantharidin, cephalotaxine and homoharringtonine on "in vitro" models of hepatitis b virus (hbv) and bovine viral diarrhoea virus (bvdv) replication characterization of dengue virus resistance to brequinar in cell culture we thank jelena kiprovskaja and laivi karu for ordering consumables and handling mtas. we thank andres merits for creating excellent infrastructure to work with viruses at tuit. the authors declare no conflict of interest. key: cord-354052-x4ckzw64 authors: li, chunhua; li, zhen; zou, yong; wicht, oliver; van kuppeveld, frank j. m.; rottier, peter j. m.; bosch, berend jan title: manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination date: 2013-08-02 journal: plos one doi: 10.1371/journal.pone.0069997 sha: doc_id: 354052 cord_uid: x4ckzw64 porcine epidemic diarrhea virus (pedv) causes severe economic losses in the swine industry in china and other asian countries. infection usually leads to an acute, often lethal diarrhea in piglets. despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. we have established a reverse genetics system for pedv based on targeted rna recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the orf3 protein. using this system, we deleted the orf3 gene entirely from the viral genome and showed that the orf3 protein is not essential for replication of the virus in vitro. in addition, we inserted heterologous genes (i.e. the gfp and renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the orf3 gene. we demonstrated the expression of both gfp and renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. the new pedv reverse genetics system will enable functional studies of the structural proteins and the accessory orf3 protein and will allow the rational design and development of next generation pedv vaccines. porcine epidemic diarrhea virus (pedv) causes diarrhea and dehydration in newborn piglets. the virus infects the epithelial cells of the small intestine resulting in severe mucosal atrophy and consequent malabsorption. pedv is common and the cause of serious problems, particularly in pigs in asia. the disease usually appears in winter during which it can cause high fatalities in suckling piglets (see for a recent review [1] ). from 2010, an outbreak of pedv has swept china with over 1 million fatalities among newborn piglets causing substantial economic losses in the swine industry [2] . the characteristics of the infection and its epidemiology were quite dramatic with morbidity and fatality approaching 100% in one-week old piglets, despite the use of commercial, inactivated vaccines. virus transmission occurs via the fecal-oral route and possibly also by vertical transmission through lactation [2] . currently there is no efficient way of treatment of the disease. prevention of the infection usually relies on vaccination with cell culture adapted live-attenuated or inactivated viruses although the efficacy of current vaccines has been questioned [2, 3] . pedv belongs to the alphacoronavirus genus within the coronavirinae subfamily of the coronaviridae family. coronaviruses are important pathogens of concern for human and animal health. they occur in almost any species, usually causing respiratory or intestinal infections. interest in these viruses has increased significantly as a result of the sars epidemic in 2002 and 2003. coronaviruses are enveloped viruses and possess a positive-sense rna genome ranging from 26 to 32 kilobases, which is the largest viral rna genome known (fig. 1a) . the 59 two-third of the viral genome contains two large open reading frames (orfs), 1a and 1b, which encode two non-structural polyproteins, pp1a and pp1ab, that direct genome replication and transcription. the remaining part of the genome contains orfs specifying structural and non-structural proteins. they are expressed via a 39-terminal nested set of subgenomic messenger rnas, the transcription of which is regulated by conserved six-nucleotides transcriptionregulating sequences (trss; in pedv xua(a/g)ac [4] ). these subgenomic mrnas encode at least four structural proteins, three membrane anchored proteins called the spike (s), membrane (m) and envelope (e) protein, and the nucleocapsid (n) protein that encapsidates the genomic rna. the non-structural proteins expressed from the subgenomic mrnas encode one or more accessory proteins, which are specific for each coronavirus genus. the genome structures of alphacoronaviruses including pedv and related members such as the human coronavirus (hcov) strains 229e and nl63 show the typical set of essential core genes but they share only one accessory gene, orf3, located between the s and the e gene (fig. 1a) . the pedv orf3 gene encodes a 224 amino acids (aa) long protein with three to four predicted transmembrane domains [5] . entry of coronaviruses into their host cells is mediated by the approximately 200 kda large s glycoprotein. trimers of s form the characteristic spikes on the viral surface which interact with the host receptor and mediate membrane fusion. pedv was reported to utilize the porcine aminopeptidase n as a receptor [6] . yet, pedv is usually propagated in vero cells, which are derived from the african green monkey kidney, indicating that pedv can utilize non-porcine receptors for cell entry. propagation of pedv in cell culture requires addition of trypsin which is believed to prime or activate the s protein for membrane fusion during virus cell entry and syncytia formation [7] recently it was demonstrated that trypsin cleavage may also play a role in detachment of the virus from infected cells [8] . interestingly, a cell culture adapted strain was reported to replicate in the absence of trypsin [9] , which suggests that the virus acquired mutations in the s protein conferring its trypsin-independence. the s protein also stimulates the induction of neutralizing antibodies and hence is an important target in developing effective vaccines. research on the molecular biology and pathogenicity of pedv has been severely hampered by the lack of a reverse-genetic system. here we report the first reverse genetic system for pedv based on targeted rna recombination. establishment of the reverse genetic system included two stages (fig. 1b) . one was the generation of the chimeric virus mpedv, a pedv derivative carrying spikes derived from the murine coronavirus mouse hepatitis virus (mhv), hence growing only in murine cells. in the second stage the mpedv virus was used as a recipient virus to reintroduce the pedv spike along with other genome alterations, in casu the deletion of the orf3 gene or the insertion of foreign, reporter genes. the generated pedv derivatives now carrying again pedv spikes could be easily selected by their regained tropism for non-murine cells. to set up a targeted rna recombination system for pedv we first created a recombinant pedv virus carrying mhv spikes (mpedv). to this end a transfer vector p-mpedv was construced ( fig. 2a) that was composed of a 59-terminal genomic cdna fragment ligated to a cdna representing the entire 39-terminal part of the genome starting within orf1b, except for the s gene. this gene was replaced by a hybrid gene encoding a chimeric s protein composed of the 1,263 aa long ectodomain from mhv s and the transmembrane domain plus cytoplasmic tail (61 aa) from pedv s. rna was transcribed from the t7 promotor of this vector and electroporated into pedv-infected vero cells after which the cells were overlaid onto a murine cell (l cells) monolayer. the recombinant mpedv virus generated during subsequent incubation was cloned by two rounds of plaque selection on l cells. the identity of purified mpedv viruses was checked at a genetic level by rt-pcr sequencing of the orf1b-s gene junction (data not shown) and at the protein level by an immunofluoresence assay (fig. 3a ). all mpedv infected cells stained positive both with the polyclonal mhv serum and with the monoclonal antibody directed against the pedv nucleocapsid protein confirming the purity and the identity of the chimeric virus. in contrast to the parental virus, mpedv displayed the ability to induce syncytia in the absence of trypsin (fig. 3a) . as predicted, cell-cell fusion mediated by mpedv could be inhibited by a mhv s specific, peptidic fusion inhibitor (fig. 3b) . the generated mpedv virus was used as a recipient virus to reintroduce by similar procedures the pedv spike along with other genome modifications by targeted rna recombination. candidate recombinant viruses carrying the pedv spikes can be selected by their regained ability to replicate in vero cells. apart from the wild-type recombinant virus (r-wtpedv) we aimed at constructing a virus lacking the orf3 gene (pedv-dorf3). a number of cell culture adapted viruses including the strain used in this study have each acquired during passaging an identical 51 nucleotide in-frame deletion in the orf3 gene, giving rise to a 17 amino acid deletion (aa 82-98) in their orf3 protein [10] . we constructed a transfer vector (ppedv-dorf3, fig. 2a ) from which the entire orf3 gene was deleted. donor rnas transcribed from the ppedv and ppedv-dorf3 transfer vectors were electroporated into mpedv-infected l cells after which we were able to recover and purify the r-wtpedv and pedv-dorf3 viruses in vero cells. rt-pcr analysis confirmed the intended loss of the orf3 gene from the viral genome ( fig. 4a ) and the genetic identity of the orf3 lacking virus was further verified by sequencing of the rt-pcr product (data not shown). the pedv-dorf3 grew unimpaired in cell culture (fig. 4b) , demonstrating that the orf3 gene product is not required for virus propagation in vitro. in addition, the successful deletion of the orf3 gene from the viral genome demonstrated the feasibility of the mpedv-based targeted rna recombination system to manipulate the 39 end of the viral genome. we next explored the possibilities of expressing heterologous proteins from the pedv genome by inserting reporter genes at different genomic positions. transfer vectors were made with the renilla luciferase gene (936 nt) and the gfp gene (720 nt) at the position of orf3, creating the ppedv-dorf3/rluc and ppedv-dorf3/gfp vectors ( fig. 2a) . these marker genes are under the transcriptional control of the trs of orf3 (ctagac) which is located in the 39end of the s gene, 46 nucleotides upstream of the orf3 gene. the renilla luciferase gene was also inserted as an extra expression cassette between the orf1b and s gene, creating the ppedv-rluc vector. to this end the otherwise overlapping orfs 1b and s were first separated and a unique bamhi restriction site was introduced (p-rpedv vector, fig. 2a and b), which did not hamper the generation of a viable virus (data not shown). the renilla luciferase gene was subsequently cloned into the bamhi site of the p-rpedv vector under control of the trs in orf1b (gtaaac) originally driving s gene expression, whereas the s gene was provided with a new trs (gtaaac; fig. 2b ). the pedv-dorf3/gfp, pedv-dorf3/rluc and pedv-rluc recombinant viruses were successfully recovered by the targeted rna recombination procedure. rt-pcr analyses confirmed the insertion of both reporter genes at the intended positions ( fig. 5a) , which was further confirmed by sequencing. we studied the luciferase expression by the 2 recombinant viruses carrying a rluc gene as well as the expression kinetics of one of these viruses, pedv-rluc, upon infection of vero cells at three different moi's. the result shows (fig. 5b ) that luciferase expression levels were linearly related to the moi during the early phase of infection until 12 hours p.i. whereas at 24 hours p.i. luciferase values converged due to reinfections. similar kinetics of luciferase expression, but to higher levels, was observed for the pedv-dorf3/rluc recombinant virus (fig. 5b ). next we studied the gfp expression of the pedv-dorf3/gfp virus upon infection of vero cells at two moi's. gfp expression in pedv-dorf3/gfp virus infected cells could be seen starting from 9 hours p.i. and became clearly evident at 12 hours p.i. (fig. 5c ). the cell adapted pedv dr13 p100 strain can propagate in the absence of trypsin in the growth medium but does not form syncytia when trypsin is absent. yet the clustered appearance of gfp-positive cells suggests that the virus predominantly spreads locally from cell to cell which may correlate with the reported cell surface attachment of progeny viruses released from infected cells in the absence of trypsin [11] . the early detection of the luciferase and gfp reporter proteins during infection can be applied to develop a more rapid pedv neutralization diagnostic test. the readout of the classical virus neutralization assay with wild-type pedv is based on the visual inspection of cytopathic effect and can only be done after a multicycle infection which takes at least 2-3 days. thus, the pedv-dorf3/gfp and pedv-dorf3/rluc virus were preincubated with dilutions of serum obtained from an experimentally pedv-infected pig and control serum, and the mixtures were subsequently added to vero cells and incubated after which the gfp and renilla luciferase expression was recorded at 9 and 6 hours p.i., respectively (fig. 5d) . in contrast to the control serum, the pedv antibody-positive serum was able to neutralize pedv infection as reflected by the reduction of gfp positive cells and luciferase activity. the results demonstrate that neutralization of the pedv-dorf3/gfp and pedv-dorf3/rluc virus can already be scored within a single replication cycle, thereby significantly speeding up the assay time. this type of assay is additionally preferred as it avoids the subjectivity that is associated with scoring of cytopathic effects. here we describe the first reverse genetics system for pedv. as we illustrate, this system now enables the manipulation of the 39proximal ,8 kilobases of the pedv genome including the structural protein genes. generation of pedv recombinants was based on the well-known high efficiency of rna recombination of coronaviruses in combination with host cell tropism switching for selection of the recombinant viruses. similar recombination systems have been successfully developed for mhv and fipv coronaviruses by the masters and rottier laboratories [12, 13] . for a number of coronaviruses genetic engineering of the full length genome has also become accomplished by the development of infectious cdna clones [14] [15] [16] [17] [18] [19] [20] . the ability to manipulate the pedv genome will be extremely valuable to study the molecular and biological features of pedv infections as well as to develop new tools and strategies for prevention and therapy of this important veterinary pathogen. unlike most other coronaviruses, the pedv genome contains only a single accessory gene, the orf3 gene, which encodes a multispanning 224-aa long membrane protein. intriguingly, propagation of pedv isolates in tissue culture cells readily leads to deletions within orf3 suggesting a dispensable role, at least for the parts deleted from the orf3 protein, for viral replication in vitro. in all these adapted viruses a shorter orf3 gene product is still translated with a minimal size of 91 amino acids [10] . the orf3 gene of the cell-adapted dr13 vaccine strain (genbank accession no.: jq023162.1) employed in our study has a 49 nucleotide deletion compared to that of the parental dr13 virus (genbank accession no.: jq023161.1), but still encodes the nterminal 81 residues long orf3 protein part including the first transmembrane domain, after which it gets out of frame due to the deletion. the deletion of the entire orf3 gene from the genome the function of the pedv orf3 product remains enigmatic. recently it was shown that the protein exhibits ion channel activity and modulates virus production [5] . sirna knockdown of orf3 gene in pedv infected cells reduced the number of particles released from the cells [5] . the question remains here why passaging of pedv in cell culture would lead to the functional loss of a gene beneficial for virus propagation in vitro, unless the 91residue truncated protein still provides that function. homologues of the orf3 protein are found in all other alphacoronaviruses. the orf3 protein of hcov-nl63 was shown to be nglycosylated at the amino terminus and incorporated into virions [21] . yet, deletion of the orf3 gene from the viral genome had little effect on virus replication in cell culture [22] . like for pedv, loss of orf3 genes of the alphacoronaviruses tgev and hcov-229e (here named orf4) is associated with unimpaired virus passaging in cell culture [23, 24] . despite a non-essential role in cell culture, the maintenance of the orf3 gene in alphacoronavirus field isolates strongly points to an important role of the orf3 protein in natural infection in the animal host. consistently, the loss of virulence of life-attenuated pedv vaccine strains has been associated with mutations in the orf3 gene resulting from cell culture adaptation [10, 25] although a contribution of the numerous additionally acquired mutations in other genes such as the spike gene can obviously not be excluded [26, 27] . the specific function of the orf3 protein (and other viral proteins in the 39 genome region) in pedv replication and pathogenesis can now be further investigated using the reverse genetics system. the introduction of foreign genes at different genomic positions without apparent great fitness loss of the virus in vitro (data not shown) once more illustrates the remarkable genome plasticity of the coronavirus genome [28, 29] . the insertion of reporter genes like for gfp and luciferase will be very useful for the study of various molecular and virological aspects of pedv infection. in addition, as we demonstrate here, these reporter properties may also be exploited for applications such as the establishment of convenient virus neutralization assays that provide answers within hours rather than days. furthermore, genomic insertion of genes encoding foreign antigens using the reverse genetics system opens avenues to the development of pedv as a vaccine vector for protection against other relevant porcine pathogens in addition to pedv. l [12] and vero ccl81 cells (purchased from atcc) were maintained as monolayer cultures in dulbecco's modified eagle medium containing 10% fetal calf serum, 100 iu of penicillin/ml, and 100 mg of streptomycin/ml (all from life technologies, ltd., paisley, united kingdom). pedv (isolated from a commercial vaccine of greencross, south korea) was propagated in vero cells in the absence of trypsin. virus was harvested by three cycles of freeze-thawing the infected cells and supernatant followed by removal of cell debris by centrifugation at 3,0006g for 20 minutes. virus infectivity in the supernatant was measured by an end-point dilution assay on vero cells and 50% tissue culture infectious doses (tcid 50 ) were calculated. mhv (strain a59) was propagated in mouse l cells as described previously [12] . the rabbit anti-mhv serum k135 raised against purified mhv has been described elsewhere [30] . the monoclonal antibody (mab) 3f12 recognizing the pedv nucleocapsid protein was obtained from bionote, korea. polyclonal pedv serum from a pig experimentally infected with pedv (strain cv777) was kindly provided by dr. kristin van reeth (gent university). pedv antibody-negative control serum was obtained from a newborn piglet deprived of colostrum. ppedv vector. a cdna clone encompassing the 39-terminal 7,832 nt part of the pedv genome starting within orf1b was obtained by reverse transcription-pcr (rt-pcr) with viral genomic rna isolated from virions as a template and primers 4922 and 4815 as plus-and minus-strand primers (for primer sequences see table 1 ), respectively. the overhang of primer 4922 and primer 4815 contained a bglii and a paci restriction site, respectively. the bglii-paci digested fragment was cloned into the bamhi-paci digested pmh54 vector [12] , creating the plasmid ppedv-1b-3t. the 59-terminal 605 nt of orf1a was amplified using primers 4884 and 4885. primer 4884 contains a t7 polymerase recognition site, as well as a bglii restriction site and primer 4885 contained a bamhi restriction site. the bglii-bamhi digested fragment was ligated into the bamhi site of the ppedv-1b-3t plasmid, resulting in the ppedv vector. p-rpedv vector. a transfer vector was constructed in which the partly overlapping orf1b and s gene were separated by introduction of a unique bamhi site to facilitate further cloning. the stop codon of orf1b was mutated to taa to knock out the overlapping atg start codon of the spike gene. first, the forward primer 5127 containing the bamhi site and a trs (taaac), and the reverse primer 4815 containing a unique paci site were used to amplify the 39 proximal 7,332 nt of the pedv genome starting with the spike gene. this fragment was cloned into the bamhi-paci site of pmh54 vector, creating the ppedv-s-3t vector. second, primers 4884 and 4885 containing a bglii and bamhi site, respectively, were used to rt-pcr amplify the orf1a fragment which was introduced into the bamhi digested ppedv-s-3t vector creating the ppedv-1a-s-3t plasmid. third, primers 4922 and 4923 that contain a bglii and bamhi in the overhang, respectively, were used to amplify the orf1b fragment by rt-pcr. this fragment was cloned into the bamhi site of the ppedv-1a-s-3t vector, creating the p-rpedv vector. p-mpedv vector. first, the plasmid ptums [31] encoding the mhv spike was used as an intermediate vector to construct a chimeric spike composed of the ectodomain of mhv and the transmembrane and cytoplasmic domain of pedv. for the construction of the hybrid gene, a styi restriction site was used that is located in both s genes at the transition between the protein's ectodomain and transmembrane domain. the forward primer 4814 (styi site in overhang) and reverse primer 4924 (eagi site in overhang) were used to amplify the 39 end of the pedv s gene and downstream sequences and cloned into the styi-eagi digested ptums plasmid, creating the ptums(mp) vector. second, to create the p-mpedv vector, the pedv s gene in the p-rpedv 0.1 and fluorescence images were taken at different times p.i. nuclei of cells were stained with dapi (blue). (d) a rapid virus neutralization assay based on recombinant pedvs expressing reporter proteins. pedv-dorf3/rluc and pedv-dorf3/gfp (8,000 tcid50) were mixed with subsequent dilutions of serum positive for pedv antibodies and a negative control serum (n.c.) for 30 minutes at room temperature. mixtures were incubated with vero cells and renilla luciferase (left panel) or gfp (right panel) expression was measured at 8 and 9 hours p.i., respectively. doi:10.1371/journal.pone.0069997.g005 vector was replaced by the chimeric mhv-pedv spike gene by cloning the bamhi-pmli digested fragment of ptums(mp) into the bamhi-pmli digested p-rpedv vector. ppedv-dorf3 vector. primers 5300 and 5301 were used to amplify the e gene and downstream sequences using the ppedv vector as a template. the forward primer 5300 contained a pmli and an ecorv restriction site and the reverse primer 5301 contained an restriction econi site to facilitate further cloning. the pmli-econi digested pcr fragment was cloned into the pmli-econi digested ppedv vector to create the ppedv-dorf3 vector. ppedv-rluc and ppedv-dorf3/rluc vector. the renilla luciferase gene was excised from the prlnull vector (promega) using enzymes nhei and xbai, blunted with dnapolymerase i large (klenow) fragment and ligated into the bamhi digested and blunted p-rpedv vector or the ecorv digested ppedv-dorf3 vector, resulting in the ppedv-rluc and ppedv-dorf3/rluc transfer vector, respectively. ppedv-dorf3/gfp vector. the gfp gene was excised from the pegfp-n1 plasmid (clontech) with enzymes ncoi and noti, blunted with dna-polymerase i large (klenow) fragment and ligated into the ecorv digested ppedv-dorf3 vector yielding the ppedv-dorf3/gfp transfer vector. the identity of all generated transfer vectors was verified by sequencing. a targeted recombination system was established for pedv in a two-stage process as outlined in fig. 1b . stage 1 generation of mpedv. introduction of the hybrid mhv-pedv s gene into the pedv genome by targeted rna recombination was carried out essentially as described previously for mhv and fipv [12, 13] . briefly, capped runoff donor rna transcripts were synthesized from the paci-linearized p-mpedv vector using a t7 rna polymerase kit (ambion) as specified by the manufacturer. donor rna was electroporated (gene pulser electroporation apparatus [bio-rad]; two consecutive pulses of 0.3 kv/975 mf) into pedv-infected (multiplicity of infection [moi] of 0.4) vero cells (2610 7 cells) at 8 hours post infection (p.i.). the electroporated cells were co-cultured in a 25-cm 2 flask with 5610 6 murine l cells. after 48-60 h of incubation at 37uc, when syncytia could be detected in the murine l cells, progeny virus in the culture supernatant was harvested and mpedv recombinant virus was purified by two consecutive cycles of plaque purification on l cells at 37uc. stage 2 generation of recombinant pedvs. the construction of pedv recombinant viruses that had regained the pedv s gene was carried out in a reverse process by using ppedv-derived donor rnas and mpedv as the recipient virus. capped runoff transcripts were synthesized from paci-linearized ppedv, ppedv-rluc, ppedv-dorf3, ppedv-dorf3/rluc, or ppedv-dorf3/gfp, respectively, with a t7 rna polymerase kit (ambion) as specified by the manufacturer. the donor transcripts were electroporated (as specified above) into murine l cells (2610 7 cells) that had been infected 4 h earlier with mpedv (moi = 1). these cells were then plated onto a monolayer of vero cells. after 4-5 days of incubation at 37uc progeny virus in the culture supernatant was harvested by freeze-thawing and candidate recombinant viruses were purified by two rounds of end-point dilutions on vero cells. recombinant genotypes were confirmed by rt-pcr on purified genomic rna and subsequent sequencing. (immuno)fluorescence microscopy l cells and vero cells were inoculated with mhv, mpedv or pedv (moi = 0.05). after 2 hours of incubation the cells were washed with pbs and incubated in culture medium. at 6.5 hours p.i., the cells were rinsed with pbs and fixed with 3.7% formaldehyde for 20 min at room temperature. the cells were washed three times with pbs and incubated with the k135 rabbita-mhv serum and the 3f12 mouse mab a-pedv-n. after 30 min at room temperature, the cells were rinsed three times with pbs and stained with goat a-rabbit fitc-conjugated and donkeya-mouse cy3 conjugated secondary antibodies (cappel). nuclei were stained with dapi (molecular probes) for 10 min at room temperature. finally, the cells were washed three times with pbs and fluorescence was viewed with an evos-fl fluorescence microscope (advanced microscopy group) at 106 magnification. the evos-fl was also used to view gfp fluorescence from pedv-dorf3/gfp infected cells after paraformaldehyde fixation. vero cell monolayers were infected as described above with the pedv-rluc and pedv-dorf3/rluc viruses at indicated moi's. at indicated times post infection, cell lysate samples were assayed for luciferase activity using the renilla luciferase assay system (promega) according to the manufacturer's instructions, and the relative light units (rlu) were determined with a berthold centro lb 960 plate luminometer. pedv-dorf3/rluc or pedv-dorf3/gfp were mixed with serial dilutions of positive or negative piglet serum or with cell culture medium. the inoculum was incubated for 30 minutes at room temperature to allow virus neutralization before inoculating vero cell monolayers as described above. cells were either lysed at 8 hours post infection and assayed for renilla luciferase activity as described above or subjected to fluorescence microscopy as described above at 9 hours post infection. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. virus genes outbreak of porcine epidemic diarrhea in suckling piglets molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea pedv leader sequence and junction sites pedv orf3 encodes an ion channel protein and regulates virus production porcine aminopeptidase n is a functional receptor for the pedv coronavirus propagation of the virus of porcine epidemic diarrhea in cell culture role of proteases in the release of porcine epidemic diarrhea virus from infected cells derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate cloning and further sequence analysis of the orf3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses enhanced cell fusion activity in porcine epidemic diarrhea virus adapted to suckling mice retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier switching species tropism: an effective way to manipulate the feline coronavirus genome engineering the largest rna virus genome as an infectious bacterial artificial chromosome reverse genetics system for the avian coronavirus infectious bronchitis virus infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus a reverse genetics approach to study feline infectious peritonitis reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus systematic assembly of a fulllength infectious cdna of mouse hepatitis virus strain a59 strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model human coronavirus nl63 open reading frame 3 encodes a virion-incorporated n-glycosylated membrane protein systematic assembly of a full-length infectious clone of human coronavirus nl63 human coronavirus 229e encodes a single orf4 protein between the spike and the envelope genes efficacy of a transmissible gastroenteritis coronavirus with an altered orf-3 gene oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus dr13 mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo coronaviruses as vectors: position dependence of foreign gene expression coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization translation of three mouse hepatitis virus strain a59 subgenomic rnas in xenopus laevis oocytes nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex key: cord-258678-0atfsivf authors: liu, hong yan; gao, xiaohu title: a universal protein tag for delivery of sirna-aptamer chimeras date: 2013-11-07 journal: sci rep doi: 10.1038/srep03129 sha: doc_id: 258678 cord_uid: 0atfsivf sirna-aptamer chimeras have emerged as one of the most promising approaches for targeted delivery of sirna due to the modularity of their diblock rna structure, relatively lower cost over other targeted delivery approaches, and, most importantly, the outstanding potential for clinical translation. however, additional challenges must be addressed for efficient rna interference (rnai), in particular, endosomal escape. currently, vast majority of sirna delivery vehicles are based on cationic materials, which form complexes with negatively charged sirna. unfortunately, these approaches complicate the formulations again by forming large complexes with heterogeneous sizes, unfavorable surface charges, colloidal instability, and poor targeting ligand orientation. here, we report the development of a small and simple protein tag that complements the therapeutic and targeting functionalities of chimera with two functional domains: a dsrna binding domain (dsrbd) for sirna docking and a ph-dependent polyhistidine to disrupt endosomal membrane. the protein selectively tags along the sirna block of individual chimera, rendering the overall size of the complex small, desirable for deep tissue penetration, and the aptamer block accessible for target recognition. more interestingly, we found that extending the c-terminal polyhistidine segment in the protein tag to 18 amino acids completely abolishes the rna binding function of dsrbd. final large complexes (typically 100 s nanometers) and the chemical composition of the nanocarriers can drastically change chimera's targeting profile, in vivo biodistribution, and clearance 23 . furthermore, it is ideal to make the aptamer loop structure exposed and the sirna block hidden for specific binding, but electrostatic condensation with cationic nanocarriers does not warrant that selectivity. as demonstrated previously, immobilizing sirna-aptamer chimeras onto cationic nanoparticles via the sirna end offers significantly improved silencing effect compared to condensing chimeras onto cationic nanoparticles through random sites 24 . this is understandable since (1) exposure of the sirna end would only increase the chances of non-specific binding and reduce the stability sirna against enzymatic degradation; and (2) interaction between cationic nanocarriers with anionic aptamers could alter aptamers' conformation and targeting capability 25 . therefore, it is of critical importance to design a delivery system that is simple for potential regulatory approval and mass production, universal for all sirnaaptamer chimera, neutral and sirna-binding specific to ensure aptamer targeting, and small to avoid major alteration of chimera's biodistribution profile. a system simultaneously achieving these features could expedite clinically translation of the highly promising sirna-aptamer chimera technology. here, we report the development of a small protein tag for efficient delivery of sirna-aptamer chimeras. as shown in figure 1 , the protein tag is composed of two functional domains: a dsrbd used as a sirna docking module and a ph-dependent polyhistidine to help disrupt the endosomal membrane. the dsrbd is the n-terminal region (20 kda) of human protein kinase that binds dsrna in a sequence-independent fashion 26, 27 . because aptamers are typically ssrna with complex secondary structures, dsrbd does not bind with them (dsrbd only tolerates small bulges) and thus will selectively bind chimera through the sirna end, leaving the aptamer end accessible. to add endosomal escape functionality, a short histidine (his) oligomer is added to the c-terminus of the dsrbd. his has been incorporated into a number gene carriers because its endosomal buffering capacity promoting drug cytoplasmic release 28, 29 . his molecules have a pka value of approximately 6. at neutral ph (such as in circulation), they are mainly deprotonated (uncharged), which is desirable over positively charged counterparts due to reduced accumulation within the res (reticuloendothelial system). in acidic compartments such as endosome, his becomes protonated and facilitates osmotic swelling that leads to cargo release, a mechanism proposed as the proton sponge effect 30 . overall, this protein tag is equally small, simple, and biodegradable as sirna-aptamer chimera, while perfectly complementing chimera's functionalities. when complexed together, they remain small in size, discrete and stable in solution, low positive charge for circulation, and simultaneously achieve therapeutic, targeting, and endosomal escaping capabilities. expression and characterization of dsbrd-his 18 protein tag. to add endosomal escape capability, a short polyhistidine peptide was added to dsrbd. the dsrbd domain comes from the first 172 amino acids of human protein kinase r (hpkr), and has two double-strand rna binding motifs (dsrbm1 and dsrbm2) for cooperative and dsrna-specific binding 31 . because dsrbm1 towards the n terminal dominates the binding with dsrna 32 , we introduced the histidine peptide towards the c terminal ( figure 1 ) to minimize impact on dsrbd's biological activity. in theory, the endosomal escape capability should increase with longer his chain; on the other hand, long his chain could potentially interfere with dsrbd protein folding and binding. to achieve a balance, dsrbd with cterminal histidines of various lengths (his n , n 5 0, 12, 18, and 24) were cloned into the pet28a (1) vector. bamh1 and xho1 restriction enzyme sites were introduced to the 59-and 39-flanking region by pcr, respectively. because all the genetic constructs contain his 6 at the n-terminal from the cloning vector (this nterminal his 6 has been previously proved to have no impact on dsrbd binding) 26 , the total numbers of his encoded by the final constructs are 6, 18, 24, and 30, respectively (sequences see methods). post expression and purification, the resulted protein tags were analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page, figure 2a ). the sizes of four protein tags show in excellent agreement with theoretical values (figure 1 ). to assess their dsrna binding activity, sirna-aptamer chimera labeled with fluorophore fam were incubated with the protein tags and probed with gel electrophoresis (1% agarose). as shown in figure 2b , the dsrna binding capability of dsrbd with his 12 at the c terminus (total his 18 ) is well preserved compared with dsrbd without a cterminus histag insertion. the minimum rna length for high affinity binding with dsrbd has been determined to be 16 base-pairs 26 . at the current rna length, the sirna segment and the adjacent short stem in the aptamer structure can bind with 1-2 copies of dsrbd. however, it has been well documented that only the first dsrbd binds to rna stably, while, at high dsrbd/rna ratio, a second copy of dsrbd can bind, but at significantly lower affinity 26, 33 . using unmodified dsrbd and sirna alone, similar dsrbd-sirna binding profiles have been observed previously by kim and coworkers, who also show that the enzymatic stability of sirna is significantly enhanced upon binding with dsrbd 34 . it is important to mention that a key difference of our technology compared to these prior works utilizing dsrbd for sirna 27, 34 is that we do not introduce highly positively charged peptides. although positively charged nanocarriers promote sirna cell entry, it is well known that they are also quickly cleared by the res, increase non-specific binding with cells and cytotoxicity 35 . furthermore, as aforementioned, avoiding positive charges in carrier design is particularly important for sirna-aptamer chimera because excessive positive charges could non-specifically interact with aptamer and affect its targeting capability. more interestingly, the gel electrophoresis experiments also reveal that extending the c-terminal his by another 6 or 12 amino acids completely abolish dsrbd's binding activity. therefore, for the following gene expression regulation studies we chose the dsrbd with a total of 18 his due to its balanced dsrna binding and endosomal escape functionalities, in comparison with the original dsrbd with no c-terminus his as a control. design, synthesis, and characterization of sirna-aptamer chimera. to evaluate the universal protein tag for sirna-aptamer chimera, we first designed and made a chimera based on the protocols described by dassie and coworkers, taking advantage of the shortened aptamer sequence for specific targeting of psma as well as the optimized sirna strands with enhanced therapeutic potency 15 . the psma targeting aptamer was kept in our chimera, because psma has been identified as one of the most attractive cell surface markers for both prostate epithelial cells and neovascular endothelial cells 36 . accumulation and retention of psma targeting probes at the site of tumor growth is the basis of radioimmunoscintigraphic scanning (e.g., prostascint scan) and targeted therapy for human prostate cancer metastasis. we replaced their sirna sequence with a sirna silencing gfp expression, because gfp is the best model for quantitative assessment of the silencing effect using optical imaging and flow cytometry. the long ssrna composed of psma aptamer and sirna antisense strand ( figure 1 ) was prepared by in vitro transcription with the presence of 29 fluoro-modified pyrimidies for improved resistance to ribonucleases. it has been shown previously that 29-f modification is compatible with dsrbd binding unlike 29-h or 29-och 3 substitutes 26, 37 . the transcript was annealed to chemically synthesized sirna sense strand. before combining the chimera with our small protein tag, we first tested the activities of the chimera. to test the targeting function of the aptamer block, psma-positive lncap and psma-negative pc3 prostate tumor cells were incubated with dye-labeled chimera. as shown in figure 2c , the chimera selectively binds and enters lncap cells indicating targeting specificity. to test the silencing effect separately, the chimera was transfected into gfp-expressing c4-2 prostate tumor cells (a derivative of lncap) using conventional transfection agents, lipofectamine. as shown in figure 2d , the silencing effect is indistinguishable with the positive control using sirna only, proving that chimera can be enzymatically processed intracellularly to generate functional sirna. targeting delivery and silencing in cells. with the biological activities of our protein tag and sirna-aptamer chimera separately characterized, we proceeded to evaluate the gene silencing effect of this simple yet functionally highly complementary protein tag in sirna-aptamer chimera delivery. gfp-expressing c4-2 cell line was used as a model because of the advantages of fluorescence imaging techniques such as microscopy and quantitative flow cytometry. figure 3a -f shows confocal images of the c4-2 cells without treatment, treated with gfp-sirna alone, chimera alone, a random sequenced sirna with the protein tag (his 18 ), chimera with protein tag (his 6 ), and chimera with protein tag (his 18 ). qualitatively, only the experimental treatment, chimera with protein tag (his 18 ), clearly shows gfp silencing, whereas none of the five control treatments leads to significant suppression of gfp expression. quantitative flow cytometry studies further confirm this result (figure 3g-l) . at the current gate value set for gfp fluorescence intensity, the original untreated cells showed a gfp-negative population of 17.4%. treating the cells with a random sequenced sirna with protein tag (his 18 ) shows virtually no change in this population (difference: 5.4% of total cell population, within error range) proving sequence-specific silencing of rnai. for cells treated with gfp sirna and chimera, the gfp negative cells only increase by 7.6% and 12.2% of the total cell population respectively. even by increasing the chimera concentration by ten times (1 mm), the total gfpnegative cell population only increase by ,20% (supplementary figure s1) , strongly suggesting the need of carrier materials. direct comparison of the chimera tagged by dsrbd-his 6 and dsrbd-his 18 shows major difference in silencing efficiency, too (14.6% and 59.6% change). taken together, these results clearly indicate that (1) chimera alone at concentration commonly used in rnai experiments does not lead to effective silencing, and (2) his 18 is remarkably more effective than his 6 in endosomal destabilization since the dsrbd block is identical in structure and function. to put the silencing efficiency of dsrbd-his 18 in the context of those of conventional rna delivery vehicles such as lipofectamine, quantitative flow cytometry was also conducted. in agreement with the microscopy results shown in figure 2d , lipofectamine reduces gfp-negative cells from the original 17.4% to 91.6% (74.2% change, supplementary figure s2 ), which is slightly more efficient than the protein tag. however, it is important to note that lipofectamine delivers chimera into cells mainly via electrostatic interactions (positively charged lipofectamine and negatively charged cell surface, non-targeted delivery), whereas our protein tag delivers chimera by cell type-specific molecular recognition (targeted delivery). it is also worth mentioning that the molar ratio of mixing chimera with protein tag is 152 because the sirna block can bind up to 2 copies of dsrbd, although the second copy has very weak binding affinity. indeed, changing the binding ratio to 1 or 4 does not affect the rnai efficiency (supplementary figure s3) . to further confirm the difference in endosomal escape capability between the two protein tags (dsrbd-his 6 and dsrbd-his 18 ), we performed a dual color imaging assay using non-fluorescence lncap cells. in this experiment, chimera was labeled with cy3 and endosome/lysosome was marked with a lysotracker (spectrally distinguishable green fluorescence). direct contrast in chimera distribution and intracellular density of endosome/lysosome was observed between the two protein tags. as shown in figure 4 , cy3labeled chimera evenly distributes inside cells when tagged by dsrbd-his 18 , whereas dsrbd-his 6 treated cells show much higher density of endosomes and lysosomes and lower level of cy3 fluorescence. this confocal imaging comparison directly explains the difference between the two protein tags in rnai efficiency, and unambiguously demonstrates the superior endosome escape capability of dsrbd-his 18 over dsrbd-his 6 . cytotoxicity. lastly, we probed the cytotoxicity of the best performing protein tag dsrbd-his 18 using a standard cell viability assay (celltiter-blueh). the assay is based on the ability of living cells to convert a redox dye (resazurin) into a fluorescent end product (resorufin). nonviable cells lose metabolic capacity and thus do not generate fluorescent signals. as illustrated in figure 5 , virtually no toxicity was detected up to a concentration four times as high as the one used in the delivery work in reference to the untreated control. this is perhaps not too surprising due to the biocompatibility of dsrbd, a small protein of human origin. more importantly, for future in vivo applications, we envision that the small protein tag would have improved clearance capability compared with synthetic polymers and inorganic nanoparticles used for sirna delivery. discussion sirna-aptamer chimera is one of the most promising approaches for cell type-specific rnai, owing to its low immunogenicity, ease of chemical synthesis and modification, small size, and the modularity of both the targeting aptamer block and the therapeutic sirna segment. more importantly, employing only rna molecules, the simple formulation of chimera-based targeted sirna therapy leads to outstanding clinical translation 15, 16 . due to the incapability of chimera to efficiently escape endosome, delivery nanocarriers are needed. however, almost all current targeted sirna delivery formulations involve cationic nanocarriers such as polymers, inorganic nanoparticles, peptides, and proteins 7, 19, 20, 27, 28, [38] [39] [40] [41] [42] [43] [44] . unfortunately, these conventional sirna nanocarriers are unsuitable for chimera delivery, and, in fact, reverse the signature property of chimera, simple formulation for regulatory approval and clinical translation 15, 16 . this is because the charge induced complex formation is basically an aggregation process, which lacks control over aggregate size, shape, stoichiometry, chimera orientation, aptamer functionality, and reproducibility during scale-up production. in addition, the final complexes often carriers positive charges as well, which is unfavorable for systemic uses 23 . as a result, first clinical trials of sirna duplexes are mainly limited to local administrations [45] [46] [47] [48] . our protein tag does not rely on high positive charge to interact with rna molecules. in fact, it only recognizes relatively long dsrnas (.16 bp) such as the sirna segment and the short stem region of the aptamer in our chimera molecule. extensive biochemistry investigations have shown that for the current length of the chimera, maximum two copies of dsrbd can bind to it with differential affinity (the first copy binds much stronger than the second copy). the gene silencing experiments conducted here reflect this effect since mixing chimera with 13 or 23 protein tags does not affect the silencing efficiency. considering the molecular weights of the chimera (28.8 kda) and the protein tag (24.8 kda), molecular weight of the final complex at 151 binding will become 53.6 kda. based on well-documented size effect for in vivo drug delivery 49 , this size is sufficiently large to reduce premature renal clearance while still small enough for deep tissue penetration. for example, by tagging sirna-aptamer chimera with a 20 kda peg, its in vivo circulating half-life has been shown to increase from approximately 30 min to 30 hours 15 ; whereas large nanoparticles (.30 nm) have been shown to be ineffective in tumor treatment except for some hyperpermeable tumors 50 . in conclusion, to solve the endosome escape problem of the highly promising sirna-aptamer chimera based therapy, we have designed a dual-block small protein by combining dsrbd and polyhistidine and identified the optimal length of polyhistidine. the resulted protein tag shares the simplicity feature of sirna-aptamer chimera, yet offers exactly complementary functionalities. the dsrbd selectively binds to the sirna block, leaving the targeting aptamer accessible. in terms of size, different from conventional cationic delivery vehicles, the dsrbd-his 18 tagged chimera remains discrete in solution rather than forming large aggregates. in terms of functionalities, chimera and dsrbd-his 18 are highly complementary to each other, and thus offer the complete set of features necessary for targeted sirna delivery (e.g., targeting, therapeutic, sirna protection, and endosomal escape). this platform is also universal, able to chaperone any chimera sequences for cell type-specific delivery. largely based on natural proteins, dsrbd-his 18 is an excellent candidate for potential clinical translation because of its simple structure and biodegradability. further development of this small protein tag with in vivo testing should raise exciting opportunities for sirna clinical translation and personalized medicine. materials. vendors for specific chemicals are listed below. in general, restriction enzymes were obtained from new england biolabs, and cell culture products were purchased from gibco/invitrogen. chimera composed of aptamer targeting psma and sirna targeting gfp. ssdna of the psma aptamer (39 nucleotides, 59-gggaggacgatgcggatca-gccatgtttacgtcactcct-39) was chemically synthesized by integrated dna technologies (idt) and used as the template to generate one strand of the sirna-aptamer chimera. for amplification, pcr was performed with 39 primer containing the anti-sense strand of gfp sirna (underlined) and 59 primer containing t7 rna polymerase promoter site (bolded). the pcr primer sequences are: 39 primer: 59-ggcaagctgaccctgaagttcttttaggagtgacgta-aac-39 59 primer: 59-taatacgactcactatagggaggacgatgcgg-39 the 81 bp pcr product was put into t-a cloning pcr 2.1 vector (invitrogen). after sequencing, positive plasmids were selected and used as the template for pcr. the resulting pcr product was separated with 2% agarose gel and recovered with qiaex ii gel extraction kit (qiagen). the purified pcr product was used as the template for in vitro transcription with megascriptt7 kit (ambion) according to manufacturer's instruction. 29 fluoro-modified pyrimidines (trilink, san diego) were added to replace ctp and utp. rna molecules generated by the transcription reaction were annealed with the sense strand of gfp sirna (chemically synthesized with or without 59-cy3 or fam by idt). the sequence is 59-(cy3 or fam)-caagcugacccugaaguucuu-39. for annealing, the transcripted rna and the synthetic sirna sense strand were mixed at molar ratio 151 in duplex buffer (idt) and incubated at 94uc for 3 min followed by slow cooling to 25uc in 1 hour. the final chimera was store at 280uc. the constructs were cloned into pet28a (1) expression vector (novagen). the constructs for dsrbd-his 6 and dsrbd-his 18 were obtained using full-length pkr gene (clone id 8068981) as pcr template, and the dsrbd-his 24 and dsrbd-his 30 constructs were made by grafting additional histidines to the dsrbd-his 18 plasmid using pcr. the restriction enzyme sites for bamh1 and xho1 were introduced in the pcr primers for cloning. dsrbd-his 6 construct was introduced with two stop codons (taa and tga) before the xho1 site. for the other three constructs, the reading frames cover the his 6 sequence in the vector at the c-terminal end before the stop codon. the pcr products and pet28a (1) single colonies were selected and grown at 37uc for 12 h in circlegrow medium containing 30 mg/ml kanamycin. overnight cultures were diluted at 15100 (v/v) into fresh medium and incubated at 37uc until the od 600 values reach 0.5-1.0. expression was induced by addition of isopropyl-b-d-thiogalactopyranoside (iptg, 1 mm), and cell growth was continued for another 4-5 hour at 30uc. cells were harvested by centrifugation (beckman ja-10 rotor) at 10,000 g for 10 min and stored at 220uc. cells were suspended in bug-buster mix (novagen) with 5 ml reagent per gram of wet cell paste. bug buster mix was added with protease inhibitor edta-free cocktail (pierce), 10% glycerol, and 1.0 mm thp (novagen). the cell suspensions were incubated on a shaker platform for 30 min at room temperature. insoluble cell debris was removed by centrifugation (beckman tl120) at 20,0003 g for 20 min at 4uc. the soluble extracts were loaded onto affinity columns with ni-charged his bind resin (novagen). following washing with binding buffer and washing buffer, the desired proteins were eluted with 6 volume elution buffer (novagen). the eluted proteins were dialyzed with pbs containing 10% glycerol and 0.1% (v/v) b-mercaptoethanol for 24 hours. purified proteins were probed using 12% sds-page and stained with coomassie brilliant blue g-250 (bio-rad). protein concentrations were determined with the bio-rad protein assay with bovine serum albumin as the standard. functional characterization of sirna-aptamer chimera. to test the functionality of the sirna block, the chimera described above and gfp sirna control (qiagen) at a final concentration of 50 nm were transfected into c4-2 prostate cancer cells stably expressing gfp using lipofectamine rnai max (invitrogen) following the instructions provided by the manufacturer. to evaluate the targeting specificity of the aptamer block, psma-positive lncap cells and psma-negative pc3 cells were treated with complex of chimera and dsrbd-his 18 (chimera/protein tag molar ratio at 152, 100 nm chimera) in serum free medium for 2 hours, followed by incubation in complete medium for another 12 h. dapi (30 nm) was added to stain cell nuclei. fluorescent images were captured on an olympus ix-71 inverted microscope equipped with 5 long-pass filters and a colored ccd camera. characterization of rna binding capability of the four protein tags. the binding capabilities of the four polyhistidine modified dsrbd proteins were evaluated by native agarose gel. the chimera was labeled with fam at the 59 end of sirna's sense strand (idt). to prepare chimera/dsrbd complex, chimera (5 mm, 10 ml) was incubated with the protein tags at protein/chimera molar ratios of 1, 2, or 4 for 1 h at 4uc. bound chimera and unbound chimera were quantified on 1% agarose gel using a macro imaging system (lightools research, ca). evaluation of endosomal escape. psma-expressing lncap cells were seeded on 35 mm glass-bottom petri dishes (matteck corp) at a density of 5 3 10 4 cells/well for 24 hours in rpmi 1640 supplemented with 10% fcs. complexes of chimera labeled with cy3 (idt) and protein tags (his 6 and his 18 ) were added to lncap cells in serum-free medium for 2 hours, followed by incubation in complete medium for 12 hours. lysotrackerh green dnd-26 (80 nm, invitrogen) was then added for 4 hours at 37uc. images were captured on a confocal laser scanning microscope (lsm 510, carl zeiss, germany). microscopy and flow cytometry studies of gene knockdown efficacy. c4-2 prostate cancer cells expressing gfp were seeded into 35 mm glass-bottom petri dishes for confocal imaging or 6-well plates for flow cytometry. cells were treated with chimera & dsrbd-his 18 and compared with five control groups including no treatment, treated with gfp-sirna alone, chimera alone, a random sequenced sirna with the protein tag (his 18 ), and chimera with protein tag (his 6 ) for 2 h in serum free media and then incubated in complete media for 60 h. confocal images were again obtained with lsm 510 confocal microscope equipped with argon (488 nm) and hene (543 nm) lasers; and quantitative flow cytometry investigation was done on a bd facscantoii flow cytometer. cytotoxicity assay. lncap cells were seeded in 96-well plate at 4 3 10 3 /well for 24 hours, and then treated with different concentrations of dsrbd-his 18 protein tag for 72 hours. celltiter-blue reagent (20 ml) was added into each well. after 4 h incubation at 37uc, cell viability was assessed by fluorescence intensity at 590 nm (excitation 570 nm) on a tecan infinite m200 microplate reader. killing the messenger: short rnas that silence gene expression approaches for the sequence-specific knockdown of mrna knocking down barriers: advances in sirna delivery visualizing a correlation between sirna localization, cellular uptake, and rnai in living cells sirna conjugate delivery systems lung delivery studies using sirna conjugated to tat(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity efficient in vivo delivery of sirna to the liver by conjugation of alpha-tocopherol therapeutic silencing of an endogenous gene by systemic administration of modified sirnas mechanisms and optimization of in vivo delivery of lipophilic sirnas selection of single-stranded-dna molecules that bind and inhibit human thrombin in vitro selection of rna molecules that bind specific ligands biochemistry -adaptive recognition by nucleic acid aptamers systemic administration of optimized aptamer-sirna chimeras promotes regression of psma-expressing tumors cell type-specific delivery of sirnas with aptamer-sirna chimeras nanotechnology and aptamers: applications in drug delivery targeted quantum dot conjugates for sirna delivery layer-by-layer assembled gold nanoparticles for sirna delivery in vivo imaging of sirna delivery and silencing in tumors engineered design of mesoporous silica nanoparticles to deliver doxorubicin and p-glycoprotein sirna to overcome drug resistance in a cancer cell line polyethyleneimine coating enhances the cellular uptake of mesoporous silica nanoparticles and allows safe delivery of sirna and dna constructs factors affecting the clearance and biodistribution of polymeric nanoparticles sirna-aptamer chimeras on nanoparticles: preserving targeting functionality for effective gene silencing systematic investigation of optimal aptamer immobilization for proteinã¢'microarray applications minor-groove recognition of double-stranded rna by the double-stranded rna-binding domain from the rna-activated protein kinase pkr efficient sirna delivery into primary cells by a peptide transduction domain-dsrna binding domain fusion protein histidine-rich peptides and polymers for nucleic acids delivery chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers the proton sponge: a trick to enter cells the viruses did not exploit structure of the double-stranded rna-binding domain of the protein kinase pkr reveals the molecular basis of its dsrna-mediated activation 2 rna-binding motifs in the double-stranded rna-activated protein-kinase, dai specific recognition of hiv tar rna by the dsrna binding domains (dsrbd1-dsrbd2) of pkr intracellular small interfering rna delivery using genetically engineered double-stranded rna binding protein domain self-assembling micelle-like nanoparticles based on phospholipidpolyethyleneimine conjugates for systemic gene delivery metastatic renal cell carcinoma neovasculature expresses prostate-specific membrane antigen nucleoside modifications modulate activation of the protein kinase pkr in an rna structure-specific manner the first targeted delivery of sirna in humans via a self-assembling, cyclodextrin polymer-based nanoparticle: from concept to clinic electrostatic ligand coatings of nanoparticles enable ligand-specific gene delivery to human primary cells rna interference in vitro and in vivo using a novel chitosan/ sirna nanoparticle system the influence of polymeric properties on chitosan/sirna nanoparticle formulation and gene silencing quantum dot-amphipol nanocomplex for intracellular delivery and real-time imaging of sirna emerging application of quantum dots for drug delivery and therapy quantum dots as a platform for nanoparticle drug delivery vehicle design inhibition of respiratory viruses by nasally administered sirna evaluation of the safety, tolerability and pharmacokinetics of aln-rsv01, a novel rnai antiviral therapeutic directed against respiratory syncytial virus (rsv) using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque small interfering rna (sirna) targeting vegf effectively inhibits ocular neovascularization in a mouse model clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats accumulation of sub-100 nm polymeric micelles in poorly permeable tumours depends on size this work was supported in part by nih (r01ca140295, ca150301), the department of bioengineering, and the office of research at university of washington. we are also grateful to drs. eva corey and bob vessella for their prostate tumor cell lines and fruitful discussions. key: cord-331414-i0oxm5mr authors: kautz, tiffany f.; jaworski, elizabeth; routh, andrew; forrester, naomi l. title: a low fidelity virus shows increased recombination during the removal of an alphavirus reporter gene date: 2020-06-19 journal: viruses doi: 10.3390/v12060660 sha: doc_id: 331414 cord_uid: i0oxm5mr reporter genes for rna viruses are well-known to be unstable due to putative rna recombination events that excise inserted nucleic acids. rna recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. to examine this, gfp was inserted into tc-83, a live-attenuated vaccine for the alphavirus venezuelan equine encephalitis virus, as well as a low-fidelity variant of tc-83, and passaged until fluorescence was no longer observed. short-read rna sequencing using clickseq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. a rapid removal of the gfp gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. eventually, these small deletions merged to fully remove the gfp gene. the removal was significantly enhanced during the passaging of low-fidelity tc-83, suggesting that increased levels of recombination are a defining characteristic of this mutant. rna viruses are notoriously error-prone due to the lack of an rna-dependent rna polymerase (rdrp) proofreading enzyme (with the unique exception of proofreading exonucleases present in coronavirus genomes). this error results in approximately one mutation per round of genome replication [1] , which due to the rapid replication of rna viruses, culminates in a broad cloud of related virus genomes, commonly referred to as viral intra-host diversity, and in specific cases as a quasispecies. if the intra-host viral population is either too diverse or too clonal, this can impact the fitness of the virus population, which commonly results in a reduction in host tropism or virulence [2] [3] [4] [5] . while the quasispecies theory has been studied extensively in silico, in vitro, and in vivo by analyzing the ratios of nucleotides at specific genomic sites [5] [6] [7] , the contribution of recombination between rna virus minority variants and the effects on genome maintenance and evolution have been comparatively understudied. rna virus recombination primarily occurs when the rdrp switches from the template rna strand to an acceptor rna strand (i.e., copy choice recombination) or, less frequently, by ligation of cleaved rna strands [8] . both homologous recombination, where the rdrp switches to a template with high sequence similarity, and nonhomologous recombination, where the rdrp switches to a template with low sequence similarity, can occur [9] . the molecular determinants of rna recombination are not well-defined, but rna recombination has been described for viruses such as poliovirus and brome-mosaic virus to arise more frequently in regions with high amounts of rna secondary structure and au-rich sequences [9, 10] . additionally, other factors, such as sequence homology between rna strands and altered replication kinetics have been implicated in changing the rate of rna recombination [9] . rates of rna recombination vary between different families of rna viruses. for example, picornaviruses have relatively high rates of recombination [11] compared to families such as flaviviridae where viable recombination is less common [12] . venezuelan equine encephalitis virus (veev) is an alphavirus from the family togaviridae. alphavirus genomes are composed of a positive-sense, monopartite single-stranded rna that is approximately 11.5 kb in length. the first two-thirds of the genome encodes the nonstructural proteins, while the last third of the genome encodes the structural proteins under a subgenomic promoter [13] . inter-strain of inter-species rna recombination is not thought to be common for members of the alphavirus genus due to superinfection exclusion [14] [15] [16] , which occurs rapidly upon cellular infection and precludes the high multiplicity infections believed to be required for frequent recombination events between co-infecting viral genomes [17] . nonetheless, alphavirus coinfection has been described [18] , and it is thought that the recombination of sindbis and eastern equine encephalitis virus-like ancestors, two different alphaviruses, produced western equine encephalitis virus [19, 20] . conversely, the formation of defective viral genomes (dvgs) of alphaviruses has been well described for numerous family members. dvgs are generated due to nonhomologous rna recombination events either intra-molecularly or between two copies of a viral genome resulting in shorter recombined versions of the parental genome missing critical regions required for autonomous replication. we have extensively characterized rna recombination in the alphavirus, chikungunya virus, and demonstrated that rna recombination events are plentiful even under low moi and low passage conditions resulting in the synthesis of dvgs de novo after viral infection [21] . beyond the role that rna recombination plays during the natural replication-cycle of rna viruses both in vitro and in vivo, it is important to characterize the role of rna recombination in the stability of recombinantly engineered strains of rna virus containing inserted reporter genes (e.g., green fluorescent protein (gfp), luciferase). increasing the understanding of where these recombination events occur may allow for a more rational design of how to insert foreign genetic material into viral genomes to increase their genetic stability, as well as how to reduce the impact that these insertions commonly have on virus fitness. here, we inserted gfp into a live-attenuated vaccine for venezuelan equine encephalitis virus, tc-83, and passaged the virus using a physiologically relevant low multiplicity of infection (moi). illumina sequencing using clickseq library preparation [22] was used to determine minority variant recombination events in the genome. we found a slow but persistent removal of the gfp gene that was highly specific to the inserted gene. additionally, we found that a low-fidelity variant of tc-83 [23] removed the gfp reporter gene much more efficiently. vero (african green monkey kidney) cells were obtained from the american type cell collection (atcc, bethesda, md, usa) and maintained in dmem (gibco, gaithersburg, md, usa) supplemented with 10% fetal bovine serum (atlanta biologicals, flowery branch, ga, usa), and 500 µg/ml gentamycin (corning, corning, ny, usa) in a 37 • c 5% co 2 incubator. a tc-83 gfp infectious clone was kindly provided by dr scott weaver (utmb), and standard cloning protocols were used to insert the gfp gene into the low-fidelity tc-83 genome. as described previously [23] , low-fidelity tc-83 contains three coding mutations to the rdrp, which were found to lower the mutation frequency produced by this mutant during replication. following this, the plasmids were transcribed, and the virus was rescued by electroporation (ep) using bhk cells. titers of the ep stock virus were determined by the plaque assay using vero cells [24] . one day prior to infection, a six-well plate was seeded with 500,000 vero cells. the day of infection, the tc-83 gfp or tc-83 3x gfp (low-fidelity tc-83) virus was diluted 10,000-fold for an moi of approximately 0.5. once diluted, media was decanted from the six-well vero cell plate, and 200 µl was used to infect each well. these infections were done using three replicates per virus. following infection, the plate was transferred to a 37 • c 5% co 2 incubator for 1 h with occasional rocking. when the incubation was complete, the inoculum was removed, and 2ml of dmem (gibco, gaithersburg, md, usa) supplemented with 2% fbs (atlanta biologicals, flowery branch, ga, usa) and 500 µg/ml gentamycin (corning, corning, ny, usa) was added to each well. the plates were then incubated for 24 h in a 37 • c 5% co 2 incubator. following this, the cell supernatant was removed, transferred to a tube with fbs (atlanta biologicals, flowery branch, ga, usa) to a final concentration of 20% fbs, and centrifuged at for 5 min at 0.8 rcf to remove debris. virus aliquots were stored at −80 • c. plaque assays were used to determine virus titer [24] . ten total passages were performed for each virus and replicate. to determine pfu:gfp, plaque assays were performed as per the usual protocol [24] . after 48 h, the cells were visualized under a fluorescent microscope, and fluorescent plaques were counted. following this, the cells were fixed and stained following the standard plaque assay protocol, and the number of plaques was counted. the number of gfp to plaque forming units (pfu) was then calculated as a ratio (i.e., # plaques/# gfp fluorescent foci) to determine the amount of gfp fluorescence lost over the course of passaging. viral rna was extracted using the zymo direct-zol rna mini kit as per the manufacturer's protocol (zymo, irvine, ca, usa). rna sequencing libraries were made following the standard clickseq protocol using 100 ng of viral rna, as previously described [22, 25] . clickseq was used instead of the traditional illumina library procedures to avoid the errant recombination that frequently occurs during the typical fragmentation and ligation steps. cdna libraries between 400-700 bp were excised and purified using the zymo research gel recovery kit (zymo, irvine, ca, usa). sequencing was performed by the university of texas medical branch next-generation sequencing core using an illumina nextseq 550 obtaining 1 × 150 bp reads. cutadapt [26] was used to remove adaptor sequences. following this, the fastx toolkit [27] was used to remove the random hexamer sequences generated during cdna synthesis, as well as sequences with a phred score below 20. bowtie [28] was used to align the virus sequences to the reference genome (accession number, l01443), allowing for one mismatch. those sequences that did not align using bowtie were processed using virema [29] , a viral recombination mapper, to determine the incidence and location of recombination events. these reads were normalized to the average number of reads aligning to the virus genome. m-fold [30] was used from nucleotide position 6501 to the end of the virus genome to determine the secondary structure of the virus genomes and how this related to the observed recombination events. raw fastq data are available on the ncbi small read archive under bioproject id prjna634635. to examine reporter gene stability, three independent replicates of tc-83 gfp and low-fidelity tc-83 gfp were passaged 10 times on vero cells using an approximate moi of 0.5 ( figure 1a ). over the course of this passaging, reduced gfp expression for tc-83 was observed beginning at passage 8 ( figure 1b ). in contrast, gfp loss was observed much earlier for the low-fidelity tc-83 replicates, typically by passage 4 ( figure 1c ). viruses 2020, 12, x for peer review 4 of 13 to examine reporter gene stability, three independent replicates of tc-83 gfp and low-fidelity tc-83 gfp were passaged 10 times on vero cells using an approximate moi of 0.5 ( figure 1a ). over the course of this passaging, reduced gfp expression for tc-83 was observed beginning at passage 8 ( figure 1b ). in contrast, gfp loss was observed much earlier for the low-fidelity tc-83 replicates, typically by passage 4 ( figure 1c ). to quantify this loss, the number of fluorescent plaques was compared to the number of overall plaques for passages 5-10. until passage 10, the majority of tc-83 plaques were fluorescent, and the largest pfu:gfp difference was approximately 1:30, which was only observed for one replicate during the final passage ( figure 1d ). in contrast to this, all low-fidelity tc-83 replicates experienced an approximately 500-1500-fold decrease in fluorescence by passage 10, which was orders of magnitude higher than the ratios observed for the tc-83 gfp passage replicates ( figure 1e ). as a low-fidelity sindbis virus mutant was previously found to produce defective interfering (di) particles at high rates [31, 32] , we hypothesized that the low-fidelity tc-83 was also recombining at higher rates than tc-83, resulting in rapid gfp gene removal. to quantify this loss, the number of fluorescent plaques was compared to the number of overall plaques for passages 5-10. until passage 10, the majority of tc-83 plaques were fluorescent, and the largest pfu:gfp difference was approximately 1:30, which was only observed for one replicate during the final passage ( figure 1d ). in contrast to this, all low-fidelity tc-83 replicates experienced an approximately 500-1500-fold decrease in fluorescence by passage 10, which was orders of magnitude higher than the ratios observed for the tc-83 gfp passage replicates ( figure 1e ). as a low-fidelity sindbis virus mutant was previously found to produce defective interfering (di) particles at high rates [31, 32] , we hypothesized that the low-fidelity tc-83 was also recombining at higher rates than tc-83, resulting in rapid gfp gene removal. clickseq was used to generate illumina rna sequencing libraries, allowing for the determination of recombination hotspots as well as the frequency of virus deletion mutants. all three tc-83 gfp replicates contained deletion variants present at a low frequency that were able to selectively remove the gfp reporter gene by passage 10 (figure 2a) . the deletion inside the gfp gene was higher than for any other gene and occurred as soon as passage 1. in support of our hypothesis, low-fidelity tc-83 gfp was much more efficient at removing the gfp gene, as the frequency of deletions within the gfp gene was far higher than those of tc-83 gfp, which was consistent among all replicates ( figure 2b ). the first replicate of each virus was chosen for further analysis, as these both had median levels of decreased gfp fluorescence in figure 1d ,e. for both viruses, peak recombination first appeared in the middle of the gfp gene, and progressively removed additional nucleotides over the course of passaging until the entire gfp gene was removed (figure 3 ). low-fidelity tc-83 was able to efficiently remove the entire gfp fragment by passage 3, while tc-83 required five additional passages to reach similar levels of gfp removal. this clearly demonstrates that low-fidelity tc-83 gfp produces recombination variants more frequently than tc-83 gfp. figure 3 . virema recombination results for tc-83 gfp and low-fidelity tc-83 gfp. each passage of the first replicate of tc-83 gfp and low-fidelity tc-83 gfp was sequenced using clickseq libraries and analyzed using virema. the x-axis shows genome position, while the y-axis shows the recombination frequency (i.e., normalized to the total reads that map to low-fidelity tc-83 gfp). below the recombination charts, a schematic shows the approximate location of the nonstructural protein, gfp, and structural genes. "p" represents passage. to determine if the regions where recombination occurred most frequently were conserved between replicates, the top 5 recombination events for each virus and replicate were identified (supplemental file 1). with few exceptions, all of the most common recombination events were deletions found within and around the gfp gene, regardless of the virus backbone. of the top five recombination events for each passage and replicate, all the replicates of low-fidelity tc-83 predominately exhibited a deletion from nucleotide positions 7899-7990 for the first five passages. this happened concurrently with another deletion event, 7894-7985, which occurred slightly less frequently than the 7899-7990 deletion. following the appearance of these two deletions, a much larger removal occurred from nucleotides 7894-8421, which encompassed the entire gfp gene ( figure 4b ). in one replicate, this deletion was found to be one of the most predominant deletions after only one passage, and in another replicate, this became the predominate deletion by passage 2. by passage 5, this 7894-8421 deletion quickly became the most common deletion event by 1-2 orders of magnitude for all low-fidelity tc-83 gfp replicates. unlike the low-fidelity mutant, tc-83 did not show the same conservation of deletions between replicates and passages, but, like low-fidelity tc-83, the 7899-7990 deletion was one of the most frequently observed. the other two deletions observed during low-fidelity tc-83 passaging were also sporadically found as one of the five most common recombination events, but not consistently between replicates. however, while not always the most common of mutations, these three mutations still occurred during tc-83 gfp passaging and at levels comparable to the low-fidelity tc-83 virus ( figure 4a) . conversely, the level of the variance observed between the replicates of tc-83 gfp and low-fidelity tc-83 gfp was large, with higher variances observed between the replicates for tc-83 gfp than for low-fidelity tc-83 gfp. other than these three deletions, all tc-83 gfp replicates produced the majority of the deletions surrounding nucleotides 7559-7574. the length and frequency of these deletions were variable but suggested that this region of the gfp gene was the most amenable to deletions and insertions of various forms. to better understand the mechanisms underlying the conserved removal of the low-fidelity tc-83 gfp gene, m-fold was used to determine the rna secondary structure of the virus genome ( figure 4c-e) . the 7894-7895 and 7899-7990 deletions were found close together in the primary rna sequence, and the sequence found between 7894-7899 was identical to the sequence found between 7895-7990, which was indicative of homologous recombination. this five base pair sequence (ggcaa) was similarly structured between 7894-7899 and 7985-7990, with three g-c nucleotides forming a small stem that led into a loop. as previously stated, once these two deletions occurred, a 7562-8421 deletion surrounding the gfp gene appeared and rose to prominence. like the other two deletions, a nearly identical sequence was found at the beginning and end of the deletion (cuaga vs. cuaaga), showing micro-homology at the site of the recombination juncture. unlike the previous deletion, these sequences were found in a loop structure and were far away from each other in the predicted secondary structure. viruses 2020, 12, x for peer review 9 of 13 figure 4 . frequency of the three most common deletion events during tc-83 gfp and low-fidelity tc-83 gfp passaging and rna structure locations of these events in low-fidelity tc-83 gfp. as tc-83 gfp (a) and low-fidelity tc-83 gfp (b) were passaged, three deletions (7894-7895, 7899-7990, and 7562-8421) rose to prominence, especially in the low-fidelity passages. frequency of the recombination events was determined by dividing the number of recombination events by the total number of sequencing reads. if one of the three deletion events depicted above was not found for a passage replicate (i.e., below the limit of detection), no data point was depicted. the rna structure of low-fidelity tc-83 was determined using m-fold (g = −1757.85) (c-e). the gfp gene (nucleotides 7581-8300) is highlighted in yellow (c). during passages 1-5, the nucleotide deletions 7894-7985 and 7899-7990 (yellow highlight) concurrently arose in all replicates (d). an identical nucleotide sequence, ggcaa, was found at the beginning and end of these initial deletions. after these deletions, a 7562-8421 nucleotide deletion (yellow highlight) arose and became the dominant deletion mutant in the population, resulting in the total excision of the gfp gene (e). a nearly identical sequence was found at the beginning and end of this deletion juncture (cuaga vs. cuaaga). figure 4 . frequency of the three most common deletion events during tc-83 gfp and low-fidelity tc-83 gfp passaging and rna structure locations of these events in low-fidelity tc-83 gfp. as tc-83 gfp (a) and low-fidelity tc-83 gfp (b) were passaged, three deletions (∆7894-7895, ∆7899-7990, and ∆7562-8421) rose to prominence, especially in the low-fidelity passages. frequency of the recombination events was determined by dividing the number of recombination events by the total number of sequencing reads. if one of the three deletion events depicted above was not found for a passage replicate (i.e., below the limit of detection), no data point was depicted. the rna structure of low-fidelity tc-83 was determined using m-fold (∆g = −1757.85) (c-e). the gfp gene (nucleotides 7581-8300) is highlighted in yellow (c). during passages 1-5, the nucleotide deletions 7894-7985 and 7899-7990 (yellow highlight) concurrently arose in all replicates (d). an identical nucleotide sequence, ggcaa, was found at the beginning and end of these initial deletions. after these deletions, a 7562-8421 nucleotide deletion (yellow highlight) arose and became the dominant deletion mutant in the population, resulting in the total excision of the gfp gene (e). a nearly identical sequence was found at the beginning and end of this deletion juncture (cuaga vs. cuaaga). it is well known that the virus reporter genes (e.g., gfp, luciferase) commonly used to answer questions related to virus lifecycles are unstable, with papers commonly reporting a 10-50% loss of fluorescence upon one virus passage [33] [34] [35] [36] [37] . however, it is not well understood how reporter gene removal occurs in virus populations because previous research has typically been limited to the use of low-throughput sequencing methods, generally by sanger sequencing of a handful of virus clones surrounding a small area of the virus genome. rnaseq library synthesis methods are often prone to the generation of chimeric short-reads, giving rise to artifactual recombination in the output data obfuscating the detailed analysis of rare and low-frequency rna recombination [22] . however, the clickseq method for rnaseq library preparation is specifically designed to reduce this artifact, thus allowing for the robust analysis of minority recombination events across the entire virus genomes. in this paper, we used clickseq to better understand how gfp is removed when inserted into an alphavirus genome under a double subgenomic promoter. this technique allowed us to visualize the thousands of recombination events that occurred during virus passaging, which were predominantly found within the inserted gfp reporter gene. we originally hypothesized that gfp removal would primarily occur across the homologous promoter sequences surrounding the gfp gene. however, while true in later passages, the first gfp deletion events actually occurred in the middle of the gfp gene via the removal of smaller segments, and thus inactivating gpf expression before spreading outwards towards the promoter sequences. this removal was enhanced in a low-fidelity tc-83 variant. perhaps counterintuitively, the specific nucleotide deletions within the gfp gene were more conserved among the low-fidelity tc-83 gfp replicates than the wild type virus. this is similar to previous research with a low-fidelity sindbis virus where a conserved deletion mutant was found at elevated levels during its passaging at a high moi of 25 [31] . the wild type sindbis virus demonstrated no such conservation under similar passaging conditions. additionally, a high-fidelity variant of poliovirus appeared to only use one type of deletion to ablate the utility of a detrimental inserted mirna, while the wild type virus displayed little conservation in the mirna sequence deletion [38] . together, these results suggest that high and low-fidelity rna virus mutants are more predictable in their methods of genome pruning than their wild type counterparts, which is ideal for the use of these mutants during live-attenuated vaccine and transgene delivery design. these results also suggest that altered recombination potential may be common among fidelity variants. this is unsurprising as altering fidelity also changes the speed and processivity of the rdrp [39] [40] [41] [42] [43] [44] [45] , which has been linked to changes in polymerase stuttering and recombination frequency [46] . however, there is evidence that at least some fidelity mutants do not exhibit large changes in recombination frequency or vice-versa [32, 47] , although this is important to reexamine using updated techniques. in the future, using clickseq to analyze virus sequence diversity and recombination would allow both of these aspects to be examined using the same dataset, which will clarify the link between replication fidelity and recombination. the mechanism of gfp removal during low-fidelity tc-83 gfp passaging primarily occurred through the formation of three deletions. the first two deletions, ∆7894-7895 and ∆7899-7990, are found within the gfp gene and were in the highest quantities for all three replicates early during passaging. the other deletion, ∆7562-8421, fully encompasses gfp and the surrounding promoters and occurred with reduced frequency until later passages. while these deletions were also found during tc-83 gfp passaging, their fixation in the population was obscured by other deletions. by later tc-83 gfp passages, almost all of the most common deletions were found between nucleotides 7559-7574, which are within the gfp subgenomic promoter. while the most frequent deletions for tc-83 gfp and low-fidelity tc-83 gfp were different, our results show that the most common deletions were not usually due to sequence homology between the two identical promotors surrounding the gfp gene but were instead due to multiple small deletions. to characterize the role of rna structure of the virus genome in rna recombination site selection, we modeled the three most common deletions that occurred at high levels during low-fidelity tc-83 gfp passaging. the first two deletions, ∆7894-7895 and ∆7899-7990, were close together in the primary sequence and shared an identical sequence and structure at the 5 and 3 ends of their shared deletion space in a clear act of homologous recombination. in the future, it would be interesting to see if disruption of the homologous recombination sites shared by ∆7894-7895 and ∆7899-7990 could help stabilize the gfp gene, as other deletions appeared to require more replication cycles to accumulate. the larger recombination event, ∆7562-8421, demonstrated microhomology, with nearly identical sequences around the 5 and 3 end of the deletion. in our study, we only examined the stability of the gfp gene when placed between two identical subgenomic promoters. in the future it would be interesting to determine how placement of the gfp gene within the virus genome could change the most common deletions within and around gfp. additionally, it would be worthwhile to examine other fidelity variants using clickseq to determine whether our observations were conserved among other fidelity mutants. however, here we demonstrate that deletions within the gfp gene are initially more common than deletions spanning the entire gfp gene, even when the gene is surrounded by identical promoter sequences. additionally, some of these deletions are common between replicates, which could be used in the future to change the secondary structure of the gfp gene while maintaining the protein sequence to aid in increasing the stability of the gene. in summary, our research has provided clear evidence for the utility of clickseq both translationally for use in understanding reporter gene stability as well as enhancing our understanding of the changes in recombination that occur during low-fidelity virus replication. funding: research reported in this publication was supported by the niaid of the national institutes of health under award numbers r01-ai095753-01a1 and r01-ai125902-01. alr and ej were supported by start-up funds from utmb. tfk was supported by a predoctoral fellowship from the utmb mclaughlin endowment as well as a predoctoral fellowship from the nih/niaid emerging and tropical infectious diseases training program (t32 ai007526). the authors wish to report no conflicts of interest. viral mutation rates selforganization of matter and the evolution of biological macromolecules quasispecies theory and the behavior of rna viruses rapid evolution of rna genomes quasispecies diversity determines pathogenesis through cooperative interactions in a viral population viral quasispecies comparative analysis of the molecular mechanisms of recombination in hepatitis c virus why do rna viruses recombine? the au-rich rna recombination hot spot sequence of brome mosaic virus is functional in tombusviruses: implications for the mechanism of rna recombination recombination and selection in the evolution of picornaviruses and other mammalian positive-stranded rna viruses a trans-complementing recombination trap demonstrates a 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defective genomes of rna viruses modulates antiviral immunity in insects sindbis virus vectors designed to express a foreign protein as a cleavable component of the viral structural polyprotein production of pseudoinfectious yellow fever virus with a two-component genome development of a rubella virus vaccine expression vector: use of a picornavirus internal ribosome entry site increases stability of expression an infectious west nile virus that expresses a gfp reporter gene analysis of in vivo dynamics of influenza virus infection in mice using a gfp reporter virus engineering attenuated virus vaccines by controlling replication fidelity a speed-fidelity trade-off determines the mutation rate and virulence of an rna virus ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications design of a genetically stable high fidelity coxsackievirus b3 polymerase that attenuates virus growth in vivo attenuation of foot-and-mouth disease virus by engineered viral polymerase fidelity sequence-specific fidelity alterations associated with west nile virus attenuation in mosquitoes vaccine-derived mutation in motif d of poliovirus rna-dependent rna polymerase lowers nucleotide incorporation fidelity coxsackievirus b3 mutator strains are attenuated in vivo signatures of nucleotide analog incorporation by an rna-dependent rna polymerase revealed using high-throughput magnetic tweezers rna recombination enhances adaptability and is required for virus spread and virulence key: cord-274210-uaaj66xq authors: ding, ning; zhao, kui; lan, yungang; li, zi; lv, xiaoling; su, jingjing; lu, huijun; gao, feng; he, wenqi title: induction of atypical autophagy by porcine hemagglutinating encephalomyelitis virus contributes to viral replication date: 2017-02-28 journal: front cell infect microbiol doi: 10.3389/fcimb.2017.00056 sha: doc_id: 274210 cord_uid: uaaj66xq autophagy is a basic biological metabolic process involving in intracellular membrane transport pathways that recycle cellular components and eliminate intracellular microorganisms within the lysosome. autophagy also plays an important part in virus infection and propagation. however, some pathogens, including viruses, have evolved unique trick to escape or exploit autophagy. this study explores the mechanism of autophagy induction by porcine hemagglutinating encephalomyelitis virus (phev) in neuro-2a cells, and examines the role of autophagy in phev replication. phev triggered autophagy in neuro-2a cells is dependent on the presence of bulk doubleor single-membrane vacuoles, the accumulation of gfp-lc3 fluorescent dots, and the lc3 lipidation. in addition, phev induced an incomplete autophagic effect because the degradation level of p62 did not change in phev-infected cells. further validation was captured using lysotracker and lysosome-associated membrane protein by indirect immunofluorescence labeling in phev-infected cells. we also investigated the change in viral replication by pharmacological experiments with the autophagy inducer rapamycin or the autophagy inhibitor 3-ma, and the lysosomal inhibitor chloroquine (cq). suppression of autophagy by 3-ma increased viral replication, compared with the mock treatment, while promoting of autophagy by rapamycin reduced phev replication. cq treatment enhanced the lc3 lipidation in phev-infected neuro-2a cells but lowered phev replication. these results show that phev infection induces atypical autophagy and causes the appearance of autophagosomes but blocks the fusion with lysosomes, which is necessary for the replication of phev in nerve cells. porcine hemagglutinating encephalomyelitis virus (phev) is a single-stranded, positive-sense rna coronavirus and is classified as a member of the coronaviridae family (andries and pensaert, 1981) . it mainly gives rise to encephalomyelitis or vomiting and wasting disease in piglets. as a neurotropic virus, it spreads via the peripheral to the central nervous system (cns), resulting in neurological damage by virus infection (andries and pensaert, 1980) . nervous lesions caused by pathogens often lead to abnormal nerve cell morphology, unnatural protein accumulation, vesicular transport or neuronal gene transcription disorders, and synaptic transmission obstacles (xilouri and stefanis, 2010) . similarly, as a virus invading the nervous system, rabies virus (rabv) can cause severe encephalitis and induce analogous cytopathic features (peng et al., 2016) . although, some studies of phev pathogenesis have been performed, the underlying mechanism of phev replication is still poorly elucidated. therefore, a more detailed description of the mechanism should be revealed, especially the biological changes of the host cells induced by infection. in eukaryotic cells, autophagy is characterized by its highly conserved as a metabolic process for preserving the homeostasis. among the intracellular membrane transport pathway, the corresponding cytoplasmic proteins and cargos are delivered to the lysosomes (levine et al., 2011) . as a cytoprotective process, autophagy is an inherent host defense mechanism against viral aggression, which helps to maintain cell homeostasis in response to multiple external stress stimuli, containing a nutrient-poor environment, endoplasmic reticulum (er) stress, especially pathogen infection (kroemer et al., 2010) . dysfunction in autophagy has been associated with many illnesses that infect humans such as neurodegeneration diseases, cancer, metabolic syndrome, lysosomal diseases, and aging (rubinsztein et al., 2011) . previous studies have indicated that autophagy not only protects cells under stressful conditions, but also plays an important part in the process of pathogen infection (shintani and klionsky, 2004) . some viruses have also developed strategies to reverse the autophagy defense pathway. us11, a late gene of herpes simplex virus-1 (hsv-1), blocks the formation and development of autophagosomes in both fibroblasts and hela cells. us11 also interacts with pkr to suppress autophagy (lussignol et al., 2013) . nevertheless, there are viruses that utilize autophagosomes for promoting their replication, such as in poliovirus infection, where the formation of autophagosomes provides a conservative platform for viral replication (suhy et al., 2000) . conversely, in the sindbis virus and tobacco mosaic virus infections, autophagy successfully suppresses viral replication (liu et al., 2005) . more interestingly, several coronaviruses can hijack the important autophagy-related protein lc3-i and endoplasmic reticulum vesicle structures for their replication (reggiori et al., 2010) using the same mechanism as rotavirus (crawford et al., 2012) . this mechanism induced by viral proteins is associated with their own pathogenic non-structural proteins, exemplified by the infectious bursal disease virus infection (hu et al., 2015) . however, whether phev, as a member of the coronavirus family, infection is associated with autophagy is unknown. in this study, we confirmed that phev infection induces atypical autophagy and leads to the accumulation of autophagosomes while blocking their fusion with lysosomes, which creates conditions for the virus to replicate within nerve cells. mouse neuroblastoma (neuro-2a) cells were cultured in high glucose dulbecco's-modified eagle's medium (dmem) (gibco, usa) containing 2 mm l-glutamine and 1.5 g/l sodium bicarbonate, added with 10% fetal bovine serum (fbs). the phev strain hev 67n (genbank: ay048917) was propagated in n2a cells. the plasmid gfp-lc3 and the tandem fluorescent monomeric red fluorescent protein (mrfp)-gfp-lc3 (ptflc3) were maintained in the laboratory. neuro-2a cells were infected with phev (10 5.43 tcid 50 /ml) in cell cultures with 2% fbs for 1 h. following a 1 h absorption period, infected cells were incubated in the complete dmem at 37 • c for the relevant times in the conformity with experimental requirements. in subsequent experiments, the optimum concentration of the corresponding drug was used and the cell viability were determined by a wst-8 cell proliferation assay (beyotime, china) according to the manufacturer's guidelines. the concentrations tested for rapamycin (selleckchem, usa) were 100, 500, and 200 µm; for chloroquine (cq) (sigma, st. louis, mo, usa) were 16, 32, 64, and 128 µm; and for 3-methyladenine (3-ma) (selleckchem, usa) were 1, 2, 3, and 5 mm. according to existing experimental procedures, the medium was removed with 100 µl of fresh medium added with 10 µl of wst-8, the sample was further incubated at 37 • c for 1 h (shao et al., 2014) . cell viability was evaluated by measuring the absorbance at 450 nm against the background control. tem is a valid and important method for monitoring autophagy induction in morphology (peng et al., 2016) . in this assay, neuro-2a cells were treated with 100 nm rapamycin in complete medium for 3 h as a positive control, and the infected cells were incubation with phev (10 5.43 tcid 50 /ml) for 24 h. the cell samples were washed three times with pbs, collected in the bottom of 1.5-ml eppendorf tubes and centrifuged at 1,000 ×g for 10 min. the cell pellets were fixed with 2.5% glutaraldehyde in pbs overnight at 4 • c and then postfixed in 1% oso 4 for 2 h. after being washed, the cells were dehydrated with a graded series of ethanol and then embedded in epoxy resin. next, ultrathin sections were prepared and stained with uranyl acetate and lead citrate as previously described (risco et al., 2012) . at last, the autophagosome-like vesicles were examined under a transmission electron microscopy (jeol, tokyo, japan). confocal fluorescence microscopy was utilized to detect the expression of lc3 in phev infected or drug-treated cells and the expression of autophagy-related proteins during phev infection. neuro-2a cells were seeded in 22.1 mm dishes with coverslips and transfected with gfp-lc3, mrfp-gfp-lc3, or control plasmids. at 24 h post-transfection, the cells were incubated with complete medium with 10% fbs, and then mock-infected, and those infected with phev were treated as required. the cells were then incubated with 50 nm lysotracker red dnd-99 (invitrogen, carlsbad, ca, usa) for 1 h, rinsed 3 times with pbs and analyzed under the olympus fv1000 confocal microscope (olympus, japan). drug treated, mock-infected or phev-infected cells were harvested at corresponding times. next, immediately lysed with ripa lysis buffer, including 1 mm pmsf (beyotime, china) for 30 min to 1 h on ice, shaking during the lysis. the clarified lysate was boiled in loading buffer for 10 min, and the proteins which in equal amounts were displayed on sds-polyacrylamide gel electrophoresis (millipore, billerica, ma, usa). afterwards, the proteins were transferred to pvdf membranes, blocked in pbst buffer added 5% nonfat milk powder for 1 h at 37 • c. the corresponding primary antibodies were incubated overnight at 4 • c, and the hrp-conjugated secondary antibodies for 1 h at 37 • c. p62, also called sqstm1, undergoes degradation in the course of autophagy. whether the complete autophagy response was activated by phev infection, we analyzed p62 degradation by western blotting or confocal immunofluorescence microscopy for the autophagic flux as previously described (bjørkøy et al., 2005) . in this test, neuro-2a cells were infected with phev and harvested at 6-48 h post-infection (hpi) using anti-p62 antibody as the primary antibody. cq can inhibit autophagy by inhibiting acidification of lysosomes and endosomes. to verify the effect of phev infection on autophagic flux, the cells were infected with phev after cq treatment, the levels of p62 and phev were detected. a tandem reporter construct, mrfp-gfp-lc3 (ptflc3), was used as an additional plasmid to monitor the autophagic flux (kimura et al., 2007) . neuro-2a cells grown to 60-70% confluency on coverslips were transfected with ptflc3 and infected with phev (10 5.43 tcid 50 /ml). at 12, 24 and 48 hpi, the cells were fixed and visualized by confocal microscopy. gfp-lc3 colocalization with the lysosome-associated membrane protein 1 (lamp1) was also investigated as d previously described. briefly, neuro-2a cells were transfected on cell slips with gfp-lc3 and infected with phev at 12, 24, and 48 h. then, the cells were incubated and examined with rabbit antibody against lamp1 (abcam, cambridge, ma, usa) under a confocal fluorescence microscope. to mark the acidic late endosomal and lysosomal structures, lysotracker red (50 nm) was added to the treated, mock-or infected cells for 1 h. following treatment or infection, cells were settled in 4% paraformaldehyde for 15 min and washed with pbs for three times, and then permeabilized using 0.1% triton x-100 for 15 min at room temperature. blocking was performed with 5% nonfat milk powder in pbs for 1 h prior to incubation with primary antibodies followed by incubation with the suitable secondary antibody. images were visualized by confocal fluorescence microscopy. real-time quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) was performed to analysis the virus copies of phev. the cells were given the appropriate treatment or infection and rna was extracted following lysis in tripure isolation reagent (roche, basel, switzerland). according to the manufacturer's protocol of the primescript r rt reagent kit (takara, japan), the corresponding treated cdna samples were synthesized. a pair of specific primers (phev-p1: agcgatgaggctattccgacta /phev-p2: ttgccagaattggctctactacg) was used in the study for phev detection, which was targeting a region to the he gene of phev. the real-time rt-pcr was performed using faststart universal sybr green master (roche). the pcr conditions were as follows: 95 • c for 3 min (1 cycle), 95 • c for 30 s, 60 • c for 30 s, and 72 • c for 30 min (40 cycles). in this study, double membrane vacuoles containing organelle and cytosolic components, which are analogous to autophagosomes, were visible in the phev infected cells compared with the mock cells ( figure 1a) . to understand how phev induces autophagy, we monitored the processing of lc3-i to its lipidated membrane-bound form lc3-ii. the neuro-2a cells were transfected with gfp-lc3 and prepared for rapamycin for 6 h or phev-infection ( figure 1b) . in the mock-infected neuro-2a cells, gfp-lc3 shows a diffuse distribution ( figure 1b) . phev infection leads to the accumulation of autophagosomes in cells, as observed by gfp-lc3 punctate redistribution. similarly, short-term treatment with rapamycin efficiently induced autophagy, resulting in gfp-lc3 puncta formation in ∼40% of cells ( figure 1c) . to further corroborate the finding that phev induces autophagy, we measured the expression of lc3-i and lc3-ii in cells by western blotting. consistent with the gfp-lc3 fluorescence and tem data, an increase of lc3-ii was detected in the rapamycin treated and phev-infected cells ( figure 1d ). to investigate the temporal regulation of autophagy by phev, a time course of phev infection showed that autophagosome accumulation could be detected by western blotting from 6 to 36 hpi, and it increased with the infection process (figure 2a ). in addition, immunofluorescence microscopy was used to detect the gfp-lc3 puncta and contrast with the cells transfected with control vector showing that, gfp-lc3 puncta steadily increased at 36 hpi ( figure 2b ). in line with these data, increased lc3-ii accumulation was also examined in phev-infected cells with the increasing time by infection ( figure 2c ). neuro-2a cells were transfected with gfp-lc3 followed by treatment at 24 h post transfection with mock treatment as a negative control, and rapamycin treatment as a positive control. the localization of gfp-lc3 positive autophagosome accumulation (green) and the s-tagged phev products (red) was visualized using a confocal microscope. (c) the quantification of cells showing gfp-lc3 puncta in phev-infected cells. in three random fields, the average number of puncta in each cell was taken from at least 80 cells in each treatment. representative results with graphs are shown in figure 1b . (d) western blotting. the turnovers of lc3-i to lc3-ii were detected for mock-treated neuro-2a cells, rapamycin-treated neuro-2a cells, and phev-infected neuro-2a cells. cells were collected at appropriate time points and detected with anti-lc3b antibody, and β-actin was used as a protein loading control. **p < 0.01; ***p < 0.001. scale bars: 10 µm. in order to examine whether the autophagy is associated with viral replication in phev infection, we performed subcellular localization of viral proteins and lc3 or lamp on phevinfected cells for the first time. as shown in the figures 3 a,b , the effective fluorescent signal is observed in the infected cells, with puncta accumulation. in addition, we also examined the fluorescence spectra of lamp1 in phev-infected cells. we found that lamp1 can is localized with the viral protein in the infected cells. it reveals that the reassignment of autophagy marker lc3 induced by phev appeared in the infected cells and the autophagy is involved in viral replication. the complete autophagy process contains the formation of autophagosomes and the fusion between autophagosomes and lysosomes (kliosnky et al., 2016) . immunoblotting showed that phev-infected cells have no significant changes in the p62 protein from 12 to 48 hpi, compared to the mock infection (figures 4a,b) . consistent with these results, the levels of the p62 protein were essentially invariant during the infection by phev determined by confocal fluorescence microscopy ( figure 4c) . furthermore, the cells were infected with phev after cq treatment, the result showed that the obvious higher accumulation levels of p62 than that of the control ( figure 4d) . figures 4e-g , cq increased the levels of lc3ii and p62 and suppressed phev infection. these results suggest that cells infected with phev suppress autophagic flux, leading to no decrease of p62 but an accumulation when treated with cq before phev infection. these results also revealed that phev induced incomplete autophagy. these data implied that phev infection may interfere with the fusion of autophagosomes with lysosomes or impair autolysosomal degradation. figure 2 | phev-induced autophagy was time-dependent. (a) immunofluorescence microscopy was used to detect the gfp-lc3-autophagosomes in phev infected cells at different post-infection time points before transfection with gfp-lc3 into neuro-2a cells. the cells were fixed at 0, 24, and 36 h post-infection, respectively, and then analyzed for gfp-lc3 positive autophagosome accumulation using a confocal microscope as described in figure 2a. (b,c) neuro-2a cells were infected with phev and mock-infected cells served as control. at 0, 12, 24, and 36 h post-infection, the cells were then lysed for western blotting analysis using an anti-lc3b and anti-β-actin antibodies. scale bars: 10 µm. according to the results presented above, phev infection induces an autophagic response, but efficient autophagic protein degradation and the complete autophagic process remains unobserved. in order to further explore the potential mystery, which of the inhibition of autophagic degradation induced by phev, we transfected the neuro-2a cells with a tandem-tagged fluorescent reporter, mrfp-gfp-lc3 (ptflc3) (figure 5a) . the gfp fluorescence from the ptflc3 fusion protein is quenched in acidic autolysosomal conditions; however, the red fluorescence signal produced by ptflc3 is not sensitive to acidic conditions. we found the gfp puncta were increased by phev during infection, as shown in figure 5a . when both rfp and gfp signals were colocalized, resulting in yellow puncta, the rfp puncta remained increased at 12 and 24 h. however, with rapamycin treated cells, ptflc3-transfected cells experienced complete autophagic flux and showed main red fluorescence with only a fuzzy green fluorescence. subsequently, we examined whether the fusion of the autolysosome with lysosomes was intact during phev infection. a marker for acidic late endosomal and lysosomal structures, lysotracker red was used in the corresponding treated cells ( figure 5b) . in mock-infected cells, a certain number of gfp-lc3 vacuoles merged with lysotracker staining. it indicated that a portion of autophagosomes had fused with lysosomes and became acidified. however, after phev infection, the autophagosomes do not fuse with acidic compartments. to exclude the autophagosomes fusion with lysosomes rather than the efficiently acidified in infected cells, we also stained the cells with an antibody for lamp1, a lysosomal marker. in accordance with the results above, the colocalization of gfp-lc3 and lamp1 was not examined in neuro-2a cells (figure 5c) . it was further clarified by western blotting, compared with the mock samples at the same time, that lamp1 showed exceptional accumulation ( figure 5d) . these data indicated that phev infection inhibits the fusion of autophagosomes with acidified lamp1 lysosomes and thereby prevents the degradation of macroautophagy substrates. collectively, these findings confirm that phev can induce autophagosome formation and activate an incomplete autophagic response. autophagy may facilitate or suppress viral replication, depending on the virus type. to investigate the possible effect of autophagy on phev replication, we treated neuro-2a cells with rapamycin, a prompter of autophagy that can stimulate complete autophagy, targeting mammalian target of rapamycin (mtor) signaling pathways. we found that the induction of autophagy with rapamycin (200 µm) for 2 h effectively upregulated the alteration of lc3-i to lc3-ii but significantly inhibited phev replication at 24 and 48 hpi in neuro-2a cells (figure 6a ). it indicated that the complete autophagy pathway was activated after treatment with rapamycin; although, phev infection blocked the smooth of autophagic flow, phev replication was also inhibited because of the rapamycin treatment. in expect to recognize the effect of autophagy induced by phev infection on viral replication, we treated the neuro-2a cells with 3-ma, the autophagic inhibitor targeting a class iii phosphatidylinositol-3-kinase (pi3k) (petiot et al., 2000) . as shown in figure 6b , there was no significant change in the conversion of lc3-i to lc3-ii and the replication of phev at 24 hpi after 3-ma treatment. however, the replication of phev was significantly increased at 48 hpi after 3-ma treatment. these results suggested that 3-ma can promote effective phev replication by inhibiting autophagy. in this study, we examined the influence of autophagy during phev infection by pharmacological alterations show the ratios of phev to β-actin, p62 to β-actin or lc3-ii to β-actin from three independent experiments of (d). *p < 0.05; **p < 0.01; ***p < 0.001. scale bars: 10 µm. of autophagy, containing rapamycin treatment, 3-ma treatment, and cq treatment. we found that the cell viability was not significant change by wst-8 assay, which provided the basis for our further explore aiming to the relationship among autophagy and viruses (figure 7) . phev is a neurotropic virus that transmits along the peripheral nerves to the cns (li et al., 2013) . the neurological dysfunction by phev infection, results in neurological symptoms, vomiting and other clinical features. some cases also accompanied with the figure 5b . (c) furthermore, the fusion of autophagosomes with lysosomes was analyzed as colocalization of the autophagosome marker gfp-lc3 with the lysosome marker lamp1. nuclear dna was stained with dapi. one of the three experiments conducted is shown. (d) western blotting analysis of lamp1 changes in phev-infected cells for 12, 24, 36, and 48 h were shown in figure 5d , compared with the relevant times in mock cells. scale bars: 10 µm. characteristics of diarrhea, but whether the infection is associated with nerve injury is not yet clear (li et al., 2016) . in a previous study, we found that the invasion of nerve cells by phev is mediated by the s protein and neural cell adhesion molecule (ncam), which is an adhesion molecule on the nerve cell surface (gao et al., 2010; dong et al., 2015) . however, the specific mechanism by which the virus invades and replicates in nerve cells remains ill-defined. autophagy plays a vital role in the replication of dna and rna viruses (dreux and chisari, 2010) . many rna viruses induce autophagy in order to produce the membrane-associated replication complexes (miller and krijnse-locker, 2008) . during the coronavirus infection, lc3 could be recruited to form double-membrane vesicles (dmvs) from the er (reggiori et al., 2010) . these dmvs appear to have a subcellular structure similar to the autophagosome, which is called a "virus factories" in a recent study. these viral factories provide a suitable platform for the replication and encourage autophagy, implying that a covered connection between dmvs and autophagy (blanchard and roingeard, 2015) . in this study, it is an initial glance that phev infection induces atypical autophagy which is involved in viral replication. here, the traditional tem assay were performed, the effectively increased number of double-or single-membrane vacuoles were displayed in phev infected cells for the first time, that is, the formation of autophagosome. we found that these single or double vesicle structure is similar to autophagosomes, but not exactly the same (pei et al., 2014) . some of these vesicles encapsulate viral particles, while others do not. it suggested that autophagy induced by phev is different from the complete autophagy in conventional sense. in addition, accumulation of gfp-lc3 puncta in phev-infected cells were observed by laser confocal microscopy. it further verified that the formation of autophagosomes. autophagy is often closely related to viral replication, and several viruses use autophagic substances or the autophagosome to escape the immune system in order to achieve viral proliferation, while others use the autophagosome or autophagic vesicle model structure as its own site for genome transcription and replication (jackson et al., 2005) . therefore, the development of autophagy often contributs to the proliferation of the virus (levine and kroemer, 2008) . several studies have determined the mechanism of virus-induced autophagy, such as influenza a virus. the virus inhibits the autophagic degradation process, and its matrix protein 2 plays a key role in this process (gannagé et al., 2009) . rotavirus also blocks the maturation of autophagosomes and inhibits autophagy maturation, both of which may offer the rotavirus as a way for escaping the antiviral function of autophagy (crawford et al., 2012) . recently, it has been reported that coronavirus membrane-associated papainlike protease plp2 (plp2-tm) could promote autophagy. in addition, the interaction between plp2-tm with beclin 1 affected the replication of the coronavirus, and further regulated the innate immune defense mechanism against viruses. (chen et al., 2014) . the similar phenomenon is shown in our results which demonstrated that phev could activate the appearance of autophagosome but influence its fusion with lysosomes. as previously mentioned, the complete autophagy process involves the appearance of autophagosomes and the fusion and degradation of autophagosomes with lysosomes. the term "autophagic flux" is used to denote the dynamic process of autophagosome synthesis, the delivery of autophagic cargoes to the lysosome, and the degradation of autophagic cargoes inside the lysosome and is a more reliable indicator of autophagic activity than measurements of autophagosome numbers. some recent studies have demonstrated that the induction of autophagy not only increases autophagosome formation and expression of autophagy proteins but also increases autophagic flux, which can be measured by detecting the levels of p62 (pankiv et al., 2007) . using several means of measuring autophagic flux, including p62 degradation, lc3-ii turnover, and gfp-lc3 lysosomal delivery and proteolysis, we confirmed that autophagic flux remained unchanged upon phev infection. the cells were infected with phev after cq treatment, the level of lc3-ii was increased, but phev infection was suppressed. the effect of cq treatment may be related to its inhibition of autophagic proteolysis by suppressing acidification of lysosomes and endosomes. cellular entry of coronaviruses is dependent on clathrin-mediated endocytosis, cq treatment may block viral genome release from the lysosome into the cytoplasm for replication (burkard et al., 2014) . this may also be another dominant effect of cq. these findings illustrate that an incomplete autophagy is activated in neuro-2a cells by phev infected, like we mentioned as "atypical autophagy" above, and this autophagy induced by phev is involved in viral replication. in some positive-strand rna viruses, there is a co-localization between their viral components and autophagic markers, which subvert the autophagy mechanism, suggesting that the assembly of the translation/replication complexes of these viruses may exist in autophagic vesicles (e.g., poliovirus, mouse hepatitis virus, and dengue virus; suhy et al., 2000; lee et al., 2008; beachboard et al., 2015) . in the present study, we examined the co-localization between autophagy-related proteins and phev virion in infected cells, including lc3, an important component in the autophagosomal membrane; lamp1, a widely used of lysosomal marker protein. we found that phev improved the rearrangement of lc3 and lamp1, suggesting that several autophagy-associated membrane proteins are related to the phev replication. in order to make a thorough inquiry the effect of autophagy on phev infection, neuro-2a cells were treated with the corresponding experimental requirements in pharmacological alterations, including autophagy inducer (rapa), the early autophagy inhibitor 3-ma and the late autophagy inhibitor cq. our data displayed that after rapamycin treatment, viral replication was inhibited by the rapamycin-treated autophagic effect. the early autophagic inhibitor 3-ma treatment could significantly promote the replication of phev, further implied that the formation of early autophagosome contributes to phev replication. at the same time, the results from the wst-8 assay we performed above proposed that the pharmacological alteration did not affect cell viability. in conclusion, we first propose that phev can induce atypical autophagy and jam the fusion of autophagosomes with lysosomes, thereby hampering the autophagy pathway degradation process. however, the specific mechanism of inhibiting autophagy maturation requires further exploration. on the other hand, such a seemingly contradictory process precisely sets the stage in the formation of early autophagy rather than the autophagolysosome. it may also be an evolutionary mechanism in which the virus escapes the immune system. our studies provide insights into phev-host interactions and may develop a theoretical basis for further study between the virus and autophagy. these insights may raise promising targets for the development of antiviral strategies against phev infection. nd, wh designed the experiments, supervised the experiments, and analyzed the data. nd, kz performed experiments and interpreted the data. yl, xl, zl, js, hl contributed reagents, materials, and analysis tools. nd, fg, wh drafted the article or revised it critically for important intellectual content. all authors agree with final approval of the version for submission. vomiting and wasting disease, a coronavirus infection of pigs immunofluorescence studies on the pathogenesis of hemagglutinating encephalomyelitis virus infection in pigs after oronasal inoculation mutations across murine hepatitis virus nsp4 alter virus fitness and membrane modifications p62/sqstm1 forms protein aggregates degraded by autophagy and has a protective effect on 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commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2017 ding, zhao, lan, li, lv, su, lu, gao and he. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-324720-acei0pn8 authors: howe, charles l; lafrance-corey, reghann g; sundsbak, rhianna s; lafrance, stephanie j title: inflammatory monocytes damage the hippocampus during acute picornavirus infection of the brain date: 2012-03-09 journal: j neuroinflammation doi: 10.1186/1742-2094-9-50 sha: doc_id: 324720 cord_uid: acei0pn8 background: neuropathology caused by acute viral infection of the brain is associated with the development of persistent neurological deficits. identification of the immune effectors responsible for injuring the brain during acute infection is necessary for the development of therapeutic strategies that reduce neuropathology but maintain immune control of the virus. methods: the identity of brain-infiltrating leukocytes was determined using microscopy and flow cytometry at several acute time points following intracranial infection of mice with the theiler's murine encephalomyelitis virus. behavioral consequences of immune cell depletion were assessed by morris water maze. results: inflammatory monocytes, defined as cd45(hi)cd11b(++)f4/80(+)gr1(+)1a8(-), and neutrophils, defined as cd45(hi)cd11b(+++)f4/80(-)gr1(+)1a8(+), were found in the brain at 12 h after infection. flow cytometry of brain-infiltrating leukocytes collected from lysm: gfp reporter mice confirmed the identification of neutrophils and inflammatory monocytes in the brain. microscopy of sections from infected lysm:gfp mice showed that infiltrating cells were concentrated in the hippocampal formation. immunostaining confirmed that neutrophils and inflammatory monocytes were localized to the hippocampal formation at 12 h after infection. immunodepletion of inflammatory monocytes and neutrophils but not of neutrophils only resulted in preservation of hippocampal neurons. immunodepletion of inflammatory monocytes also preserved cognitive function as assessed by the morris water maze. conclusions: neutrophils and inflammatory monocytes rapidly and robustly responded to theiler's virus infection by infiltrating the brain. inflammatory monocytes preceded neutrophils, but both cell types were present in the hippocampal formation at a timepoint that is consistent with a role in triggering hippocampal pathology. depletion of inflammatory monocytes and neutrophils with the gr1 antibody resulted in hippocampal neuroprotection and preservation of cognitive function. specific depletion of neutrophils with the 1a8 antibody failed to preserve neurons, suggesting that inflammatory monocytes are the key effectors of brain injury during acute picornavirus infection of the brain. these effector cells may be important therapeutic targets for immunomodulatory or immunosuppressive therapies aimed at reducing or preventing central nervous system pathology associated with acute viral infection. viral infection of the central nervous system (cns) may induce clinically relevant outcomes that range from coma, paralysis, and death to persistent cognitive impairment, seizures, and epilepsy [1] . many viral infections of the cns are acute, with viral clearance mediated by the adaptive arm of the immune system. however, the relationship between the delayed adaptive antigen-specific t and b cell-mediated response that eventually controls and eliminates the viral pathogen and the rapid but largely non-specific innate immune system response is poorly understood. indeed, different viruses and different hosts exhibit disparate relationships between the innate and adaptive response to infection. for example, mcgavern and colleagues have shown in mice infected with lymphocytic choriomeningitis virus that pathogenic neutrophils and inflammatory monocytes are recruited to the brain by antiviral cd8 + t cells. depletion of the cd8 + t cell response reduced the neutrophil and inflammatory monocyte burden in the cns and delayed pathogenesis [2] . in contrast, bergmann and colleagues observed that neutrophils and inflammatory monocytes were the first leukocytes to infiltrate the brain in mice infected with the non-lethal neurotropic jhm strain of mouse hepatitis virus (jhmv) [3] , and depletion or blockade of monocytes impaired subsequent t cell infiltration into the brain parenchyma [4] . lane and colleagues also observed a very early neutrophil response to cns infection with jhmv, and in contrast to the bergmann et al. findings implicating monocytes, they found that blocking neutrophil entry into the cns resulted in impairment of the subsequent t cell response [5] . finally, lokensgard and colleagues observed an early neutrophil and inflammatory monocyte response in the brain following infection with herpes simplex virus 1 and this response preceded lymphocyte infiltration by a week [6] . overall, the common denominator in all of these studies was the ensuing neuropathology triggered by infiltrating neutrophils and inflammatory monocytes. our previous experience with the theiler's murine encephalomyelitis virus (tmev) model of acute picornavirus infection of the cns [7] indicated that brain pathology and the functional sequelae of such injury occur as a result of an early, preadaptive immune response. indeed, hippocampal pyramidal neurons were clearly injured at 1 day after infection [8] . we have hypothesized that neutrophils and inflammatory monocytes, as part of an early wave of first responders to infection, are responsible for hippocampal injury and loss of memory function observed in tmev-infected mice, but the relative contribution of each population to this injury was unclear. in the present study we phenotyped the brain-infiltrating leukocyte population at several acute time points after infection with tmev. we found that neutrophils and inflammatory monocytes were present in the brain within 12 h of infection, indicating that in this model system infiltration of such innate effectors is a hyperacute response. furthermore, we found that the absence of an inflammatory monocyte response but not the absence of a neutrophil response resulted in neuroprotection and cognitive preservation. at 5 to 8 weeks of age, mice were infected by intracranial injection of 2 × 10 5 plaque-forming units (pfu) of the daniel's strain of the theiler's murine encephalomyelitis virus (tmev) in 10 μl rpmi (the media used to grow the virus) [9] . when relevant, sham-infected mice received intracranial injection of 10 μl virus-free rpmi. mice c57bl/6/j (no. 000664) mice aged 4 to 6 weeks were acquired from the jackson laboratories (bar harbor, me, usa). upon arrival, mice were acclimatized for at least 1 week prior to use. breeding pairs of lysm:egfp mice [10] were kindly provided by dr. david sacks (national institutes of health/national institute of allergy and infectious diseases) and bred in house. mice were group housed in the mayo clinic college of medicine research vivarium under conventional conditions with ad libitum access to food and water. sex was mixed for all experiments. all animal experiments conformed to the national institutes of health and mayo clinic institutional animal care and use committee guidelines. following intraperitoneal injection of a terminal dose of pentobarbital (100 mg/kg), mice were perfused via intracardiac puncture with 50 ml of 4% paraformaldehyde in phosphate-buffered saline (pbs). for paraffin sections, the brain was postfixed in 4% paraformaldehyde at 4°c for 24 h and then blocked via coronal cuts at the level of the optic chiasm and infundibulum. tissue blocks were embedded in paraffin, sectioned at 5 microns, mounted on charged slides, rehydrated, and stained with hematoxylin and eosin. for vibratome sections, the brain was postfixed in 4% paraformaldehyde at 4°c for 6 h and then blocked to isolate the hippocampal field. tissue blocks were embedded in agar and sectioned at 80 microns. free floating sections were blocked in pbs plus 10% normal donkey serum for 1 h, incubated overnight at 4°c with primary antibodies diluted 1:100 in block, washed, incubated with fluorescently tagged secondary antibody diluted 1:200 in pbs, washed, and mounted on gelatin-subbed slides. cd45 was detected with clone 30-f11 (bd biosciences, san jose, ca, usa). cd11b was detected with clone m1/70 (bd biosciences). ly6c/g was detected with clone gr1, rb6-8c5 (bd biosciences). ly6g was detected with clone 1a8 (bd biosciences). our published protocol was followed with slight modification [9] . briefly, homogenized brain material was centrifuged through a 30% percoll gradient at 7,800 g ave for 30 minutes without braking. the washed and strained cell suspension was then centrifuged on a 1.100 g/ml percoll layer for 20 minutes at 800 g ave . the interface containing neutrophils and inflammatory monocytes was collected, washed, and used for flow cytometry. flow cytometry buffer contained 1% bovine serum albumin and 0.02% sodium azide in pbs. blocking buffer contained flow cytometry buffer, supernatant from the 2.4g2 hybridoma (fc block; anti-cd16/32; american type culture collection, manassas, va, usa no. , and fetal bovine serum at a ratio of 10:5:1. after isolation, cells were blocked at 4°c for 30 minutes. primary antibodies were used at 1:200 and incubated for 30 minutes at 4°c. stained cells were washed three times in flow cytometry buffer and fixed in 2% paraformaldehyde prior to flow cytometric analysis on a bd facscalibur (bd biosciences). files were analyzed offline using flowjo 7.5 (windows version; tree star, inc., ashland, or, usa). cd45 was detected with clone 30-f11 (bd biosciences no. 557235). cd11b was detected with clone m1/70 (bd biosciences no. 553312). f4/80 was detected with clone bm8 (ebiosciences no. 53-4801-82). ly6c/g was detected with clone gr1, rb6-8c5 (bd biosciences no. 553129). ly6g was detected with clone 1a8 (bd biosciences no. 551467). ly6b was detected with clone 7/4 (caltag no. rm6504). ly6c was detected with clone al-21 (bd biosciences no. 560595). cognitive performance was assessed beginning at 14 days postinfection using our previously published methodology [1] . all graphs show mean ± 95% confidence intervals. cell counts in the depletion experiments were assessed by one-way analysis of variance (anova). morris water maze performance was assessed by two-way anova. pairwise analyses, when appropriate, used the student-newman-keuls method. all tests utilized α = 0.05, β = 0.2. immune cells rapidly infiltrate the brain of tmev-infected mice but not sham-infected mice c57bl/6j mice at 5 to 8 weeks of age were infected by intracranial injection of 2 × 10 5 pfu of the daniel's strain of tmev in a volume of 10 μl. sham-infected mice received identical injections of vehicle lacking virus. histological analysis of the brain at 24 h post infection (hpi) revealed marked inflammation and large numbers of infiltrating cells in the infected mice ( figure 1 ). inflammation was most concentrated in the hippocampal region ( figure 1b) , with high density in the corpus callosum and alveus and at the hippocampal fissure ( figure 1b , d). infiltrating cells were also observed within the pyramidal neuron layer of cornu ammonis 1 (ca1) in the hippocampus ( figure 1d , i). in shaminfected mice, despite receiving a needle stick through the calvarium, no evidence of inflammation was ever observed in the brain. we also never observed an inflammatory response in mice injected with uv-inactivated tmev (data not shown). we conclude that productive infection of the brain with tmev results in the rapid induction of immune cell infiltration that is concentrated in the white matter above the hippocampus and at the hippocampal fissure. given the conflicting observations that exist in the literature regarding temporal staging of innate and adaptive immune cell infiltration into the acutely infected brain [2] [3] [4] [5] 11] , we sought to determine the identity of the cells observed in figure 1 . using our established protocol for isolating a highly enriched population of leukocytes from the brain [9] , we observed a robust population of cd45 hi cells in tmev-infected mice at 18 hpi ( figure 2b ) that are not present in the brain of sham-infected ( figure 2a ) or uninfected mice (data not shown). further phenotyping revealed that most of the cd45 hi cells in the brain-infiltrating leukocytes (bils) were cd11b positive, albeit at two different intensity levels. we routinely distinguished cd11b ++ and cd11b + ++ populations ( figure 2c , d). critically, these populations could also be distinguished by levels of surface staining with the gr1 antibody ( figure 2c ) and the 1a8 antibody ( figure 2d ), such that cd11b ++ cells were positive for gr1 but negative for 1a8 and cd11b +++ cells were positive for both markers ( figure 2c , d). likewise, the population of cd45 hi cells could be distinguished by surface f4/80 expression, with some f4/80 + figure 1 histological evidence of rapid immune cell infiltration into the hippocampus of mice acutely infected with theiler's murine encephalomyelitis virus (tmev). c57bl/6j mice were injected intracerebrally with 2 × 10 5 pfu tmev in 10 μl of rpmi (b, d, h-j) or with 10 μl rpmi (sham) (a, c, e-g). the animals were killed by intracardiac perfusion of 4% paraformaldehyde 24 h after infection. brain was processed for paraffin embedding and sectioning, and stained with hematoxylin and eosin. robust inflammatory infiltrate is present above the hippocampus and throughout the corpus callosum in the tmev-infected mice (b, d). sham-infected mice exhibit no signs of infiltrate (a, c). higher magnification confirms the presence of infiltrate throughout the corpus callosum and alveus (h), within the ca1 pyramidal neuron cell layer (i), and within the perivascular space along the hippocampal fissure (j) in virus-infected mice but not in sham-infected mice (e-g). scale bar in b is 500 μm and refers to (a); scale bar in (d) is 100 μm and refers to (c); scale bar in (j) is 20 μm and refers to (e-i). abbreviations: alv = alveus; ca1 and ca3 = cornu ammonis fields of the hippocampus; cc = corpus callosum; dg = dentate gyrus; fi = fimbria; hf = hippocampal fissure. findings are representative of more than 20 animals per group. cells positive for gr1 but no f4/80 + cells positive for 1a8. cd11b is a component of the α m β 2 integrin complex that is found on the surface of monocytes, granulocytes, macrophages, and natural killer cells [12] . the f4/ 80 antigen is a member of the epidermal growth factor 7 transmembrane family that is found on various macrophage populations including tissue resident macrophages such as microglia [13, 14] . the gr1 antigen is a mixture of ly6c and ly6g that is expressed on the surface of circulating monocytes and neutrophils [15] . in contrast, the 1a8 antigen is explicitly ly6g and is only observed on neutrophils and granulocytes but not on monocytes or macrophages [15, 16] . on the basis of our findings, we have established the following definitions, which are also elaborated below: cd45 hi cd11b ++ f4/80 + gr1 + 1a8cells are inflammatory monocytes; cd45 hi cd11b +++ f4/ 80 -gr1 + 1a8 + cells are neutrophils. we find that approximately 60% of the cd45 hi cell population in the brain at 18 hpi are inflammatory monocytes and approximately 20% are neutrophils ( figure 2g ). in order to corroborate these working definitions, we carefully examined the forward and side scatter profiles of the 18 hpi bils population from a large number of mice. gating of the scatter profiles as shown in figure 3a yielded three distinct cell populations that consistently displayed almost pure populations of neutrophils (fraction 1, figure 3b ), inflammatory monocytes (fraction 2, figure 3c ), or microglia (resident macrophages; cd45 mid cd11b + gr1 -1a8 -) (fraction 3, figure 3d ) using the flow cytometric definitions provided above. flow sorting to isolate each of these three populations followed by histological examination of the cells further supported our definitions (data not shown) [17, 18] . confusion exists in the literature regarding the use of ly6 antigens as markers of neutrophils and inflammatory monocytes. historically, gr1 staining of the ly6c/g heteroantigen was used to define neutrophils. more recently, it has become clear that gr1 marks both neutrophils and a population of inflammatory monocytes. the availability of the 1a8 marker, which only recognizes ly6g and only labels neutrophils, now allows for better resolution of these populations [15, 19] . in order to further validate our phenotype definitions, we also examined the ly6 series in blood at 18 hpi and compared the profile to bils from the same animal ( figure 4 ). in general, the staining for gr1 (ly6c/g) ( figure 4a -d) and 1a8 (ly6g) ( figure 4e -h) was higher on the brain-infiltrating cells as compared to the same populations in the blood. of note, brain-infiltrating inflammatory monocytes exhibited elevated gr1 staining compared to brain-infiltrating neutrophils ( figure 4b ). we were unable to identify a population of inflammatory monocytes in the blood in the infected mice, suggesting either that these cells are not circulating at this time or that so many have been recruited to the brain that the figure 2 flow cytometric assessment of the infiltrate present in the brain of mice acutely infected with theiler's murine encephalomyelitis virus (tmev). tmev-infected (b-g) or shaminfected (a) mice were killed at 18 h post infection (hpi). brain was collected fresh and processed for the isolation of brain-infiltrating leukocytes (bils). bils were stained with antibodies against cd45, cd11b, f4/80, gr1 (ly6c/g) and 1a8 (ly6g) and analyzed by flow cytometry. no cd45 hi cells were observed in sham-infected mice (a). in contrast, tmev-infected mice exhibited a large population of cd45 hi cells at 18 hpi. analysis of the cd45 hi cells in bils from infected mice revealed the presence of cd11b ++ cells that were also strongly positive for the gr1 antigen (c) and cd11b +++ cells that were positive for both the gr1 antigen (c) and the 1a8 antigen (d). in addition, a population of the gr1 + cells was also positive for f4/80 (e); no 1a8 + cells were f4/80 + (f). combined gating revealed that cd45 hi cd11b ++ gr1 + cells were also f4/80 + while cd45 hi cd11b +++ gr1 + cells were f4/80 -. quantitation of neutrophils (cd45 hi 1a8 + gr1 + cd11b +++ f4/80 -) and inflammatory monocytes (cd45 hi 1a8 -gr1 + cd11b ++ f4/80 + ) in bils from 10 individual mice revealed that about 80% of the cd45 hi cells belonged to one of these populations (g). error bars in (g) represent 95% confidence intervals; black circles represent individual animals; 10 mice per group were analyzed and the flow plots in (a-f) are representative. howe et al. journal of neuroinflammation 2012, 9:50 http://www.jneuroinflammation.com/content/9/1/50 blood levels are reduced to below detection ( figure 4a , e, i, m). surface 1a8 labeling was increased on neutrophils that had entered the brain ( figure 4f ) as compared to circulating neutrophils ( figure 4e ), suggesting that this molecule may be upregulated as neutrophils home to target tissues. finally, inflammatory monocytes exhibited higher surface levels of 7/4 (ly6b-specific) and al-21 (ly6c-specific) staining as compared to neutrophils in the brain ( figure 4j , l, n, p). based on these observations, in addition to differential surface cd11b, gr1, and 1a8 staining, we can further distinguish brain-infiltrating monocytes from brain-infiltrating neutrophils on the basis of surface expression of the 7/4 and al-21 antigens. brain-resident microglia/macrophages were consistently negative for all ly6 antigens (not shown). we further confirmed the presence of neutrophils and inflammatory monocytes in the brain following infection using lysm:gfp reporter mice. these animals express enhanced green fluorescent protein (gfp) at the lysozyme m locus, yielding gfp hi neutrophils and gfp mid monocytes [10] . no other cells express gfp in these mice, making them an excellent tool for tracking neutrophils and inflammatory monocytes. we observed robust and clearly distinguished populations of gfp hi and gfp mid cells in the bils at 18 hpi ( figure 5b ). gating into gfp hi , gfp mid , and gfp neg populations confirmed that our cd11b +++ gr1 + 1a8 + neutrophil population was almost exclusively gfp hi ( figure 5d , g), while our cd11b ++ gr1 + 1a8inflammatory monocyte population was almost exclusively gfp mid ( figure 5e, h) . the gfp neg population in the 18 hpi bils exhibited a cd11b + gr1 -1a8immunophenotype consistent with microglia (resident macrophages) (figure 5f , i). the clear distinction between gfp + and gfpcells in the bils population was further exploited by assessing the physical location of infiltrating gfp + cells in vibratome sections of brain from 18 hpi mice ( figure 6 ). we found that the majority of gfp + cells were located in proximity to the hippocampal formation ( figure 6a) , with high concentrations of labeled cells just superior and lateral to the hippocampus proper. higher magnification also showed dense clusters of gfp + cells in the corpus callosum and alveus ( figure 6b , c, e) and in the hippocampal fissure (figure 6d , e). this suggests that the white matter tracts overlying the hippocampus and the vasculature-rich hippocampal fissure are primary sites for infiltration of neutrophils and inflammatory monocytes. these observations are consistent with the histological analyses shown in figure 1 and further support a model in which neutrophils and inflammatory monocytes are rapid responders to brain infection. of note, the robust localization of these cells to the hippocampal formation is consistent with our previously published observations that the hippocampus is the primary site of pathology following tmev infection and that this damage is not a direct result of the virus but is rather a bystander phenomenon [8] . bils were prepared from uninfected mice and at 12, 18, and 24 hpi (figure 7 ) in order to determine how rapidly neutrophils and inflammatory monocytes enter the brain. we found a robust population of inflammatory monocytes present at 12 hpi ( figure 7e, f) , suggesting that the cells had started infiltrating even earlier. analysis of bils at 6 hpi showed a small but inconsistent population of inflammatory monocytes (data not shown), indicating that infiltration began in earnest between 6 and 12 hpi. a small population of neutrophils was present in the bils by 12 hpi (figure 7e , f). by 18 hpi the density of both neutrophils and inflammatory monocytes had increased ( figure 7h , i). by 24 hpi the number of neutrophils continued to increase while the number of inflammatory monocytes decreased relative to 18 hpi ( figure 7k , l). cell counts and relative percent of each cell population are provided in table 1 . immunostaining brain sections at these time points revealed a growing population of cd45 + cells in the hippocampal fissure and in the superior white matter tracts ( figure 8a , b, f, g, k, l, p, q). many of these cells were cd11b + ( figure 8c , h, m, r) and a strong population of gr1 + cells was observed at 24 hpi ( figure 8n ) but was greatly decreased by 48 hpi ( figure 8s ). likewise, 1a8 + cells were clearly present in the hippocampus at 12, 24, and 48 hpi ( figure 8j, o, t) . the relative differences in number of cells labeled with each of these markers is hard to interpret, especially given the overall difficulty in successfully immunostaining for several of the markers. nonetheless, figure 8 indicates that the hippocampus is a target for the rapid infiltration of however, inflammatory monocytes were only detected in the bils. consistent with the analyses performed above, the cd45 hi cd11b ++ f4/80 + population of inflammatory monocytes was positive for gr1 (ly6c/g), 7/4 (ly6b), and al-21 (ly6c) but negative for 1a8 (ly6g). the cd45 hi cd11b +++ f4/80neutrophil population was positive for all four ly6 series antigens. of note, cd45 hi cd11b ++ f4/80 + inflammatory monocytes in the bils expressed higher levels of surface ly6c/g (b, d) and ly6b (j, l) as compared to cd45 hi cd11b +++ f4/80neutrophils and expressed 10-fold higher levels of the al-21 antigen (n, p). the cd45 + cd11b + macrophage population in the blood was negative for all ly6 series antigens. results are representative of five mice. cd45 + cd11b + gr1 + and cd45 + cd11b + 1a8 + cells, confirming the findings at 18 hpi in the lysm:gfp mice ( figure 5, figure 6 ) and providing anatomic localization to the observations in figure 7 . we have previously hypothesized that the innate immune response to acute tmev infection mediates subsequent hippocampal injury that leads to cognitive deficits [8] . in order to distinguish the role of inflammatory monocytes from neutrophils in this injury, we immunodepleted these populations and assessed hippocampal pathology ( figure 9 ) and cognitive performance ( figure 10 ). neutrophils and inflammatory monocytes were simultaneously depleted by daily treatment from -2 dpi to +2 dpi with intraperitoneal injection of purified rb6-8c5 (500 μg/day; rat anti-gr1). neutrophils but not inflammatory monocytes were depleted by daily injection from -2 dpi to +2 dpi of purified 1a8 (500 μg/ day; mouse anti-ly6g). controls received daily injections of pbs. bils prepared at 18 hpi from control mice showed 4,898 ± 191 inflammatory monocytes and 1,515 ± 192 neutrophils ( figure 9a, d) ; 22.9% ± 1.0% of the bils were cd45 hi . control mice showed robust hippocampal pathology at 7 dpi as assessed by hematoxylin and eosin (h&e) histology ( figure 9g ) and immunostaining for the neuronal marker neun ( figure 9j ). regions of no or weak neun staining in the ca1 layer in figure 9j represent neuronal dropout or pyknotic neurons. in contrast, rb6-treated mice had only 106 ± 34 inflammatory monocytes and 11 ± 3 neutrophils in the 18 hpi bils (figure 9b , e) and only 6.7% ± 0.4% of the bils were cd45 hi . moreover, the hippocampus was completely preserved in these mice at 7 dpi ( figure 9h , k). the thick, multi-cell-layered neun staining in the ca1 layer in figure 9k is indistinguishable from figure 5 assessment of the green fluorescent protein (gfp) + brain infiltrate in theiler's murine encephalomyelitis virus (tmev)infected lysm:egfp mice. flow cytometric analysis of brain-infiltrating leukocytes (bils) collected at 18 hpi from lysm:egfp reporter mice (lys: gfp) and from wild-type littermates (wt) (c) revealed the presence of distinct gfp hi and gfp mid populations in the reporter mice (b). further characterization of cd11b, gr1, and 1a8 staining showed that the gfp hi population was composed of cd11b +++ gr1 + 1a8 + neutrophils, the gfp mid population was composed of cd11b ++ gr1 + 1a8inflammatory monocytes, and the gfp neg population contained cd11b + gr1 -1a8microglia (resident macrophages). (d-i) are derived from the gated populations in (b). these findings confirm our other observations and indicate the presence of both neutrophils and inflammatory monocytes in the brain at 18 hpi. results are representative of more than 10 mice. essentially no gr1 + (b) or 1a8 + cells (c) were observed in uninfected mice and the forward scatter (fsc) vs side scatter (ssc) plot showed a clear absence of the larger, more granular cells present at later time points (a). in contrast, tmev-infected mice exhibited both cd11b ++ gr1 + inflammatory monocytes and cd11b +++ 1a8 + neutrophils as early as 12 hpi (d-e), with inflammatory monocytes outnumbering neutrophils (e, f). both of these populations increased in number by 18 hpi (g-i). neutrophils continued to increase in the infiltrate by 24 hpi (k, l) while inflammatory monocytes began to decrease at this time point (k, l). the granular profile of neutrophils was also clearly present by fsc and ssc at 24 hpi (j). results are representative of more than 20 mice at each timepoint. table 1 quantitation of brain infiltrating inflammatory monocytes and neutrophils (brain-infiltrating leukocytes (bils)) at 12, 18, and 24 h post infection (hpi) rb6: q(28,3) = 19.923, p < 0.001). however, while treatment with 1a8 almost completely abrogated the neutrophil response in the 18 hpi bils (66 ± 24 neutrophils; control vs 1a8: q(28,2) = 17.917, p < 0.001) ( figure 9f ) there was an actual increase in the number of inflammatory monocytes (6,251 ± 237 inflammatory monocytes; control vs 1a8: q(28,2) = 19.194, p < 0.001) ( figure 9c ). the percentage of cd45 hi cells in the bils following 1a8 treatment was 23.4% ± 1.3% (control vs 1a8: q (28,2) = 1.064, p = 0.458; rb6 vs 1a8: q(28,3) = 34.188, p < 0.001). critically, the hippocampus was robustly injured in the 1a8-treated mice ( figure 9i, l) . finally, the absence of hippocampal injury in the rb6-treated mice led to the preservation of cognitive function as assessed by morris water maze ( figure 10 ). infected, rb6-treated mice showed the same ability to learn in the maze as uninfected mice (f(2,28) = 15.508, p < 0.001; uninfected vs rb6-treated: q(28,2) = 1.089, p = 0.441) while infected, control-treated mice were completely unable to learn to navigate the maze (uninfected vs infected, control-treated: q(28,2) = 14.826, p < 0.001; rb6-treated vs control-treated: q(28,3) = 15.519, p < 0.001) [1, 8] . we interpret these findings as strong support for our hypothesis that neutrophils and inflammatory monocytes are first responders to tmev infection in the brain and that inflammatory monocytes are primarily responsible for hippocampal damage and loss of cognitive function. in contrast to other viral models in which neutrophils apparently do not infiltrate the cns until recruited by other immune populations such as lymphocytes [2] , our findings suggest that neutrophils are one of the earliest responders in the tmev model. as in other viral model systems [4] [5] [6] , we predict that these cells will serve to prime the way for the adaptive response. in addition, our time course findings suggest that inflammatory monocytes precede neutrophils into the brain and the 1a8 depletion experiments show that inflammatory monocytes are competent to enter the cns in the absence of neutrophils. indeed, in the absence of neutrophils there was a significant increase in the number of inflammatory monocytes in the brain at 18 hpi, suggesting that neutrophils may exert a regulatory effect that slows or reverses inflammatory monocyte accumulation in the brain. the depletion experiments also show that inflammatory monocytes are required for the loss of ca1 pyramidal neurons that occurs in the first few days of infection [8] while neutrophils appear to be dispensable for this injury and the downstream consequences. ongoing experiments will assess the impact of inflammatory monocytes versus neutrophils in the recruitment of the adaptive immune system and eventual control of the virus. likewise, ongoing experiments in chemokine receptor knockout hosts will determine the relative impact of different chemotactic pathways in the separate and integrated neutrophil and inflammatory monocyte responses. our findings indicate that neutrophils and inflammatory monocytes rapidly and robustly respond to tmev infection by infiltrating the brain. we hypothesize that these effector cells, and inflammatory monocytes in particular, may be important therapeutic targets for immunomodulatory or immunosuppressive therapies aimed at reducing or preventing cns pathology associated with acute viral infection. figure 10 immunodepletion of inflammatory monocytes preserves cognitive function in the morris water maze. uninfected mice, untreated theiler's murine encephalomyelitis virus (tmev)-infected mice (no tx), and infected mice treated with rb6-8c5 to deplete inflammatory monocytes and neutrophils (rb6 tx) were assessed in the morris water maze starting at 14 dpi. following 3 days of training on a visible platform, latency to hidden platform acquisition was measured over six trials each day for 6 days. pretesting latency to the hidden platform is shown (pre) and compared to the latency to acquisition during the sixth and final trial of each day (day1 to day6). the uninfected mice readily learned the maze and navigated to the escape platform in less than 10 s by day 5. rb6-8c5 immunodepleted mice performed as well as uninfected controls. in contrast, the infected mice that were not depleted were unable to learn the maze at any point during the testing phase. these observations are consistent with the hippocampal pathology findings in figure 9 . results are from 10 mice in each group. disrupted spatial memory is a consequence of picornavirus infection myelomonocytic cell recruitment causes fatal cns vascular injury during acute viral meningitis coronavirus infection of the central nervous system: host-virus stand-off monocytes regulate t cell migration through the glia limitans during acute viral encephalitis a protective role for elr + chemokines during acute viral encephalomyelitis prolonged microglial cell activation and lymphocyte infiltration following experimental herpes encephalitis picornaviruses and cell death apoptosis of hippocampal pyramidal neurons is virus independent in a mouse model of acute neurovirulent picornavirus infection isolation of brain-infiltrating leukocytes insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages inflammation on the mind: visualizing immunity in the central nervous system the ins and outs of leukocyte integrin signaling f4/80: the macrophagespecific adhesion-gpcr and its role in immunoregulation a function for the macrophage f4/80 molecule in tolerance induction use of ly6g-specific monoclonal antibody to deplete neutrophils in mice absence of cross-reactivity between murine ly-6c and ly-6g normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive cd4+ t cells compared subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response characterization of the five novel ly-6 superfamily members encoded in the mhc, and detection of cells expressing their potential ligands inflammatory monocytes damage the hippocampus during acute picornavirus infection of the brain this work was supported by grant r01 ns64571 from the nih/ninds and by a kind gift from don and fran herdrich. authors' contributions clh designed all experiments, analyzed all data, and executed all of the microscopy. rlc prepared all of the animals and executed all of the experiments except immunostaining and mwm. rss sectioned the tissue and performed the immunostaining. sjl performed the morris water maze experiments. all authors read and approved the final version of the manuscript. the authors declare that they have no competing interests. key: cord-002320-m99amd4y authors: mathur, kalika; anand, abhishek; dubey, sunil kumar; sanan-mishra, neeti; bhatnagar, raj k.; sunil, sujatha title: analysis of chikungunya virus proteins reveals that non-structural proteins nsp2 and nsp3 exhibit rna interference (rnai) suppressor activity date: 2016-11-30 journal: sci rep doi: 10.1038/srep38065 sha: doc_id: 2320 cord_uid: m99amd4y rnai pathway is an antiviral defence mechanism employed by insects that result in degradation of viral rna thereby curbing infection. several viruses including flaviviruses encode viral suppressors of rnai (vsrs) to counteract the antiviral rnai pathway. till date, no vsr has been reported in alphaviruses. the present study was undertaken to evaluate chikungunya virus (chikv) proteins for rnai suppressor activity. we systematically analyzed all nine chikv proteins for rnai suppressor activity using sf21 rnai sensor cell line based assay. two non-structural proteins, namely, nsp2 and nsp3 were found to exhibit rnai suppressor activity. we further validated the findings in natural hosts, namely in aedes and in mammalian cell lines and further through emsa and agrobacterium infiltration in gfp silenced transgenic tobacco plants. domains responsible for maximum rnai suppressor activity were also identified within these proteins. rna binding motifs in these domains were identified and their participation in rnai suppression evaluated using site directed mutagenesis. sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. further validation of rnai suppressor activity of these proteins awaits establishment of specific virus infection models. are transmitted through arthropods, and display differential replication pattern in the two hosts. while invertebrate hosts like mosquitoes and ticks behave as maintenance hosts where the viruses persists in low levels 19, 20 , vertebrate hosts get infected to high titers with efficient virus replication 21 and this alteration between hosts has been shown to play an important role in evolution of arboviruses 22 . until recently, it had been speculated that these arbovirus do not possess rnai suppressor activity 23 unlike other insect-specific viruses like fhv and crpv 24, 25 . however, recent studies have established that flaviviruses encode for vsrs 26, 27 . alphaviruses are member of arboviruses containing 27 recognised members 28 with a very wide geographic distribution and several geographical variants on the basis of which they have been classified as old world and new world viruses 29 . grouped into seven complexes based on serological cross-reactivity, they infect a variety of host including birds, fishes, mammals including humans and are maintained in natural cycles by transmission between susceptible vectors and vertebrate hosts 30 . alphaviruses have a single plus-stranded rna genome encapsulated by capsid proteins. the approx. 12 kb genome consists of two open reading frames that encodes four non-structural proteins (nsp1 to nsp4), three structural proteins (capsid, envelope glycoproteins e1 and e2) and two small cleavage products (e3 and 6k). while the structural proteins are involved in mediating entry and fusion of the virus into the hosts, the non-structural proteins (nsps) form the replication complex 31 . the non-structural proteins that are initially translated as one or two polyprotein from the full-length genomic viral rna are processed exclusively by the virus-encoded protease, nsp2. upon cleavage, each of the mature proteins as well as the intermediates actively participates in virus replication. as of date, some properties of these nsps are known 32 : nsp1 is membrane-associated and possess both guanine-7-methytransferase and guanyltransferase activity, nsp2 exhibit several enzymatic activities including helicase, protease activity, nsp3 possess phosphatase and rna-binding activity 33, 34 and nsp4 is a rna-dependent rna polymerase (rdrp) 35 . even though much information is available from the above and other studies, it is expected that these proteins have more novel roles in viral replication. the current study undertaken to identify viral encoded suppressors of rnai in chikungunya virus (chikv) has thrown light to possible rnai suppressor activity in two non-structural proteins of chikv. systematic analysis of all chikv proteins using a sf21 rnai sensor cell line based assay revealed that non-structural proteins nsp2 and nsp3 exhibited rnai suppressor activity. we further validated this finding in its natural hosts, namely, an aedes and mammalian cell line. after dissecting the domain responsible for suppressor activity, we evaluated the suppressor strength by mutating the rna binding motifs in these domains. aligning known sequences of nsp2 and nsp3 across all alphavirus species, we observed that these rna binding motifs are conserved across species thereby confirming the importance of these motifs in alphavirus-host interactions vis-à-vis rnai pathway. recombinant alphavirus with an endogenous rnai suppressor have shown an increase in alphavirus replication in aedes mosquitoes 36 . while it was previously thought that flaviviruses do not possess any rnai suppressors, it was recently proved that these viruses do possess vsrs that are active in both insect and mammalian cells 26, 27 . in case of alphaviruses, reports have suggested that rnai suppressors in this class of viruses may either be weak or undetected due to inappropriate assay conditions 4, 37 . we undertook the present study to systematically evaluate chikv proteins (fig. 1a) for possible rnai suppressor activity using a sf21 rnai sensor cell line developed in our lab 16 . using this rnai sensor cell line, in which gfpshrna is expressed constitutively to silence gfp in a gfp sf21 cell line; we checked for reversion of gfp due to rnai suppressor activity of the cloned chikv genes in pib vector using flow cytometry ( fig. 1b and c) . post 72 h transfection, it was observed that nsp2, nsp3 and nsp4 showed a reversion in gfp expression of 60%, 55% and 40% respectively (p values < 0.0001). the other genes showed reversion similar to empty vector. denv rnai suppressor ns4b and flock house virus protein b2 served as positive controls. previous studies have emphasised the importance of testing the efficiency of vsrs in relevant host systems 17 . having checked suppressor activity of all chikv genes using an established cell line based assay, we next checked for their suppressor activity in its natural hosts, namely a mosquito and mammalian system. for this purpose, we utilised an aedes cell line, aag2 and the mammalian cell line, hek293t. using transient transfection protocols in which gfpshrna in pizt/v5-his and pgfp vrs plasmid were co-transfected with nsp2/nsp3 pib/ pcdna3.1+ plasmids in aag2 and hek293t respectively (fig. 2a) , we evaluated suppressor activity of all nine genes. overall, it was seen that transfection worked better in hek as compared to aag2 owing to better transfection efficiency in the mammalian system. with respect to gfp reversions, it was observed that nsp2 and nsp3 showed significant reversion (p value < 0.001) in both aedes and hek cell lines ( fig. 2b and c) . in case of nsp4, however, rnai suppressor activity was not evident in both the natural hosts and only around 25% gfp reversions were seen in the cells upon transfections with nsp4. based on these results, we identified chikv_nsp2 and chikv_nsp3 to possess rnai suppressor activity and further proceeded to validate these findings. nsp2 and nsp3 are suppressors of rnai. having established that nsp2 and nsp3 show rnai suppressor activity, we proceeded to detect the level of gfp protein due to effect of the putative vsrs. western analysis also revealed increase in gfp expression in nsp2 and nsp3 transfected cells as compared to sf21 rnai sensor cells (fig. 3a) . in addition to the above mentioned in vitro assays, we utilised transgenic nicotiana leaf based assay to validate rnai suppressor activity of nsp2 and nsp3 under in vivo conditions 38 . the putative vsrs were cloned into the plant binary vector pbi 121 and reversal of silencing by agro-infiltration-mediated transient ectopic expression of chikv nsp2 and chikv nsp3 was assessed in transgenic tobacco leaf tissues in which gfp is silenced. the infiltrated leaves showed reversal of gfp activity at 3 days post-infiltration in nsp2 and 5 days post-infiltration in nsp3 as seen under uv light ( fig. 3b and c) . in the infiltration experiments, fhvb2 plasmid was used as positive control and a mutated fhvb2 expressing plasmid as negative control. gfp reversal was further confirmed by analysing gfp transcript levels and the depletion of small rna population of gfp in the leaves using northern hybridisation. 18 s rna and 28 s rrna were used as loading controls for mrna and small rna northerns respectively. (fig. 3d) . while there was an increase in the gfp mrna levels in leaves infiltrated with nsp2 and nsp3, gfp small rnas showed 50% and 80% decrease in their population in the leaves infiltrated by nsp2 and nsp3 respectively thereby providing evidence that chikv nsp2 and nsp3 are influencing small rna production post-transcription. even as the assays in different systems provided enough clarity as to presence of rnai suppressor activity in chikv nsp2 and nsp3, the first strong proof for any rnai suppressors is its role in interfering with the rnai machinery. the most well known mechanism is the ability of vsrs to bind to dsrna/sirna population thereby preventing risc from acting upon them. this phenomenon has been evaluated by studying the binding of suppressor protein to double stranded rna population resulting in a shift in their mobility efficiency 7, 16 . for this purpose, we expressed both nsp2 and nsp3 in the heterologous e.coli system. while we were able to produce nsp3 in its natural folded form, nsp2 aggregated into inclusion bodies and could not be purified, most probably owing to its large size. to overcome this problem, we expressed both chikv_nsp2 and chikv_nsp3 in sf21 cell line by transient transfection (supplementary fig. s2 ). using cell lysate of the transfected cell expressing nsp2 and nsp3, we evaluated their ability to bind double stranded rna by using [γ 32p] labeled shrna of gfp and running the bound complex in a 6% polyacrylamide gel (fig. 3e) . binding of shrna to nsp2 and nsp3 proteins were evident by complex formation and shift that increased proportionally to protein concentration (fig. 3e -lane no. 2, 3, 4, 5) . addition of cold shrna probe served as competitive inhibitor of binding reaction and the decrease in the intensity of the shift proved the binding specificity of the complex. untransfected sf21 lysate in the presence of non-specific inhibitor (salmon sperm dna) of varying concentrations and gfpshrna served as negative the y-axis shows % gfp reversion (mean ± sd). statistical significance was analysed using student's t-test and fisher's least significant difference (lsd) test using empty vector as control. the *symbol indicates a statistically significant difference in terms of the p value (p < 0.001). nsp2, nsp3 and nsp4 were found statistically highly significant from lsd test (p value < 0.0001 each) when compared with the vector (negative control) (alpha level of the test = 0.05). 8, 9, 10) . these results clearly proved nsp2 and nsp3 bound to dsrna. taken all these findings together, we conclude that chikv nsp2 and chikv nsp3 are suppressors of rnai. suppressor proteins interfere with the host rnai components to inhibit the pathway. vsrs may block the dicing activity and thereby reduce formation of sirnas. alternately, vsrs may also impede loading of sirnas into the risc and consequently block functionalization of sirna loaded risc 37 .a direct way to assess effect of vsrs on sirna formation is to estimate dicing enzyme activity in vitro in the presence of the vsrs. unfortunately, our attempts to purify chikv nsp2 in its active form were unsuccessful, hence we resorted to cell based facs assays to gain preliminary insight into possible suppression mechanism. using sf21 gfp cell line, we transfected dsrna or sirna-gfp and subsequently co-transfected with the vsrs to estimate the level of gfp reversal. we reasoned that gfp reversal was an indication of where the vsrs may act -if they acted during dicing, then the reversal would be evident upon co-transfection with dsrna+ vsrs and if they acted at the time of sirna loading into risc, it would be evident while using sirna+ vsrs. our results showed there was > 50% gfp reversion with both dsrna and sirna for both nsp2 and nsp3 ( fig. 4a and b) , thus highlighting that their mode of action may be downstream of risc loading in the rnai pathway. domain mapping of nsp2 and nsp3 reveal domain specific rnai suppressor activity. the non-structural proteins, nsp2 and nsp3 of chikv participate in several functions; nsp2 has been shown to have multiple functions including helicase, protease and ntpase activities [39] [40] [41] [42] . this protein is 90 kda and consists of four domains, the 180 aa long n-terminal domain, 240 aa long domain that exhibits helicase activity, 200 aa long protease domain and a 160 aa long c terminal domain 43 (fig. 5a) . similarly, nsp3 exhibits phosphatase activity and also consists of a macrodomain that has been well characterised 32 . this is 57 kda and consists of three domains (fig. 5c) . the macrodomain, present on the n-terminus, is a globular domain 162 aa long and is evolutionary highly conserved. most of the activities exhibited by the protein are associated with this domain. the sequence of central part of nsp3 (162 aa) is conserved only among alphaviruses and hence is termed alphavirus unique domain (aud). the c-terminal domain (204 aa) is termed hypervariable region, since it has no sequence similarity even among alphaviruses. having confirmed that both nsp2 and nsp3 exhibit rnai suppressor activities, efforts were taken to characterise the proteins better and to identify those specific domains that were and ns4b (dengue virus) were taken as positive controls and empty vector was used as negative control. statistical significance was analysed using student's t-test and fisher's least significant difference (lsd) test using empty vector as control. the *symbol indicates a statistically significant difference in terms of the p value (p < 0.001) and # symbol represents p-value > 0.001. nsp2 (p-value = 0.0003) and nsp3 (p-value = 0.0007) were found statistically highly significant, while nsp4 was found not significant (p-value = 0.035) (alpha level of the test = 0.05). (c) fluorescent microscopic images of hek293t cell line based assay. hek293t cells were transfected with gfp plasmid and gfpshrna plasmid in separate wells. for gfp reversion assay gfpshrna and individual vsr plasmid were co-transfected and cells were checked for fluorescence under microscope at 72 h post transfection. scientific reports | 6:38065 | doi: 10.1038/srep38065 responsible for rnai suppressor activity. for this purpose, each of the domains was cloned individually in pib/ v5-his topo vector and transfected into the sf21 sensor line. for convenience, the domains were labelled as n2d1, n2d2, n2d3 and n2d4 in nsp2 and n3d1, n3d2 and n3d3 in nsp3. in case of nsp2, n2d2, the domain that is responsible for helicase activity showed maximum reversion of gfp expression and in nsp3, n3d1, i.e., the macrodomain showed maximum reversion ( fig. 5b and d) . rna binding motifs are conserved across alphaviruses. it is well known that most of the vsrs possess certain sequence characteristics to function as an rnai suppressor. presence of rna binding motifs and presence of gw/wg motifs has been shown to be important features required for binding to dsrna/sirna and argonaute loading 44 . to understand the sequence structure of the domains that showed maximum suppressor activity in nsp2 and nsp3, we identified the rna binding motifs within the domains showing maximum reversions in both the vsrs (fig. 6a and c) . furthermore, we hypothesized that these features are important amongst all species of alphavirus. to test this hypothesis, we aligned the sequences of all known alphaviruses nsp2 and nsp3. to date, complete sequence information is available for 15 alphavirus species. as expected, all rna binding motifs were conserved in both nsp2 and nsp3 proteins across all species (fig. 6b and d) . additionally, we observed the presence of one gw motif in domain 2 of nsp2 in all species. taking all these aspects, we hypothesize the rnai suppressor activity of chikv nsp2 and nsp3 may be conserved across all alphaviruses. rna binding motifs contribute to vsr activity of chikv nsp2 and nsp3. having identified the conserved rna binding motifs in nsp2 and nsp3, we proceeded to evaluate the impact these motifs have in rnai suppressor activity in the vsrs. substituting all or most of the amino acids in motifs with alanine through site directed mutagenesis, we generated mutants. three mutants were generated in nsp2 with different permutations and combinations of substitutions, while two mutants were generated in nsp3. schematic representation of mutant generation is depicted in fig. 7a and b. gfp reversal in these mutants were evaluated using sf21 rnai sensor line and the results are shown in fig. 7c . further, we performed mobility shift assays to evaluate the binding capacities of the domains and the mutants generated using [γ 32 p] labeled shrna of gfp and running the bound complex in a 6% polyacrylamide gel (fig. 7d) . complex formation due to dose dependent binding of shrna to the mutants of nsp3 and nsp2 are evident (lane 3, 4, 14 and 15 respectively). in the same manner, binding efficiencies of the domains to shrna are demonstrated in lane 6, 7, 11 and 12 respectively. the results clearly show the involvement of the rna binding motifs in the binding to dsrna and we conclude that these motifs play a direct role in rnai suppressor activity. nsp1-sf21 lysate in the presence of non-specific inhibitor (salmon sperm dna) and gfpshrna served as negative controls (lane 1, 9, 10). alphaviruses are maintained in nature by alteration between mosquitoes and human host. while their infections in mosquitoes are mainly asymptomatic and persistent, in vertebrate hosts, the infection is acute and self limiting 31 . efficiency of virus replication in both vector and host depends not only on the expression of its own proteins but also on its ability to counteract upon host antiviral defence. mosquito innate immune system poses a serious challenge to virus infection and activates different defence pathways including toll pathway, imd (immune deficiency) pathway, jak stat pathway and rnai pathway 45 . rnai pathway is a major antiviral defence mechanism employed by insects that results in degradation of viral rna thereby curbing infection [1] [2] [3] [4] . viruses employ numerous strategies to evade host rnai response, including mutations in or near the target to bring conformational changes rendering risc inaccessible 46 and vsrs which inhibit or compete with dicer/other rnai components, thus decreasing the amounts of viably loaded risc complexes 7,46,47 . earlier reports have suggested that arboviruses may possess weak suppressors that have been yet undetected due to inappropriate assay conditions 4, 37 . the speculations of absence of vsrs in arboviruses were eliminated when denv proteins showed suppressor activity 48 . however, since alphaviruses do not cause as severe illness as the flavivirus, it was assumed that vsr activity against rnai defence might be detrimental for the host, thus reducing virus transmission 36, 49 . using an in-house developed gfp reversion assay 16, 26 , we report that chikv nsp2 and nsp3 are rnai suppressors. certain vsrs such as geminivirus nuclear protein ac2 have been previously shown to transactivate host genes 50 . we confirmed the specificity of the gfp reversion by checking nsp2 and nsp3 for non-specific promoter activity (supplementary fig. s1 ). the non-structural proteins, nsp2 and nsp3 of chikv participate in several functions; nsp2 has been shown to have multiple functions including helicase, protease and ntpase activities 30, [40] [41] [42] . nsp2 has been shown to interact the most with host factors than any other chikv protein, mainly due to its dual role as key component of viral replication complex and also as an important virulence factor inhibiting the host antiviral response 51 . in our study, nsp2 has shown stronger vsr activity in comparison to nsp3. earlier reports have shown this protein exhibits anti-ifn activity 52 , emphasising on the overlapping nature of interferon and rnai pathway. domain mapping of this protein revealed the domain that possessed helicase activity showed maximum activity for rnai suppression. interestingly, helicases such as heif of dictyostelium, b2 of wuhan nodavirus and ns3 of dengue virus has been proven to exhibit vsr activity 15, 48, 53 . of interest is the presence of a gw motif, conserved across all alphavirus species studied, that is known to be important for binding to dsrna/sirna and argonaute loading 44 . the crystal structure of chikv nsp3 macrodomain reveals that this domain possesses rna-binding activity 33 . this protein is also known to modulate pathogenicity in mice 54 . our study has provided evidence of an additional role of the macrodomain and clearly elucidates it role in pathogenicity as a viral suppressor of rnai. the existence of both nsp2 and nsp3 in both the nucleus as well in the cytoplasmic fraction 55,56 may help in their interactions with the various components of the risc complex. alphavirus species exhibit sequence conservation amongst several of their proteins. motifs that are involved in direct interactions with both the vector as well as the host have been shown to be conserved even in the otherwise non-conserved regions 57 . through our study we have established all rna binding motifs of chikv nsp2 and nsp3 domains that possess vsr activity are conserved across alphavirus species. it is speculated that these proteins may participate in similar functions in the other alphavirus species as well. taken together, the current study has identified chikv proteins nsp2 and nsp3 to exhibit rnai suppressor activity in the in-vitro system that was further demonstrated in its natural hosts, namely, an aedes and mammalian cell line. the domains responsible for suppressor activity was further identified and their suppressor strength evaluated based on mutations introduced in the rna binding motifs of these domains. finally, these rna binding motifs were found to be conserved across alphavirus species. it is to be noted however, the vsr activities of nsp2 and nsp3 exhibited by in-vitro assays could be strengthened further by a direct demonstration employing in-vivo infection system. supplementary table s1 . to find the putative rnai suppressors in chikv, a previously developed rnai sensor cell line constitutively expressing gfpshrna was used. briefly, 0.2 million sf21 gfpshrna cells were seeded in a 24 well plate in complete media and transfected with 2 μ g of chikv gene plasmids using cellfectinii reagent (invitrogen) as per manufacturer's protocol to transiently express the chikv proteins. empty pib/v5-his topo vector was used as negative control. previously published dengue virus suppressor ns4b and flock house virus protein b2 (fhvb2) were used as positive controls. facs analysis was done after 72 h for gfp expression in transfected cells. sf21 cell line with and without gfp were used as facs gating control. gfp specific dsrna and sirna mediated reversion assay was carried out similarly in sf21 gfp stable cell line in 24 well plate. briefly, 2 μ g vsr plasmid was co-transfected with 1.5 μ g gfp dsrna plasmid and 2.5 pmole sirna in separate wells. scrambled sirna was used as gfp silencing negative control. facs analysis was done similarly after 72 h. rnai suppression assay in natural host cell lines. aag2 (aedes aegypti) cells were transfected using attractene reagent (qiagen). briefly, 0.2 million aag2 cells were co-transfected with gfpshrna plasmid and chikv-pib/v5-his topo plasmid. gfp levels were analyzed with facs after 72 h. gfp reversion assay in human host was done using hek293t cells. each chikv-pcdna3.1+ plasmid was co-transfected with gfpshrna plasmid using jet prime reagent (polyplus transfection) in jet prime buffer, followed by facs analysis after 72 h. activated cell sorting (facs) with facs calibur (bd biosciences). media from cells was removed and the cells were washed and resuspended in 500 μ l of facs grade pbs. respective cell lines without gfp were used specifically for gating the region of negative control. cell quest software (becton-dickinson, franklin lakes, nj, usa) was used to analyse the percentage of gfp reversion in 20,000 cells against fl1-h.percentage gfp reversion data was represented as mean ± sd. the experiments were repeated at least four times in triplicates. statistical analysis of experimental data was conducted using graphpad prism (version 6). two tailed student's t test and fisher's least significant difference (lsd) test were performed to check significance of the data and p-values < 0.001 were considered significant. validation of rnai suppression in nicotiana leaves. nsp2 and nsp3 genes were separately cloned in pbi 121 vector for transformation in agrobacterium. the secondary culture was incubated in fresh yem and acetosyringone for 1 h, followed by infiltration in transgenic nicotiana leaves which has gfpshrna stably integrated. the culture was infiltrated on dorsal side of the leaves. the leaves were analysed in uv transilluminator 3, 5, 7 and 10 days post infiltration (dpi). a previously known suppressor fhvb2 was used as positive control and mutated fhvb2 was used as negative control. fected with all 9 genes of chikv cloned in pib vector, nsp2/m3 pib, nsp3/m2 pib and vsr domains cloned in pib plasmids separately. the cells were harvested after 72 h and lysed in lysis buffer (50 mm tris hcl and 300 mm nacl) containing protease inhibitor cocktail (roche). whole cell lysate concentration was determined using bradford protein assay (bio-rad laboratories). protein extracts (60 μ g) were subjected to 12% reducing page and western blotting was done using anti-his (mice) primary antibody (sigma), followed by ap conjugated (mice) secondary antibody. western blotting to detect changes in gfp expression levels. sf21sensor cell line was transfected with nsp2 pib and nsp3 pib plasmids separately and cells were harvested after 72 h. 60 μ g total cell lysate was run on 12% sds page and western blotting was done using anti-gfp primary antibody (santa cruz) and ap conjugated secondary antibody. levels of gapdh proteins were used as housekeeping control. electrophoretic mobility shift assay. gfpshrna binding ability of of different proteins was evaluated using electrophoretic mobility shift assay (emsa). sf21 cells transfected with nsp2, nsp3, mutants and domains plasmids were analysed along with untransfected sf21 cell line as negative control. gfpshrna probe was synthesised from sigma as a self complementary 49 bp dna oligonucleotide with a hairpin loop. binding reaction was setup with different concentrations of cell lysate, 1x binding buffer (30 mm hepes and 100 mm nacl), [γ −32 p] atp labelled shrna probe (20,000 cpm per reaction) and 2 μ g of salmon sperm dna (thermo scientific). unlabelled shrna was added as cold probe to check specificity of the binding. the complex was resolved on 6% polyacrylamide non-denaturing gel (pre-run for 1 h at 4 °c) in 1x tbe buffer at 100 v at 4 °c. gels were dried and exposed for auto-radiography. the scanning was performed using typhoon 9210 variable mode imager (amersham biosciences). northern blotting. nicotiana leaves infiltrated with vsr plasmids were harvested and total rna was isolated using gitc method. for mrna northern. 20 μ g total rna was run on 1% agarose gel. rna was transferred onto hybond-n + membrane (ge healthcare) overnight by capillary transfer. rna was immobilised on the membrane by uv cross linking at 1,200 × 100 μ j. northern blotting for mrna was performed using [γ −32 p] atp labelled shrna probe. for small rna northern. rna was isolated using gitc method followed by small rna enrichment. 50 μ g rna was run on 15% page containing 8 m urea. rna was transferred onto a hybond-n + membrane (ge healthcare) for 60 min at 15 v and immobilised by uv cross-linking at 1,200 × 100 μ j. gfp probe was prepared by dig labelling kit (roche, usa). northern hybridisation and development was performed according to instruction manual. small rna marker (neb) lane was cut from the blot and radioactively labelled with [γ −32 p] atp separately. domain mapping of the chikv rnai suppressors in sensor cell line. sf21 sensor cell line was transfected with 2 μ g of each domain plasmid using cellfectin ii reagent, followed by facs analysis after 72 h. complete gene plasmid and fhvb2 were used as positive controls and empty vector was used as negative control. alignment of rna binding motifs of alphaviruses. nsp2 and nsp3 sequences of 14 other alphaviruses were fetched from ncbi and aligned with chikv new delhi strain ind-10-del9 using mega5 software. the rna binding motifs were predicted using bindn software and checked for conservation amongst alphaviruses. site directed mutagenesis in nsp2 and nsp3 pib/v5-his topo plasmids. nsp2 and nsp3-pib clones were mutated using quickchange multi site directed mutagenesis kit (agilent technologies) for mutating different sites in the same plasmid simultaneously. briefly, 50ng plasmid was mutated using different primers binding to the same dna strand with pcr, followed by dpni digestion and transformation. the mutations were confirmed by sequencing from macrogen. gfp reversion was assayed in sf21 sensor cell line. wild type vsr plasmids were used as positive controls. rna-based antiviral immunity suppression of gene silencing: a general strategy used by diverse dna and rna viruses of plants rnai function, diversity, and loss in the fungal kingdom mosquito rnai is the major innate immune pathway controlling arbovirus infection and transmission dicing of viral replication intermediates during silencing of latent drosophila viruses small silencing rnas: an expanding universe the rna silencing endonuclease argonaute 2 mediates specific antiviral immunity in drosophila melanogaster the rest is silence dicing and slicing: the core machinery of the rna interference pathway the many faces of rnai the pirna pathway in flies: highlights and future directions. current opinion in genetics & development antiviral immunity directed by small rnas double-stranded rna binding may be a general plant rna viral strategy to suppress rna silencing mechanism of induction and suppression of antiviral immunity directed by virus-derived small rnas in drosophila rna binding by a novel helical fold of b2 protein from wuhan nodavirus mediates the suppression of rna interference and promotes b2 dimerization suppression of rna silencing by flock house virus b2 protein is mediated through its interaction with the paz domain of dicer novel drosophila viruses encode host-specific suppressors of rnai present and future arboviral threats advances in dissecting mosquito innate immune responses to arbovirus infection induction and suppression of tick cell antiviral rnai responses by tick-borne flaviviruses arbovirus evolution in vivo is constrained by host alternation mosquitoes put the brake on arbovirus evolution: experimental evolution reveals slower mutation accumulation in mosquito than vertebrate cells the role of rnai and micrornas in animal virus replication and antiviral immunity the structure of the flock house virus b2 protein, a viral suppressor of rna interference, shows a novel mode of double-stranded rna recognition cricket paralysis virus antagonizes argonaute 2 to modulate antiviral defense in drosophila role of rna interference (rnai) in dengue virus replication and identification of ns4b as an rnai suppressor noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells alphaviruses (togaviridae) and flaviviruses (flaviviridae the old world and new world alphaviruses use different virus-specific proteins for induction of transcriptional shutoff evolution of rna viruses the alphaviruses: gene expression, replication, and evolution functions of alphavirus nonstructural proteins in rna replication the crystal structures of chikungunya and venezuelan equine encephalitis virus nsp3 macro domains define a conserved adenosine binding pocket a structural and functional perspective of alphavirus replication and assembly mapping of rna-temperature-sensitive mutants of sindbis virus: assignment of complementation groups a, b, and g to nonstructural proteins suppression of rna interference increases alphavirus replication and virus-associated mortality in aedes aegypti mosquitoes the long and short of antiviral defense: small rna-based immunity in insects the 7a accessory protein of severe acute respiratory syndrome coronavirus acts as an rna silencing suppressor processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the c-terminal half of nsp2 and functions both in cis and in trans proteolytic processing of semliki forest virus-specific non-structural polyprotein by nsp2 protease identification of the active site residues in the nsp2 proteinase of sindbis virus site-specific protease activity of the carboxyl-terminal domain of semliki forest virus replicase protein nsp2 evasion of the innate immune response: the old world alphavirus nsp2 protein induces rapid degradation of rpb1, a catalytic subunit of rna polymerase ii viral rna silencing suppressors (rss): novel strategy of viruses to ablate the host rna interference (rnai) defense system the host defense of drosophila melanogaster rna interference against viruses: strike and counterstrike virus counterdefense: diverse strategies for evading the rna-silencing immunity dengue ns3, an rnai suppressor, modulates the human-mirna pathways through its interacting partner alphavirus-derived small rnas modulate pathogenesis in disease vector mosquitoes suppression of rna silencing by a geminivirus nuclear protein, ac2, correlates with transactivation of host genes mapping of chikungunya virus interactions with host proteins identified nsp2 as a highly connected viral component chikungunya virus nonstructural protein 2 inhibits type i/ii interferon-stimulated jak-stat signaling helf, a putative rna helicase acts as a nuclear suppressor of rnai but not antisense mediated gene silencing the nsp3 macro domain is important for sindbis virus replication in neurons and neurovirulence in mice different types of nsp3-containing protein complexes in sindbis virusinfected cells nuclear localization of semliki forest virus-specific nonstructural protein nsp2 sh3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsp3 promotes viral rna replication this work was supported by the financial grant from department of biotechnology (bt/pr14725/ agr/36/672/2010). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. km acknowledges council of scientific and industrial research (csir, india) for the fellowship. we thank dr. alain kohl for providing aag2 cell line. we are grateful to dr. sunil mukherjee for the discussions and beneficial scientific inputs. we also thank pavan kakumani for providing the ns4b-pib construct and sudhanshu sekhar das for fhvb2 and fhvb2m constructs. geetha sundaresan's involvement in agrobacterium infiltration assays is acknowledged. key: cord-345651-admlzeu4 authors: wang, gang; liang, rui; liu, ziwei; shen, zhou; shi, jiale; shi, yuejun; deng, feng; xiao, shaobo; fu, zhen f.; peng, guiqing title: the n-terminal domain of spike protein is not the enteric tropism determinant for transmissible gastroenteritis virus in piglets date: 2019-03-30 journal: viruses doi: 10.3390/v11040313 sha: doc_id: 345651 cord_uid: admlzeu4 transmissible gastroenteritis virus (tgev) is the etiologic agent of transmissible gastroenteritis in pigs, and the n-terminal domain of tgev spike protein is generally recognized as both the virulence determinant and enteric tropism determinant. here, we assembled a full-length infectious cdna clone of tgev in a bacterial artificial chromosome. using a novel approach, the clustered regularly interspaced short palindromic repeat (crispr)/crispr-associated protein 9 (cas9) systems efficiently and rapidly rescued another recombinant virus with a 224-amino-acid deletion in the n-terminal domain of the tgev spike gene (s_ntd224), which is analogous to the n-terminal domain of porcine respiratory coronavirus. s_ntd224 notably affected the tgev growth kinetics in pk-15 cells but was not essential for recombinant virus survival. in animal experiments with 13 two-day-old piglets, the tgev recombinant viruses with/without s_ntd224 deletion induced obvious clinical signs and mortality. together, our results directly demonstrated that s_ntd224 of tgev mildly influenced tgev virulence but was not the enteric tropism determinant and provide new insights for the development of a new attenuated vaccine against tgev. importantly, the optimized reverse genetics platform used in this study will simplify the construction of mutant infectious clones and help accelerate progress in coronavirus research. coronaviruses (covs) are single-stranded, positive-sense rna viruses closely related to animal and human health [1] [2] [3] . covs belong to the coronaviridae family, which consists of the alpha-, beta-, gamma-and deltacoronavirus genera [4] . since 2003, covs, including severe acute respiratory syndrome cov (sars-cov), middle east respiratory syndrome cov (mers-cov), and porcine epidemic diarrhea virus (pedv), have swept across the world and caused considerable global economic losses [5] [6] [7] [8] [9] . as the largest rna genome viruses, covs have at least six typical overlapping open reading frames pk-15 cells were cultured in dulbecco's modified eagle's medium (gibco, waltham, ma, usa) supplemented with 10% fetal bovine serum at 37 • c with 5% co 2 . tgev strain wh-1 (genbank accession number hq462571) was propagated at 37 • c in a 5% co 2 incubator in dulbecco's modified eagle's medium (gibco, waltham, ma, usa) supplemented with 2% fetal bovine serum (gibco, waltham, ma, usa). all the experiments using live viruses were performed under biosafety level (bsl) 2 conditions. total rna was extracted from virus-infected cultures using trizol reagent (invitrogen, carlsbad, ca, usa), and cdna was reverse transcribed with reverse transcriptase (takara, amv, kusatsu, japan) using random primers (takara, kusatsu, japan, 6 mer). all the fragments were amplified by polymerase chain reaction (pcr) with phanta super fidelity dna polymerase (vazyme, nanjing, jiangsu, china). the natural complete genome of tgev wh-1 was determined by sequencing (genscript, nanjing, china) the overlapping pcr products cloned into the corresponding vectors in triplicate. compared with the parental tgev wh-1 from ncbi (bethesda, md, usa), several site mutations, including t6299c, g11123t, g25943c, c26094t, and c26336g, were observed, and these mutations were maintained during the cloning of the tgev full-length genome. a point mutation, a4553t, was introduced by overlap extension pcr to remove the natural van91i site and maintained as the rescue marker. as described in previous research, the egfp gene was inserted into the tgev genome [46, 47] to replace the original sequence positioned from 24,826 to 28,580. the virus genome was divided into six continuous fragments (a to f), and each fragment was amplified from the total cdna using specific primers (available upon request). fragments a, b, d, and e were cloned into the pmd18-t vector (takara, kusatsu, japan). fragment a was cloned with the saci and van91i sites, and fragment e was cloned with the van91i and kasi sites. notably, fragment a was cloned to contain a saci site (gagctcgtttagtgaaccgt) [48] located in the 5 terminal of the tgev genome sequence. fragments b and d were cloned with van91i sites. fragment c was cloned into a bac plasmid (kindly provided by prof. cao gang) that was modified from pbelobac11 to include a van91 site [36] . fragment f was also cloned into the bac plasmid with saci and kasi sites introduced at the 5 and 3 termini of the tgev genome, respectively. as the final recipient bac vector, subclone f also contained the synthesized essential element sequences, such as the cmv promoter, the poly(a) tail sequence (25a), the hdv rz sequence (hepatitis delta virus self-cleaving ribozyme sequence, rz), and the bovine growth hormone (bgh) transcription terminal signal (genscript, nanjing, china) [49] . after the six subclones were sequenced in their corresponding vectors, subclones a and e were first digested with saci and kasi, respectively, and then treated with calf intestinal alkaline phosphatase (ciap, scientific). all the subclones were then digested with van91i except subclone f, which was digested with saci and kasi. subsequently, all the digested products were purified with a gel extraction kit (omega, norcross, ga, usa), and fragments a to f were ligated for more than 2 h at 16 • c and transformed into chemically competent dh10b cells (biomed, beijing, china). after determination of all the fragments by bacterial pcr, the positive clones were further determined by restriction fragment length polymorphism with kpni, and the correct clone was designated ptgev-gfp bac after sequencing (genscript, nanjing, china). pk-15 cells were seeded in a six-well plate and incubated for 12 h, and the recovery of tgev-gfp or tgev-gfp-∆s_ntd was then performed by transfecting 5 µg of the corresponding bac into pk-15 cells with 8 µl of lipofectamine 2000 (invitrogen, carlsbad, ca, usa). at 48 h post-transfection, the collected virus progenies were purified once by fluorescent plaques. subsequently, the purified virus clone was amplified and stored until use at −80 • c. for the design of a sgrna to mediate cleavage of the targeted site, a constant reverse primer (ssdna-r) and two forward primers (ssdnaa-f and ssdnab-f) specific for sites a and b were synthesized as shown in table 1 , similar to the protocol described in a previous report [45] . to anneal the primers ssdnaa-f and ssdnab-f with the reverse primer ssdna-r, pcr was conducted for 30 cycles at 95 • c for 15 s, 55 • c for 15 s, and 72 • c for 20 s using 2× utaq mastermix (zoman). the pcr products were then purified with cp buffer (omega, norcross, ga, usa) and transcribed using a t7 transcription kit (neb, ipswich, ma, usa) according to the manufacturer's instructions to produce the targeted sgrna a and sgrna b. the purity of the sgrna products was analyzed by electrophoresis on agarose gels using 0.5 µg of each sgrna product. to modify the sequence of tgev s_ntd224, ptgev-gfp bac was digested using the targeted sgrnas. specifically, ptgev-gfp bac was digested in a 50 µl reaction mixture with 5 µg of ptgev-gfp bac, 5 µl of cas9 (neb, ipswich, ma, usa), 10 µg of sgrna, and 5 µl of nuclease reaction buffer incubated at 37 • c for more than 2 h or, preferably, overnight. for purification of the digested ptgev-gfp bac, an equivalent volume of solution i (plus rnase; omega, norcross, ga, usa) was first added to digest the sgrna at room temperature for 3 min, and the cp buffer (omega, norcross, ga, usa) was then applied to recycle the digested bac according to the manufacturer's instructions. the pcr products with the 672-bp deletion were constructed by two-cycle pcr. first, the primers rec-672sf and δs-ntdr or the primers rec-672sr and δs-ntdf (table 2 ) were used to amplify the primary pcr products from the template of the ptgev-gfp bac. second, the two primary pcr products were annealed to produce pcr products of the 672-bp deletion using the primers rec-672sf and rec-672sr (table 2 ). homologous recombination was then performed using the clonexpress ii one step cloning kit (vazyme, nanjing, jiangsu, china) according to the manufacturer's instructions using 200 ng of recycled linearized ptgev-gfp bac, 45 ng of pcr products of s_ntd and two pairs of primers (rec-672sf/δs-ntdr and rec-672sr/δs-ntdf) ( table 2) . subsequently, a pair of primers (primerf/primerr) ( table 2 ) was designed to amplify the sequence of the modified s_ntd224 area for sequencing (genscript, nanjing, china). the recombinant virus corresponding to the correct ptgev-gfp-∆s_ntd bac was then recovered as described above. pk-15 cells in six-well plates were inoculated with 10-fold serially diluted recombinant virus. after virus adsorption for 30 min, monolayer cells were washed three times with pbs and overlaid with a mixture of 2% low-melt agarose and 2 times the concentration of dmem (invitrogen, carlsbad, ca, usa) supplemented with 4% fetal bovine serum (gibco). the overlay was then solidified at 4 • c for 10 min. subsequently, the plates were cultured at 37 • c in a 5% co 2 incubator, and 4 days post-infection, the fluorescent plaques were visualized by fluorescence microscopy. thirteen 2-day-old piglets from a tgev-free sow were randomly divided into three groups and fed fresh liquid milk diluted in warm water every 4 h. all piglets were confirmed to be free of tgev, pedv, porcine delta coronavirus (pdcov), and rotavirus (rv) through a rt-pcr assay of piglet feces before viral challenge. the piglet weights were measured and recorded at the beginning of the challenge. the piglet challenge group was intranasally and orally inoculated with 500 µl (1 × 10 5 tcid 50 ) of chimeric virus, and the mock-infected control group was intranasally and orally inoculated with 500 µl of dmem. the piglets were monitored for their clinical status every 4 h. any piglet exhibiting moribund signs were euthanized. at 7 days post-inoculation, all surviving piglets were euthanized consecutively to reduce the stress of the other piglets. before necropsy, the weight of each piglet was recorded. at necropsy, five sections of the duodenum, jejunum, ileum, colon and stomach were collected, fixed in 10% formalin for histopathological examination and stained with hematoxylin and eosin (he). after necropsy, samples of jejunal contents and lung tissue were collected for virus detection by nested rt-pcr using the specific primers f1/r1 and f2/r2 (table 2) [50]. the animal experiments were performed according to the protocols approved by the scientific ethics committee of huazhong agricultural university (permit number: hzausw-2017-007). the animal care and maintenance protocols complied with the recommendations detailed in the regulations for the administration of affairs concerning experimental animals made by the ministry of science and technology of china. to construct an infectious clone of tgev, six overlapping cdna fragments designated a to f were generated by reverse transcriptase pcr (rt-pcr) using total rna extracted from pk-15 cells infected with tgev wh-1 ( figure 1a ,b). fragments a, b, d, and e were cloned into the pmd18-t vector, and fragments c and f were cloned into the bacterial artificial chromosome (bac) to produce the corresponding subclones. subclone f was also constructed as the final recipient bac vector by inserting the cytomegalovirus (cmv) promoter at the 5 terminus of fragment f and a 25-bp poly(a) tail (25a) followed by the hepatitis delta virus ribozyme (rz) and bovine growth hormone (bgh) transcription terminal signal sequences at the 3 terminus of fragment f ( figure 1b ). to more conveniently observe the chimeric virus and exclude the influence of the accessory gene orf3 on tgev enteric tropism and virulence, the gene encoding orf3 at the genome position from 24,826 to 28,580 was replaced by the egfp gene ( figure 1a ,b). through the one-step assembly of fragments a to f, we successfully obtained a full-length cdna infectious clone of tgev, designated ptgev-gfp bac ( figure 1b ,c). the full-length ptgev-gfp bac was verified by sequencing. after propagation for more than 200 generations in e. coli dh10b cells, ptgev-gfp bac digestion with the kpni enzyme, which produced six different fragment products, also confirmed the correct ptgev-gfp bac clone ( figure 1c ,d). in other words, the cloning of fragment c into the bac yielded no toxic sequences in any of the experiments. produce the corresponding subclones. subclone f was also constructed as the final recipient bac vector by inserting the cytomegalovirus (cmv) promoter at the 5′ terminus of fragment f and a 25-bp poly(a) tail (25a) followed by the hepatitis delta virus ribozyme (rz) and bovine growth hormone (bgh) transcription terminal signal sequences at the 3′ terminus of fragment f ( figure 1b) . to more conveniently observe the chimeric virus and exclude the influence of the accessory gene orf3 on tgev enteric tropism and virulence, the gene encoding orf3 at the genome position from 24,826 to 28,580 was replaced by the egfp gene ( figure 1a ,b). through the one-step assembly of fragments a to f, we successfully obtained a full-length cdna infectious clone of tgev, designated ptgev-gfp bac ( figure 1b ,c). the full-length ptgev-gfp bac was verified by sequencing. after propagation for more than 200 generations in e. coli dh10b cells, ptgev-gfp bac digestion with the kpni enzyme, which produced six different fragment products, also confirmed the correct ptgev-gfp bac clone ( figure 1c,d) . in other words, the cloning of fragment c into the bac yielded no toxic sequences in any of the experiments. to rescue the recombinant cov of tgev-gfp (corresponding to ptgev-gfp bac), the ptgev-gfp bac was transfected into pk-15 cells using lipofectamine 2000. sporadic green fluorescence was observed 24 h post-transfection, as depicted in figure 2a , but the infected pk-15 cells grew and showed a normal morphology. however, compared with the mock-infected group, obvious green fluorescence and the cytopathic effect (cpe) could be observed 48 h post-transfection ( figure 2b ). western blot and rt-pcr assays were performed to further confirm the recombinant virus tgev-gfp. the 43.5-kilodalton band of tgev n protein ( figure 2c ) and the marker mutation at position 4553 in the tgev genome ( figures 1b and 2d ) confirmed the recovery of the tgev-gfp recombinant virus. to rescue the recombinant cov of tgev-gfp (corresponding to ptgev-gfp bac), the ptgev-gfp bac was transfected into pk-15 cells using lipofectamine 2000. sporadic green fluorescence was observed 24 h post-transfection, as depicted in figure 2a , but the infected pk-15 cells grew and showed a normal morphology. however, compared with the mock-infected group, obvious green fluorescence and the cytopathic effect (cpe) could be observed 48 h post-transfection ( figure 2b ). western blot and rt-pcr assays were performed to further confirm the recombinant virus tgev-gfp. the 43.5-kilodalton band of tgev n protein ( figure 2c ) and the marker mutation at position 4553 in the tgev genome ( figures 1b and 2d ) confirmed the recovery of the tgev-gfp recombinant virus. spike with n-terminal 224 aa deletion in tgev wh1 is analogous to the spike of a reported natural prcv strain ( figure 3a ). to verify whether s_ntd224 is the enteric tropism determinant for tgev, we used the crispr-cas9 system to efficiently manipulate the tgev gene. briefly, two specific enzyme sites encompassing the sequence of s_ntd224 were selected. we then synthesized two types of single-stranded dna forward primers (ssdnaa-f or ssdnab-f) and a constant reverse primer (ssdna-r) ( table 1) corresponding to the two enzyme cutting sites, designated sites a and b ( figure 3c ). after annealing pcr using the forward and reverse primers, the purified pcr products of short dna fragments were transcribed by t7 rna polymerase ( figure 3b ). the transcribed products corresponding to sites a and b (designated sgrna a and sgrna b) ( figure 3c ) were incubated with the nuclease cas9 to digest the ptgev-gfp bac in vitro, and the digestion yielded a linearized bac and a~2.1-kb dna fragment that included the sequence of s_ntd224 ( figure 3a,d) . tgev, we used the crispr-cas9 system to efficiently manipulate the tgev gene. briefly, two specific enzyme sites encompassing the sequence of s_ntd224 were selected. we then synthesized two types of single-stranded dna forward primers (ssdnaa-f or ssdnab-f) and a constant reverse primer (ssdna-r) ( table 1) corresponding to the two enzyme cutting sites, designated sites a and b ( figure 3c ). after annealing pcr using the forward and reverse primers, the purified pcr products of short dna fragments were transcribed by t7 rna polymerase ( figure 3b ). the transcribed products corresponding to sites a and b (designated sgrna a and sgrna b) ( figure 3c ) were incubated with the nuclease cas9 to digest the ptgev-gfp bac in vitro, and the digestion yielded a linearized bac and a ~2.1-kb dna fragment that included the sequence of s_ntd224 ( figure 3a,d) . to construct the mutant infectious clone of the s_ntd224 deletion (designated ptgev-gfp-δs_ntd bac) from the ptgev-gfp bac, we produced a 672-nucleotide deletion-specific mutation pcr product using two pairs of primers (rec-672sf/δs-ntdr and rec-672sr/δs-ntdf) ( table 2 ). the mutation pcr products were then recombined into the linearized bac vector cleaved from the full-length ptgev-gfp bac ( figure 3d ). after the recombination products were transformed into dh10b competent cells, all 10 monoclonal colonies were identified as positive clones by pcr using the primer pair rec-672sf/rec-672sr ( figure 3e ). the sequencing of three randomly selected monoclonal colonies also confirmed the positive ptgev-gfp-δs_ntd bac, and we then constructed an infectious clone with the corresponding 224-aa deletion in the n-terminus of the tgev-gfp s protein by sequencing one entire genome of the three positive clone ( figure 3e ). to construct the mutant infectious clone of the s_ntd224 deletion (designated ptgev-gfp-∆s_ntd bac) from the ptgev-gfp bac, we produced a 672-nucleotide deletion-specific mutation pcr product using two pairs of primers (rec-672sf/δs-ntdr and rec-672sr/δs-ntdf) ( table 2 ). the mutation pcr products were then recombined into the linearized bac vector cleaved from the full-length ptgev-gfp bac ( figure 3d ). after the recombination products were transformed into dh10b competent cells, all 10 monoclonal colonies were identified as positive clones by pcr using the primer pair rec-672sf/rec-672sr ( figure 3e ). the sequencing of three randomly selected monoclonal colonies also confirmed the positive ptgev-gfp-∆s_ntd bac, and we then constructed an infectious clone with the corresponding 224-aa deletion in the n-terminus of the tgev-gfp s protein by sequencing one entire genome of the three positive clone ( figure 3e ). we rescued the recombinant virus tgev-gfp-∆s_ntd from pk-15 cells as previously described, and sporadic and more obvious green fluorescence was observed at 24 and 48 h post-transfection, respectively ( figure 4a ). we then verified the modified virus tgev-gfp-∆s_ntd by rt-pcr using the primers primerf and primerr (table 2 ). an obvious deletion of approximately 600 bp was observed in tgev-gfp-∆s_ntd in comparison with tgev-gfp ( figure 4b ). subsequently, the rt-pcr product was sequenced using the primers primerf and primerr ( figure 4c) . comparison with the ptgev-gfp bac showed that all the modified nucleotides were correct, as depicted by the model shown in figure 4d . to validate whether s_ntd224 determines the enteric tropism of tgev, 13 two-day-old piglets were randomly divided into three groups, with five piglets in each virus-infected group and three piglets in the mock-infected control group. the piglets in the two virus-infected groups were to further evaluate the role of s_ntd224 in tgev, we measured the growth kinetics of the wild-type virus, tgev-gfp and tgev-gfp-∆s_ntd. the replication kinetics of the tgev-gfp and wild-type viruses were comparable to each other and considerably different from those of tgev-gfp-∆s_ntd ( figure 4e ). twelve hours after inoculation, the titer of tgev-gfp was more than 10-fold greater than that of tgev-gfp-∆s_ntd ( figure 4e ). to further identify the effect of s_ntd224 on tgev, we also analyzed the fluorescent viral plaques. the plaque size of tgev-gfp-∆s_ntd was notably different from that of tgev-gfp ( figure 4f ), which also indicated that tgev-gfp infected cells more effectively than tgev-gfp-∆s_ntd. to validate whether s_ntd224 determines the enteric tropism of tgev, 13 two-day-old piglets were randomly divided into three groups, with five piglets in each virus-infected group and three piglets in the mock-infected control group. the piglets in the two virus-infected groups were inoculated intranasally and orally at a dose of 1 × 10 5 50% tissue culture infective dose (tcid 50 ) with the respective chimeric virus, and the mock-infected control piglets were inoculated with dulbecco's modified eagle's medium (dmem). all the piglets in the tgev-gfp group exhibited severe clinical symptoms and weight loss, and those in the tgev-gfp-∆s_ntd group showed ameliorated but still obvious clinical symptoms ( figure 5a,b) . in addition, the piglets in the tgev-gfp and tgev-gfp-∆s_ntd groups appeared moribund within 3 days postinoculation, whereas the piglets in the mock-infected control group remained healthy ( figure 5c ). in addition, the piglets in the tgev-gfp group showed a higher mortality rate (as high as 100%) and presented earlier symptoms compared with those in the tgev-gfp-∆s_ntd group, which showed 40% mortality at 3 days postinoculation ( figure 5a,c) . to better detect the presence of the inoculated virus in the euthanized piglet intestine, the presence of both tgev-gfp and tgev-gfp-∆s_ntd in intestinal tissue was detected by nested pcr using the primer pairs f1/r1 and f2/r2 (table 2) . tgev-gfp and tgev-gfp-∆s_ntd were detected in intestinal tissue from the moribund piglets ( figure 5d ), but no chimeric virus was detected in the two piglets in the tgev-gfp-∆s_ntd group that were euthanized at 7 days post-inoculation ( figure 5d ). tgev-gfp-δs_ntd groups appeared moribund within 3 days postinoculation, whereas the piglets in the mock-infected control group remained healthy ( figure 5c ). in addition, the piglets in the tgev-gfp group showed a higher mortality rate (as high as 100%) and presented earlier symptoms compared with those in the tgev-gfp-δs_ntd group, which showed 40% mortality at 3 days postinoculation ( figure 5a ,c). to better detect the presence of the inoculated virus in the euthanized piglet intestine, the presence of both tgev-gfp and tgev-gfp-δs_ntd in intestinal tissue was detected by nested pcr using the primer pairs f1/r1 and f2/r2 (table 2) . tgev-gfp and tgev-gfp-δs_ntd were detected in intestinal tissue from the moribund piglets ( figure 5d ), but no chimeric virus was detected in the two piglets in the tgev-gfp-δs_ntd group that were euthanized at 7 days post-inoculation ( figure 5d ). the postmortem of the moribund piglets in the tgev-gfp and tgev-gfp-δs_ntd groups revealed that the small intestines were filled with watery contents. in particular, the intestinal walls in the jejunal section of the intestines of these piglets were clearly thinner and more transparent compared with those of the mock group ( figure 6a) . he staining compared with the normal mock group also revealed that the tgev-gfp chimeric viruses caused more severe intestinal tissue the postmortem of the moribund piglets in the tgev-gfp and tgev-gfp-∆s_ntd groups revealed that the small intestines were filled with watery contents. in particular, the intestinal walls in the jejunal section of the intestines of these piglets were clearly thinner and more transparent compared with those of the mock group ( figure 6a ). he staining compared with the normal mock group also revealed that the tgev-gfp chimeric viruses caused more severe intestinal tissue damage than tgev-gfp-∆s_ntd ( figure 6b ). more severe villous atrophy was observed in the small intestine, particularly the jejunum and ileum, of the piglets in the tgev-gfp and tgev-gfp-∆s_ntd groups compared with those of the mock group ( figure 6b ). collectively, these results suggested that s_ntd224 has not altered the enteric tropism for tgev but exerts a mild influence on tgev virulence. the n-terminal domain of spike protein is recognized as the tgev enteric tropism determinant in piglets, as demonstrated through comparisons of the sequences of natural tgev isolates or those obtained after continuous passage in cell culture [13, 51, 52] , but more direct evidence is still needed. in this study, based on a dna-launched infectious clone, we used a novel cov gene editing method to efficiently perform cov targeted gene editing. using reverse genetics, we found that s_ntd224 was not the enteric tropism determinant for tgev. the relevant insights regarding the novel cov targeted gene editing method and tgev s_ntd224 are discussed below. because covs are the viruses with the largest rna genomes, the construction of a cov infectious clone is hampered by two main challenges: large full-length cdna and toxic sequences in the bacterial clone [31, 53] . although the problem of cov cdna sequence instability has been overcome by several methods, the manipulation of the large full-length cov genome remains a considerable challenge. until now, the direct editing of the full-length cdna of covs has not been reported. in this study, we constructed a tgev-gfp infectious clone by ligating six fragments in one the n-terminal domain of spike protein is recognized as the tgev enteric tropism determinant in piglets, as demonstrated through comparisons of the sequences of natural tgev isolates or those obtained after continuous passage in cell culture [13, 51, 52] , but more direct evidence is still needed. in this study, based on a dna-launched infectious clone, we used a novel cov gene editing method to efficiently perform cov targeted gene editing. using reverse genetics, we found that s_ntd224 was not the enteric tropism determinant for tgev. the relevant insights regarding the novel cov targeted gene editing method and tgev s_ntd224 are discussed below. because covs are the viruses with the largest rna genomes, the construction of a cov infectious clone is hampered by two main challenges: large full-length cdna and toxic sequences in the bacterial clone [31, 53] . although the problem of cov cdna sequence instability has been overcome by several methods, the manipulation of the large full-length cov genome remains a considerable challenge. until now, the direct editing of the full-length cdna of covs has not been reported. in this study, we constructed a tgev-gfp infectious clone by ligating six fragments in one step [49] . the crispr/cas9 system was then used to finish the construction of ptgev-gfp-∆s_ntd. to our knowledge, this study provides the first demonstration of the direct in vitro manipulation of full-length coronavirus cdna. to edit specific cov genes, targeted cleavage of the bac was completed by cas9 protein through a reaction mediated by two types of sgrna transcribed together or separately ( figure 3b,d) . sgrna can be easily obtained by annealing pcr and transcription using an available kit. moreover, regardless of the exonuclease trimming activities of cas9 [45] , in the experiment, we were able to insert the mutated fragments in the linearized bac with 200-bp overlapping sequences through homologous recombination. notably, numerous mutation fragments can be inserted efficiently into the linearized bac at the same time ( figure 4d ), which is perfect for the construction of a viral mutant library [54] [55] [56] . similar to traditional plasmid manipulation, we edited the targeted gene by recombination in vitro with overlapping pcr products (e.g., mutations, deletions, or insertions). moreover, as little as 100 ng of linearized, digested bac was sufficient to complete the recombination reaction. to determine the mutation of the targeted bac, we only needed to amplify the fragments by bacterial pcr using a pair of primers in duplicate, and this assay can be used to sequence the site of recombination and the modified fragment area. furthermore, almost any area of the targeted bac can be simply cleaved by the crispr/cas9 system with two types of specific sgrna. throughout the process, we accomplished recombinant virus recovery using only one plasmid of bac in a single week ( figure 3) . namely, once an infectious clone was constructed, the recombinant coronaviruses was more efficiently and conveniently rescued in this study than in previous research, and the proposed approach thus greatly accelerates the gene editing speed of large rna virus rescue. moreover, the simple manipulation of a bac vector and modification of the specific small region throughout the procedure would theoretically lower the mutation probability of the full-length cov cdna. thus, this method is not only cost-effective but also reduces the probability of introducing additional mutations during the bac modification procedure. the spike gene of tgev has been shown to alter tgev virulence or enteric tropism. however, recombinant tgev with s protein n-terminal amino-acid deletion was constructed through targeted recombination and passaged several times, which might cause other locus mutations in addition to the s protein deletion [20] . in particular, an early study reported that a 224-residue deletion in prcv corresponding to the n-terminal domain of the tgev s protein, as depicted in figure 3a , is likely responsible for the loss of replication observed in the enteric tract [21] . no other studies have provided direct evidence demonstrating that only the n-terminal region of the spike gene changes the tgev virulence or enteric tropism [57] . here, we emphasize the importance of the n-terminus of the tgev s protein for the enteric tropism of the virus. to that end, we constructed a recombinant virus with an s protein analogous to that of prcv ( figure 3a) , tgev-gfp-∆s_ntd, and this recombinant virus showed titers and fluorescent plaque sizes that greatly differed from those of tgev-gfp ( figure 4e,f) . these results indicated that the 224 amino acids of the n-terminal of the tgev spike protein are not essential for viral survival but important for viral replication or infection, which was analogous to the findings obtained for pedv and other covs [58] [59] [60] [61] . our animal experiments revealed that tgev-gfp-∆s_ntd caused 40% mortality in piglets and obvious intestinal tissue damage, which indicates that s_ntd224 has a mild influence on virulence but does not alter the enteric tropism of tgev [62] . using reverse genetics, we confirmed that changes in s_ntd224 alone altered, albeit not completely, the virulence of tgev. one reason explaining no detection of tgev-gfp-∆s_ntd in the two piglets euthanized at 7 days post-inoculation might be related to immunity of recovering piglets. the role of s_ntd224 might be analogous to that of the 197-amino-acid region in the n-terminus of the pedv s gene when used as a viral virulence marker. consistent with previous reports, our experiments also detected tgev-gfp and tgev-gfp-∆s_ntd in the jejunal contents tissue by nested rt-pcr [63, 64] , which indicates that changes in s_ntd224 alone do not alter tgev enteric tropism in vivo. and it is also possible that other genes in addition to the 224 amino acids of the n-terminal of the tgev spike protein might regulate changes in tgev tissue tropism [20, 58] . additional research is needed to determine the detailed mechanism of tgev enteric tropism in vivo. in summary, using the reverse genetics method, we have provided direct evidence showing that the n-terminal domain of spike protein is not the determinant of tgev enteric tropism in piglets, although s_ntd224 exerts a mild influence on tgev virulence. these results provide new insights into the 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gastroenteritis virus from chronically infected experimental pigs isotype-specific antibody-secreting cells to transmissible gastroenteritis virus and porcine respiratory coronavirus in gut-and bronchus-associated lymphoid tissues of suckling pigs the authors declare no conflict of interest. key: cord-317142-qd61qvch authors: muramatsu, tomonari; kim, yong‐tae; nishii, wataru; terada, takaho; shirouzu, mikako; yokoyama, shigeyuki title: autoprocessing mechanism of severe acute respiratory syndrome coronavirus 3c‐like protease (sars‐cov 3cl (pro)) from its polyproteins date: 2013-03-27 journal: febs j doi: 10.1111/febs.12222 sha: doc_id: 317142 cord_uid: qd61qvch like many other rna viruses, severe acute respiratory syndrome coronavirus (sars‐cov) produces polyproteins containing several non‐structural proteins, which are then processed by the viral proteases. these proteases often exist within the polyproteins, and are excised by their own proteolytic activity (‘autoprocessing’). it is important to investigate the autoprocessing mechanism of these proteases from the point of view of anti‐sars‐cov drug design. in this paper, we describe a new method for investigating the autoprocessing mechanism of the main protease (m (pro)), which is also called the 3c‐like protease (3cl (pro)). using our method, we measured the activities, under the same conditions, of the mature form and pro‐forms with the n‐terminal pro‐sequence, the c‐terminal pro‐sequence or both pro‐sequences, toward the pro‐form with both n‐ and c‐terminal pro‐sequences. the data indicate that the pro‐forms of the enzyme have proteolytic activity, and are stimulated by the same proteolytic activity. the stimulation occurs in two steps, with approximately eightfold stimulation by n‐terminal cleavage, approximately fourfold stimulation by c‐terminal cleavage, and 23‐fold stimulation by the cleavage of both termini, compared to the pro‐form with both the n‐ and c‐terminal pro‐sequences. such cleavage mainly occurs in a trans manner; i.e. the pro‐form dimer cleaves the monomeric form. the stimulation by n‐terminal pro‐sequence removal is due to the cis (intra‐dimer and inter‐protomer) effect of formation of the new n‐terminus, whereas that by c‐terminal cleavage is due to removal of its trans (inter‐dimer) inhibitory effect. a numerical simulation of the maturation pathway is presented. severe acute respiratory syndrome (sars) is a highly lethal infectious disease. it was first discovered in guangdong province, china, in november 2002, and spread to 26 countries with 8098 cases, including 774 deaths, over 8 months until 5 july 2003, when the world health organization reported that the last human chain of transmission in that epidemic had been broken [1] . sars coronavirus (sars-cov) is the etiological agent of this disease. the 3c-like protease (3cl pro , also referred to as the main protease, m pro ) of the virus is a key enzyme, as it cleaves several sites to produce non-structural proteins that are essential for genome replication and virion production, such as an rna-dependent rna polymerase, a helicase, ribonucleases and 3cl pro itself, from two types of polyproteins (pp1a and pp1ab) [2] . rna viruses encode polyproteins containing several non-structural proteins, which are then processed by the viral proteases. however, these proteases exist within the polyproteins, and are excised by their own proteolytic activity ('autoprocessing'). these proteases often function as homo-dimers. sars-cov 3cl pro exists as a homodimer, in which each protomer has an active site [3] . precise processing of its n-terminus is important for its proteolytic activity [4] [5] [6] . indeed, expression of enzymes with two to five additional vector-derived amino acid residues or without the first amino acid residue displayed reduced activity (1-40%) [5, 6] . this is because the processed n-terminus of one protomer is required for formation of the oxyanion hole in the active site of the other protomer in the homo-dimer [3] . although the mature protease cleaves both the n-and c-terminus of the 3cl pro precursor from the replicase polyproteins, it is not clear how the first 3cl pro molecule is excised and becomes mature after sars-cov infection of human cells. the difficulty in investigating the autoprocessing mechanism lies in the ability to measure the activity of each pro-form of the enzyme, because (a) a chain reaction exists, in which the processed and activated enzyme (product) reacts with the precursor molecule (substrate), and (b) the enzyme protomer dimerizes with a substrate protomer, even if the catalytically inactive mutant pro-form of the enzyme (such as c145a or h41a [3] ) is used as the substrate. we developed a new method to investigate autoprocessing of sars-cov 3cl protease, which completely eliminated these problems. in this paper, we describe the activities, measured under the same conditions, of the mature form and pro-forms with the n-terminal pro-sequence, c-terminal pro-sequence and both pro-sequences toward the pro-form with both n-and c-terminal pro-sequences. furthermore, by use of two types of substrates, we determined that the first n-terminal processing from the polyprotein occurs in a trans manner, i.e. the pro-form dimer cleaves the monomeric form of the pro-form, and determined the inhibitory modes of the n-and c-terminal prosequences, i.e., the n-terminal pro-sequence has a cis (intra-dimer and inter-protomer) effect, and the c-terminal pro-sequence has a trans (inter-dimer) effect. in vitro 3cl pro autoprocessing system first we developed a sars 3cl protease autoprocessing system by use of the escherichia coli cell-free protein synthesis system, with the n-and c-terminal 10 amino acid pro-sequences accompanied by an s tag and a his tag, respectively (fig. 1a) . the s tag is a 15 amino acid residue peptide tag derived from nuclease s, which can be detected by the s protein, derived from the other part of nuclease s. although, stretches of more than several hundred amino acid residues are associated with the 3cl pro region on both sides of the polyprotein in infected cells, we assumed that the effects of elements upstream and downstream of the 10 amino acid regions tested are minimal. over the course of the cell-free protein synthesis reaction (30°c, 4 h), the n-and c-terminal processing sites of the catalytically active construct (wild-type, wt in fig. 1a) were completely cleaved by the enzyme's endogenous proteolytic activity, as shown in fig. 1b ,c (wt). the 34 kda band of the catalytically active construct (wt) in fig. 1b was stained using an antibody against sars-cov 3cl pro , but not by those against either the his or s tag. this mature form was excised from the gel, and its n-terminal amino acid sequence was determined to be sgfrkmafp. its molecular mass was determined by maldi-tof mass spectrometry as 33 792 da, which agreed well with its calculated molecular mass (33 845 da). however, the catalytically inactive form (c145a), in which the active residue cys145 was converted to ala, was unable to process either the n-terminal pro-sequence or the c-terminal pro-sequence, and resulted in the 41 kda band in fig. 1b (calculated molecular mass 40 525 da), which reacted with antibodies against the his and s tags (fig. 1c) . these observations are consistent with those previously reported [7] . as 3cl pro requires a glutamine residue at the p1 position of the substrate [8] , mutation of q-1 to n and q306 to n resulted in loss of cleavage at these sites ( fig. 1a,b) . however, interestingly, the n-terminal uncleavable mutant q-1n was able to cleave its own c-terminal processing site, and the c-terminal uncleavable mutant, q306n, was able to cleave its own n-terminal processing site. the processed site for q-1n was assumed to be the same as the c-terminal processing site for wt, and that for q306n was assumed to be the same as the n-terminal processing site for wt, based on their molecular weights estimated by sds/page (fig. 1b) . measuring the activity of 3cl pro by the trans-processing system to investigate the activities of the pro-forms of sars-3cl pro in detail, we developed a 'trans-cleaving assay', using a substrate in which the core region of the 3cl protease was exchanged with gfp (green fluorescent protein) ( fig. 2a) . this substrate comprises the s tag, the n-terminal pro-sequence of 10 amino acid residues, the n-terminal sequence of 10 amino acid residues, gfp, the c-terminal sequence of 10 amino acid residues, the c-terminal pro-sequence of 10 amino acid residues, and the 6xhis tag. the substrate molecule (gfp substrate) and the enzyme molecule (3cl pro , mature or pro-form) were each synthesized using the e. coli cell-free protein synthesis system at 30°c for 4 h, as described above. the protein solutions were then mixed together and incubated for 1 h at 30°c. the mature and pro-enzymes (q-1n, q306n and q-1n/q306n) cleaved the n-and c-terminal processing sites in the substrate molecule, and thus we were able to estimate the activities of the proenzymes toward these processing sites. the enzyme solution synthesized by the cell-free protein synthesis reaction was serially diluted (2-, 4-, 8-, 16-, 32-fold, etc) , mixed with the substrate molecule (1 : 1), and incubated for 1 h (fig. 2b) . we estimated the activity of the enzyme (or pro-enzyme) toward the n-and c-terminal processing sites in the gfp substrate on the basis of the dilution ratio at which 50% cleavage was achieved (fig. 2c) . the calculated molecular masses of the gfp substrate and the pro-form of 3cl with both the n-and c-terminal pro-sequences (q-1n/q306n) are 35 723 and 40 526 da, respectively. however, under some conditions, the mobility of gfp-fused proteins in sds/page is smaller than the calculated values, i.e. the estimated molecular masses are higher [9] , presumably as a result of the covalently formed chromophore and partially formed secondary/tertiary structures. in our experiments, the migration of the gfp substrate and its cleaved products represented masses approximately 10 kda greater than their actual molecular masses, and approximately 5 kda greater than those of the corresponding pro-3cl pro species, as confirmed by western blotting (fig. 2c) . kinetic parameters for the mature 3cl pro toward the gfp substrate we attempted to measure the k cat and k m values using a gel-based assay. the gfp substrate and the mature 3cl pro enzyme were prepared as described above, and their concentrations were estimated by coomassie brilliant blue g-250 staining of the gel, using bsa as a standard. the substrate (41.8 lm) was diluted 1-, 2-or 4-fold, and was mixed with 256-fold diluted mature 3cl pro enzyme solution (0.088 lm) (1 : 1). after 0, 15, 30 and 60 min incubations at 30°c, the n-terminal processing rates were estimated by sds/page, and the data from the 15 min incubation were used for estimation of the initial velocities (data not shown). although the k m value could not be estimated, because it is much larger than the concentrations of the substrates used, the k cat /k m per protomer was estimated as 6800 m à1 ás à1 from the lineweaver-burk plot (fig. 2d ). strictly speaking, kinetic parameters must be measured under conditions where the employed substrate concentration is comparable to the k m value. however, in this case, the substrate concentrations were quite low compared to the k m value. therefore, the k cat /k m value obtained is not precise, but may be used as an apparent value. the mature form of sars-cov 3cl pro exists as a homo-dimer, and the k cat /k m value of the dimer is 13 600 m à1 ás à1 . considering the differences in the substrates, this value is comparable to that reported by xue et al. [5] (26 500 m à1 ás à1 ). they used a synthetic peptide as a substrate, whereas we used proteins synthesized by , synthesized by the e. coli cell-free protein synthesis system, were mixed together, incubated for 1 h at 30°c, and analyzed by sds/page. in (c), the red arrows and asterisk indicate the gfp substrate, the blue arrows and asterisk indicate the n-terminally processed substrate, and the purple arrows and asterisk indicate the substrate processed at both termini, as confirmed by western blotting analysis using an antibody against gfp. the c-terminal processed substrate did not appear, but its expected positions are indicated by black dashed arrows. the activity of the enzyme for a processing site was estimated on the basis of the dilution ratio at which 50% cleavage was achieved. this value was then converted to the k cat /k m value using the concentration of the enzyme species. the standard k cat /k m value was obtained by the slope of the plot shown in (d). (d) lineweaver-burk plot for the wild-type (mature form of 3cl pro ) toward the n-terminal processing site of the gfp substrate. the enzyme, the mature (wild-type) 3cl pro and the gfp substrate were prepared by the e. coli cell-free protein synthesis system. the substrate solution was diluted, mixed with 256fold diluted mature 3cl pro enzyme solution, and incubated at 30°c for 15 min. the enzyme, substrate and product concentrations were measured by quantification of the respective bands on an sds/page gel stained with coomassie brilliant blue g-250. the cell-free translation system. to our knowledge, this is the first report of the k cat /k m value for sars-cov 3cl pro toward a protein substrate rather than a peptide substrate. activities of the mature and pro-forms of the 3cl pro enzyme toward the gfp substrate and the inactive 3cl pro pro-form (c145a) the mature enzyme, its pro-forms, the gfp substrate and the catalytically inactive pro-form of 3cl pro (c145a) [pro-3cl(c145a)] were produced by the e. coli cell-free protein synthesis system, as described above. in each experiment, the reactions were fractionated by sds/page (figs 1b and 3, and data not shown), and then the concentration of each species was analyzed using the band stained by coomassie brilliant blue g-250 (fig. 3a) , with bsa as a standard, as described above and in experimental procedures. typically, the molar concentration of each species determined by its calculated molecular mass is 22.6 lm for the mature enzyme, 14.4 lm for the pro-form with the n-terminal pro-sequence (q-1n), 17.3 lm for the proform with the c-terminal pro-sequence (q306n), 18.7 lm for the pro-form with both n-and c-terminal pro-sequences (q-1n/q306n), and 41.8 lm for the gfp substrate. these values were used for calculation of the k cat /k m values, as described below. the activities of the enzyme and its pro-forms were estimated on the basis of the dilution ratios at which 50% cleavage of the substrates was achieved at 30°c and 1 h (fig. 3b ). under these conditions, the velocities of the cleavage reactions were the same, and thus the dilution ratios are proportional to the velocities of the cleavage reactions catalyzed by a certain amount of the enzyme. in turn, they are proportional to the v max /k m values, because the k m is much higher than the concentration of the substrate used (fig. 2c) . these v max /k m values may be converted to k cat /k m values using the concentrations of the enzyme species estimated by the sds/page analyses described above, and the activities are indicated by their k cat /k m values in table 1 and fig. 4 . all molecules had processing activities towards both the n-and c-terminal processing sites of the gfp substrate (fig. 4) : the pro-form with the n-terminal pro-sequence (q-1n) had approximately 19% activity, the pro-form with the c-terminal pro-sequence (q306n) had approximately 33% activity, and the pro-form with both pro-sequences had approximately 4% activity as compared to the mature form (wt). the cleavability of the c-terminal processing site (vtfq↓gkf) of the gfp substrate was approximately half of that of the n-terminal processing site (avlq↓sgf) (0.56 by the mature enzyme, 0.60 by q-1n, 0.50 by q306n, and 0.49 by q-1n/q306n). these values are in good agreement with that obtained in a previous study of the mature enzyme (0.41) toward synthetic peptides [10] . these values also explain the order of cleavage of the two processing sites in the catalytically inactive pro-3cl pro mutant (c145a) with the mature enzyme [7] : first at the n-terminal site, and then at the c-terminal site. the values may also be explained by the difference at the p2 position (leu for the n-terminal site, phe for the c-terminal site), according to recent detailed analyses by chuck et al. [11] . they estimated the substrate specificity by mutating each of the residues, one by one, at the p5-p3′ positions of the n-terminal processing site. therefore, there is no difference in accessibility between the n-and c-terminal processing sites in the gfp substrate. trans or cis reaction? exact processing of the n-terminus of sars 3cl pro is required for efficient cleavage activity [5] . however, as shown in fig. 1 , the pro-forms of 3cl pro synthesized by the e. coli cell-free protein synthesis system had sufficient proteolytic activity to process their own autoprocessing sites. moreover, they were able to process a substrate in which the core region was replaced with the gfp protein. although sars-cov 3cl pro forms a homo-dimer [3] , this gfp-replaced substrate molecule does not dimerize with 3cl pro . therefore, these proforms efficiently process the autoprocessing sites in a trans manner on the gfp substrate (figs 2, 3b, and 4) . these pro-forms also efficiently processed the autoprocessing sites on the pro-form of a catalytically inactive 3cl pro (c145a) mutant [pro-3cl(c145a)] (figs 3c and 4) . the fact that 3cl pro species with the n-terminal pro-sequence (q-1n and q-1n/q306n) cleaved the autoprocessing site of 3cl pro (c145a) as efficiently as the gfp-replaced substrate suggests an autoprocessing mechanism in which the pro-3cl pro dimer (enzyme) cleaves the site in the monomeric form of the pro-form (trans reaction). although an x-ray crystallographic analysis revealed that the n-terminus of the mature form in the dimeric structure of 3cl pro is buried inside the molecule [3] , this protease exists in equilibrium between the dimer and monomer in solution [7, 12] . the pro-sequences enhance dissociation of the dimer [7, 12] , and both the n-and c-terminus of the monomer molecule are exposed [13] [14] [15] . therefore, it is plausible that the pro-form also exists in a dimer and monomer equilibrium, and that the dimer molecule easily processes the monomer molecule. the presence of either the n-terminal pro-sequence (q-1n) or the c-terminal pro-sequence (q306n) reduces the activity toward the gfp substrate (fig. 4) . however, there are some differences in the processability of the substrate molecules. we used two substrates: pro-3cl(c145a), the model substrate of the pro-form, and the gfp substrate, in which the core region is replaced by the gfp protein ( fig. 2a) . the difference between these two substrates is the ability to dimerize with the enzyme protomer (the mature and pro-forms of 3cl pro ). the gfp substrate acts only as a substrate, whereas pro-3cl(c145a) acts as both substrate and protomer in a dimer. as each protomer of the sars 3cl pro dimer has an active site, the concentration of the active site in the reaction solution does not change when using the pro-3cl(c145a) substrate, even if the active protomer dimerized with the pro-3cl(c145a) substrate. therefore, the difference in reactivity between the gfp substrate and the pro-3cl (c145a) substrate reflects the influence of the n-or c-terminal pro-sequence of 3cl pro (c145a) on the active site of the catalytically active protomer with which it dimerizes. the fact that the activity of the pro-form with the c-terminal pro-sequence (q306n) toward the pro-3cl (c145a) substrate was lower than that toward the gfp substrate (fig. 4) indicates that the pro-3cl (c145a) substrate dimerized with the pro-form (q306n), and its n-terminal pro-sequence inhibited the activity. in contrast, there was no difference in the activities of the pro-form with both the n-and c-terminal pro-sequences (q-1n/q306n) toward the pro-3cl(c145a) substrate and the gfp substrate (fig. 4) . as this pro-form (q-1n/q306n) has the n-terminal pro-sequence, dimerization with the pro-3cl(c145a) substrate did not affect its activity further through the n-terminal pro-sequence. it is worth mentioning the minimal difference in the activities of the pro-form with the n-terminal prosequence (and without the c-terminal pro-sequence) (q-1n) between the two substrates (fig. 4) . as there is an activity difference between the mature form (wt) and the pro-form with the c-terminal pro-sequence (q306n) toward the gfp substrate, the c-terminal pro-sequence has an inhibitory effect. however, this inhibitory effect is not achieved through either dimerization or an inter-protomer interaction in the dimer. two possibilities may be considered. one is that the c-terminal pro-sequence of the protomer reduces its own activity ('intra-protomer'). the other is that the c-terminal pro-sequence inhibits another enzyme dimer, e.g. through competitive inhibition ('interdimer'). the latter proposal is more plausible, as judged from the physical relationship between the c-terminus and the active site of a protomer in the crystal structure of the mature enzyme [3] . hsu et al. [7] reported dissociation constants (k d values) for dimerization of the wild-type (mature form) and mutants (c145a) with extra n-or c-terminal amino acids (10aa-c145a and c145a-10aa) as 0.35, 17.2 and 5.6 nm, respectively. the catalytically active pro-forms q-1n and q306n, for which the activities were measured in our study, had almost the same structures as 10aa-c145a and c145a-10aa. the differences are that our preparations have enzymatic activity (i.e., they are not the c145a mutants), q-1n (gln to asn) and/or q306n (gln to asn) mutations, and s and/or his tag moieties. in our study, the activity of each proform was based on the concentration of the pro-form enzyme at which 50% cleavage of the substrate was achieved. these concentrations were 47.7, 263.9 and 135.3 nm for the mature form, q-1n and q306n, respectively. using the k d values (mentioned above) from hsu et al. [7] , the populations of the dimer of each species were estimated as 94%, 84%, and 87%, respectively. therefore, we assumed that the major part of each of protomer species exists as a homo-or heterodimer under our experimental conditions. although the pro-form of 3cl pro with both the n-and c-terminal 10 amino acid residue prosequences processed both processing sites itself (fig. 1b,c, wt) , the paths from the pro-form to the mature form are complicated. there may be four types of protomer molecules (pro-form with n-and c-terminal pro-sequences, pro-form with the n-terminal pro-sequence, pro-form with the c-terminal pro-sequence, and the mature form), and they form several types of homo/hetero-dimers with each other. thus, during the course of maturation, several types of dimer species co-exist and cleave each other's pro-sequences, thus changing the types of dimeric forms and finally generating the mature form. however, we can simulate this pathway, as described below. during maturation, 10 types of dimeric forms exist, and their activities toward the n-and c-terminal processing sites were estimated (table 2 ) from the data in table 1 . based on the assumption that, between any dimeric protomer species, the monomer/dimer exchange rate is so fast that the population of each dimer at each time is proportional to the population of the protomers forming the dimer, the maturation process was simulated as shown in fig. 5a . this assumption is supported by the difference in the inhibitory effects of the n-terminal pro-sequences of the two substrates, the pro-3cl(c145a) and gfp substrate, on the n-terminally processed enzyme/proenzyme (wt or q306n; fig. 4 ). as described in results, this difference indicates that dimerization of the enzyme protomer (wt or q306n) and the substrate pro-3cl(c145a), i.e. inter-exchange of a protomer between the enzyme dimer and the substrate pro-3cl(c145a) dimer, occurs under our experimental conditions. this conversion must occur through the monomeric form. at time t, the population of each dimer was calculated from the population of protomers forming the dimer. the n-terminal processing activity [k cat /k m (n)] and the c-terminal processing activity [k cat / k m (c)] were then calculated using the estimated (table 2 ). (a) four types of protomers were assumed to be present (the mature type, the protomer with the n-terminal pro-sequence, the protomer with the c-terminal pro-sequence, and the protomer with both pro-sequences). at time t, the population of each dimer was calculated from the population of protomers forming the dimer. the n-terminal processing activity [k cat /k m (n)] and the c-terminal processing activity [k cat /k m (c)] were then calculated by using the estimated k cat /k m value of each dimer ( table 2) . using these values, the population of protomers at time t + dt was calculated. at time 0, all protomers were the protomer with both pro-sequences, i.e. all dimers were dimer 1. k cat /k m value for each dimer (table 2) . using these values, the population of protomers at time t + dt was calculated. at time 0, only one dimer species, dimer 1, is assumed to exist (indicated by '1'; fig. 5a ). the other species (dimers 2-10; indicated by '2'-'10') emerge as the processing reaction continues, and finally all of the molecules become the mature form, dimer 10. the total activities toward the n-and c-terminal processing sites are also indicated. although the time scale changes according to the initial concentration of dimer 1 at time 0, the shape and size of each curve are independent of the initial concentration. based on these simulated data, the conversion paths of the dimers are indicated in fig. 5b . hsu et al. [7] investigated the processing of a model molecule (trx-10aa-3cl pro (c145a)-10aa-gst), by the mature 3cl protease, and found that n-terminal processing occurred before c-terminal processing. this is consistent with the higher cleavability of the n-terminal processing site than the c-terminal processing site, as reported by fan et al. [10] and found in the present study. they proposed a mechanism for autoprocessing of polyproteins in which the first mature 3cl pro molecule is formed: first, cleavage of the n-terminal processing site occurs, the dimeric structure forms as the mature 3cl pro , and c-terminal processing then occurs in an inter-molecular manner [7] . they also solved the crystal structure of an inactive mutant (c145a) of the mature form of 3cl pro , in which the c-terminal moiety of a protomer in the dimer was bound to the active site of a protomer in the adjacent asymmetric unit, and proposed that this structure represented that just after c-terminal processing had occurred [7] . thus, the most important step in the maturation process of 3cl pro appears to be n-terminal autoprocessing of the 3cl pro moiety in the polyprotein [7, 12] . as protomer dimerization had been assumed to be essential for the proteolytic activity of 3cl pro , hsu et al. [7] proposed that the 3cl pro moiety of the polyprotein dimerized in a different manner to dimerization of the mature form, in which the n-terminal processing site of a protomer was cleaved by the active site of the other protomer (cis cleavage). after cleavage of both n-termini of the protomers, rearrangement of the protomers occurs to form the same dimeric structure as that of the mature enzyme. subsequently, cleavage of the c-terminal processing site of the other molecule of the n-terminally processed dimer, as well as cleavage of the n-and c-terminal processing sites in other polyprotein molecules, occurs. however, the possibility of a first cis cleavage of the n-terminus of the polyprotein dimer is based only on the fact that the n-terminus of a protomer is near the active site of the other protomer in the crystal structure of the mature enzyme [3] . this possibility was supported by chen et al. [16] , who demonstrated that mutants of the 3cl pro pro-form that exist in the monomeric form, as determined by a gel-filtration assay, had n-terminal processing activities. in contrast, li et al. [12] proposed a trans mechanism for the first n-terminal processing of the polyprotein, in which the 3cl pro moiety forms the dimeric structure of the mature form, and cleaves the n-terminus of another polyprotein molecule. they used two types of pro-3cl pro molecules with an n-terminal pro-sequence of six amino acids, accompanied by cfp and yfp at the n-and c-termini, respectively: one contains an active (wild-type) protease site and and inactive n-terminal processing site (q-1e) (enzyme), and the other contains an inactive protease site (c145a) and an active (wild-type) n-terminal processing site (substrate). they demonstrated cleavage of the processing site in the substrate molecule (c145a). however, this approach did not eliminate the cis reaction by which the enzyme molecule and the substrate molecule dimerize. although our results also did not completely eliminate the possibility of the cis reaction, they indicated that the trans reaction is sufficiently robust for the autoprocessing, and is the main path for the first n-terminal processing of the 3cl pro moiety in the polyprotein. liberation of 3cl pro from the polyprotein at the er membrane sars-cov contains rna(+) as its genome in the virion particle [8] . after infection of cells, this rna(+) is introduced into the cytosol and translated to form two large polyproteins, pp1a (4382 amino acids) and pp1ab (7073 amino acids), the latter of which is produced by a à1 translational frameshift occurring just upstream of the stop codon of pp1a. these polyproteins are processed by pl2 pro and 3cl pro to form proteins nsp1-nsp11 from pp1a, and proteins nsp1-nsp10 and nsp12-nsp16 from pp1ab (nsp5 is 3cl pro ). these proteins form a multi-subunit protein complex called the 'viral replicase complex', which generates a nested set of viral sub-genomic segments by complicated reactions [8] . only three cleavages (between nsp1 and nsp2, nsp2 and nsp3, and nsp3 and nsp4) are performed by pl2 pro , and the other 11 sites are cleaved by 3cl pro . however, the cleavages by pl2 pro are suggested to occur before those by 3cl pro [17] , and proteolysis between nsp3 and nsp4 by pl2 pro may occur before 3cl pro activation. at this stage, 3cl pro (nsp5) is located between nsp4 and nsp6. both nsp4 and nsp6 are membrane proteins. oostra et al. reported that the n-and c-termini of both the nsp4 and nsp6 proteins are on the cytoplasmic side of the er [17, 18] , so the 3cl pro moiety is on the cytoplasmic side. moreover, they proposed the membrane topology for polyprotein pp1a, in which all of the cleavage sites cleaved by 3cl pro , including its own n-and c-terminal processing, are on the cytoplasmic side, and are accessible by mature 3cl pro and the 3cl pro moiety in the polyprotein [18] . using our new method, we were able to analyze the enzymatic activities of the mature form and three proforms (with the n-terminal pro-sequence, the c-terminal pro-sequence, or both pro-sequences) of the 3cl protease towards the n-and c-terminal processing sites, under the same conditions. we identified the inhibitory effect of the c-terminal pro-sequence, by which the c-terminal pro-sequence of a dimer molecule inhibits the activity of another enzyme dimer molecule (inter-dimer manner). the assay system developed here is a convenient method to investigate the autoprocessing mechanisms of other viral proteases. the dna fragment of part of the sars-cov cdna, encompassing the n-terminal proximal 10 amino acids, the coding region of 3cl pro , and the c-terminal proximal 10 amino acids, was prepared by pcr using oligonucleotides 5′-cgtggatcccagacatcaatcacttctgctgttc tgcagagtggttttaggaaaatggcattccc-3′ and 5′-ggtgctcgagagtgcccttaacaattttcttgaa cttaccttggaaggtacaccagagcattgtc-3′), and inserted into the bamhi and xhoi sites of the pet29a vector (merck kgaa/novagen, darmstadt, germany). the encoded protein ([s tag]-gs-qtsitsavlq-[3cl pro ]-gkfkkivkgt-le-hhhhhh), in which gs and le are derived from the bamhi (ggatcc) and xhoi (ctcaga) sites of the pet29a vector, and qtsitsavlq and gkfkkivkgt are from the adjacent pro-sequences in the sars-cov polyprotein, was expressed by the e. coli cellfree protein synthesis system. the gln-1 to asn (q-1n), gln306 to asn (q306n) and cys145 to ala (c145a) mutations were introduced by quik-change mutagenesis (agilent technologies/stratagene, santa clara, ca, usa) using with the oligonucleotides 5′-caatcacttctgctgttctga acagtggttttaggaaaatggc-3′ and 5′-gccatt ttcctaaaaccactgttcagaacagcagaagtga ttg-3′ for q-1n, 5′-gctctggtgttaccttcaacg gtaagttcaagaaaattg-3′ and 5′-caattttcttg aacttaccgttgaaggtaacaccagagc-3′ for q30 9n, and 5′-ggttctttccttaatggatcagctggta gtgttggttt taac-3′ and 5′-gttaaaaccaacac taccagctgatccattaaggaaagaacc-3′ for c14 5a. the gfp substrate was created by replacing the region encoding amino acid residues 11-296 with that encoding the gfp protein, by pcr using four primers (5′-ttctcct ttactcattgacgggaatgccattttcctaaaacc actc-3′, 5′-gatgagctctacaaagttagacaatgc tctggtgttaccttccaag-3′, 5′-gcattcccgtca atgagtaaaggagaagaacttttc-3′, and 5′-gca ttgtctaactttgtagagctcatccatgccatg-3′). the protein expressed from this plasmid is [s tag]-gs-qtsitsavlqsgfrkmafps-[gfp]-vrqcsgvtfqg kfkkivkgt-le-hhhhhh. the plasmids were amplified in the e. coli dh5a strain, purified using a purelink hipure plasmid filter midiprep kit (life technologies/ invitrogen, carlsbad, ca, usa), and used in the e. coli cell-free protein synthesis system. the e. coli cell-free protein synthesis system has been described previously [19, 20] . a 60 ll reaction solution, containing 55 mm hepes/koh buffer (ph 7.5), 16 lgáml à1 plasmid dna, 1.7 mm dithiothreitol, 1.2 mm atp, 0.8 mm each of ctp, gtp and utp, 80 mm creatine phosphate, 250 lgáml à1 creatine kinase, 0.64 mm 3′,5′-cyclic amp, 68 lm l(-)-5-formyl-5,6,7,8-tetrahydrofolic acid, 175 lgáml à1 e. coli total trna, 210 mm potassium glutamate, 27.5 mm ammonium acetate, 10.7 mm magnesium acetate, 1.0 mm each of standard 20 amino acids, 93 lgáml à1 t7 rna polymerase, and e. coli s30 extract at a final attenuance at 260 nm of standard approximately 60, was placed in a dialysis cup, slide-a-lyzer mini dialysis unit (10k molecular weight cut-off; thermo scientific, waltham, ma, usa). dialysis was performed at 30°c for 4 h with moderate shaking against 1.0 ml of the same reaction solution, but lacking dna, trna, creatine kinase, t7 rna polymerase and s30 extract (dialysis buffer). the enzyme molecule (3cl pro , mature or pro-form) was synthesized by the e. coli cell-free protein synthesis system and diluted using the dialysis buffer described above. the substrate molecule [3cl pro (c145a) or the gfp substrate] was also synthesized by the e. coli cell-free protein synthesis system. the two solutions were mixed together at a 1 : 1 ratio, incubated for 1 h at 30°c, and analyzed by sds/page. sds/page analyses of proteins were performed using 12.5% or 15% polyacrylamide mini-gels (10 cm 9 6 cm) in laemmli's system [21] . after electrophoresis, the gels were stained with coomassie brilliant blue g-250 using quick-cbb plus (wako pure chemical industries, osaka, japan), and each band on the gel was quantified using imagej (http://rsb.info.nih.gov/ij/) software. background subtractions were performed for each of the bands using the control value (-dna in fig. 3a) of the corresponding region of the same area. mouse monoclonal antibody against his-tag (novagen), mouse anti-sars-cov 3cl pro monoclonal antibody (genesis biotech inc., taipei, taiwan), and mouse monoclonal antibody against gfp (medical & biological laboratories co. ltd, nagoya, japan) were used as the primary antibodies for his-tag, 3cl pro and gfp detection, respectively, and the s-protein horseradish peroxidase conjugate (merck kgaa/novagen, darmstadt, germany) was used for s tag detection. the protein sample was excised from the sds/page gel and analyzed by maldi-tof mass spectrometry using sinapinic acid (3,5-dimethoxy-4-hydroxy cinnamic acid) as the maldi matrix and a life technologies/applied biosystems (carlsbad, ca, usa) voyager biospectrometry workstation, according to the manufacturer's instructions. the n-terminal amino acid sequence of the protein was analyzed using life technologies/applied biosystems procise 494-ht protein sequencer, according to the manufacturer's instructions. who guidelines for the global surveillance of severe acute respiratory syndrome (sars). world health organization molecular biology of severe acute respiratory syndrome coronavirus the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor characterization of sars main protease and inhibitor assay using a fluorogenic substrate production of authentic sars-cov m pro with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction a structural view of the inactivation of the sars coronavirus main proteinase by benzotriazole esters mechanism of the maturation process of sars-cov 3cl protease mechanisms and enzymes involved in sars coronavirus genome expression aequorea green fluorescent protein. expression of the gene and fluorescent characteristics of the recombinant protein biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase profiling of substrate specificity of sars-cov 3cl pro maturation mechanism of severe acute respiratory syndrome (sars) coronavirus 3c-like protease mutation of gly-11 on the dimer interface results in the complete crystallographic dimer dissociation of severe acute respiratory syndrome coronavirus 3c-like protease. crystal structure with molecular dynamics simulations mechanism for controlling the dimer-monomer switch and coupling dimerization to catalysis of the severe acute respiratory syndrome coronavirus 3c-like protease two adjacent mutations on the dimer interface of sars coronavirus 3c-like protease cause different conformational changes in crystal structure liberation of sars-cov main protease from the viral polyprotein: n-terminal autocleavage does not depend on the mature dimerization mode localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning preparation of escherichia coli cell extract for highly productive cell-free protein expression cell-free production and stable-isotope labeling of milligram quantities of proteins cleavage of structural proteins during the assembly of the head of bacteriophage t4 we thank hideaki tanaka key: cord-353467-wbtzvm4i authors: lambert, carsten; thomé, nicole; kluck, christoph j.; prange, reinhild title: functional incorporation of green fluorescent protein into hepatitis b virus envelope particles date: 2004-12-05 journal: virology doi: 10.1016/j.virol.2004.09.031 sha: doc_id: 353467 cord_uid: wbtzvm4i the envelope of hepatitis b virus (hbv), containing the l, m, and s proteins, is essential for virus entry and maturation. for direct visualization of hbv, we determined whether envelope assembly could accommodate the green fluorescent protein (gfp). while the c-terminal addition of gfp to s trans-dominant negatively inhibited empty envelope particle secretion, the n-terminal gfp fusion to s (gfp.s) was co-integrated into the envelope, giving rise to fluorescent particles. microscopy and topogenesis analyses demonstrated that the proper intracellular distribution and folding of gfp.s, required for particle export were rescued by interprotein interactions with wild-type s. thereby, a dual location of gfp, inside and outside the envelope, was observed. gfp.s was also efficiently packaged into the viral envelope, and these gfp-tagged virions retained the capacity for attachment to hbv receptor-positive cells in vitro. together, gfp-tagged virions should be suitable to monitor hbv uptake and egress in live hepatocytes. hepatitis b virus (hbv), a human hepadnavirus, is an enveloped dna virus that infects hepatocytes and causes acute and chronic liver disease. despite considerable understanding of the details of hepadnaviral genome replication, fundamental insights into the early and late steps of an hbv infection are still lacking. infection initiates by virus attachment to the hepatocyte and is determined by the hbv envelope. among the three related large l, middle m, and small s envelope proteins, l has been shown to play the key role in receptor recognition and cell attachment (le seyec et al., 1999; neurath et al., 1986) . the route of subsequent hbv uptake is a yet poorly identified process and may proceed by direct fusion of the viral envelope with the plasma membrane, receptor-mediated endocytosis, or alternate mechanisms (for review, see cooper et al., 2003) . in favor of a fusion mechanism at the cell surface is the previous demonstration of a ph-independent internalization of hbv in primary human hepatocytes (hagelstein et al., 1997) . by contrast, infectivity studies performed with the related duck hbv in duck liver cells have demonstrated that viral entry depends on endocytosis (breiner et al., 1998) . in the late stages of an hbv infection, progeny virions are formed by budding of the pre-assembled cytosolic nucleocapsids, enclosing the partially double-stranded dna genome 3.2 kb in length and the viral polymerase through intracellular membranes accommodating the viral envelope proteins (for review, see nassal, 1996) . during this process, an excess of envelope proteins is not incorporated into virion envelopes but self-assembles into secreted subviral lipoprotein particles referred to as hepatitis b surface antigen (hbsag) particles or empty envelopes. these particles have been shown to mature by budding into intralumenal cisternae of post-endoplasmic reticulum (er)-pre-medial-golgi compartments and to exit the cell by the constitutive secretory pathway (huovila et al., 1992) . in contrast, the intracellular budding site of viral particles has not been defined to date. the three l, m, and s envelope proteins contribute differently to subviral and viral particle formation. assembly and secretion of subviral particles are solely driven by the s protein and are initiated by co-translational integration of s molecules into the er membrane (simon et al., 1988) . the current models for the transmembrane structure of s assume a lumenal disposition (i.e., external in the mature particles) of both the n-and c-termini and four membranespanning segments (berting et al., 1995; stirk et al., 1992) . following dimerization, about 100 transmembrane s monomers then self-assemble with host-derived lipids into spherical empty envelopes, 20 nm in diameter, which are secreted from the infected liver and transfected cell lines with high efficiency. this process is also sustained by the m protein that shares the sequence of s but differs in its nterminal pres2 extension. by contrast, the l protein along with its n-terminal pres1 plus pres2 domains blocks subviral particle production but is the key player in virion formation (bruss and ganem, 1991; chisari et al., 1986) . the pivotal role of the l protein in the viral life cycle is related to its dual transmembrane topology (bruss et al., 1994; ostapchuk et al., 1994; , as its internal (cytosolic) pres domain is needed for envelopment of cytosolic nucleocapsids , while the same external (lumenal) pres domain mediates receptor binding during host cell attachment (le seyec et al., 1999) . in the present study, we addressed whether the hbv envelope could be tagged with the green fluorescent protein (gfp) without impairing its functionality. we reasoned that such gfp-tagged particles should be a promising tool to study the early and late stages of the hbv life cycle in live cells. for tagging of the hbv envelope with gfp, the s protein was chosen as the target because this protein is an essential and abundant constituent of the virion envelope. two chimeras were constructed by fusing either a fluorescenceenhanced gfp in frame to the n-terminus of s (gfp.s) or the yellow-shifted yfp variant to the c-terminus of s (s.yfp) (fig. 1a) . the fusion constructs were transiently expressed in cos-7 cells and analyzed by gfp-specific western blotting. the unfused s protein with a c-terminal ha-tag (sha) was included as reference. as shown in fig. 1b , lysates of cells contained sha in its characteristic doublet of a non-glycosylated (p24) and single-glycosylated (gp27) form as a consequence of partial modification at asn-146. n-linked glycosylation was confirmed by treatment with peptide/n-glycosidase f (pngase f), which converted the gp27 form of s to its faster migrating p24 form. the gfp.s and s.yfp fusion proteins were stably expressed as 51-kda polypeptides in accordance with the molecular masses calculated for their non-glycosylated forms (p51) (fig. 1b) . as evidenced by enzymatic deglycosylation, both fusion proteins appeared in glycosylated forms (gp54) in addition, indicating that they were fig. 1 . the n-terminal gfp.s fusion is co-secreted with wild-type s. (a) schematic representation of the hbv s protein and the gfp.s and s.yfp fusion constructs. numbers below the domains refer to the corresponding amino acid positions of s (hatched bar) and gfp/yfp (white bars). for gfp.s, the two domains are linked by the amino acid sequence sglrsraqasn and terminated by the extra sequence wdppdldn, while s and yfp are interspersed by the sequence dppvat. (b) synthesis, glycosylation, and secretion of sha and the fusion constructs. lysates (c) from transfected cos-7 cells were divided into two portions and either left mock treated or digested with pngase f, as indicated. cellular supernatants (m) were analyzed without enzymatic de-glycosylation. proteins were processed by sds-page and ha-specific (lanes 1-3) or gfp-specific (lanes 4-9) immunoblotting. nonglycosylated (p) and glycosylated (gp) forms are indicated on the left with the numbers referring to the molecular masses of the polypeptides. (c) secretion of gfp.s is rescued by coexpressed wild-type s molecules. supernatants of cells synthesizing gfp.s in the absence (à) or presence (+) of s were assayed by gfp-specific western blotting. competent for co-translational integration of the s region into the er lumen. however, unlike wild-type sha, each chimera failed to be secreted from transfected cells into the supernatants (fig. 1b) . although these results implicated some misfolding of gfp.s and s.yfp, we nonetheless assessed whether their block to secretion could be abrogated by co-expression of wild-type s. cos-7 cells were co-transfected with each fusion construct, and the wild-type vector at a ratio of 1:1 and cellular supernatants were assayed by gfp-specific immunoblotting. the s.yfp fusion turned out to act in a trans-dominant-negative manner and even inhibited export of co-expressed wt s chains (data not shown). by contrast, gfp.s was secreted in the presence of s (fig. 1c) , and thus was chosen for further analysis. for quantitative evaluation of the secretion efficacy of gfp.s, lysates and supernatants of gpf.s + s co-transfected cells were analyzed with a gfp-specific elisa. this determination revealed that 68 f 5.2% of the intracellular gfp.s was released into the supernatant when co-expressed with wt s. in order to get insights how s rescued the secretion of gfp.s, we comparatively examined the intracellular distribution of gfp.s in the presence and absence of sha. when expressed alone, gfp.s surprisingly yielded an intense micropunctate fluorescence (fig. 2a) . these bdotsq partially overlapped with er structures, as shown by co-staining of the cells with antibodies to the er-resident protein disulfide isomerase (pdi) (figs. 2b and c) . although the nature of these structures remained to be determined, they might present aggregates of malfolded gfp.s destined for degradation. importantly, however, on co-expression of gfp.s with sha, the chimera now exhibited a widely dispersed membranous staining pattern together with a significant colocalization with the er marker pdi (figs. 2d-f ). under the same conditions, a nearly 100% co-localization of gfp.s with sha was observed when cells were stained with haspecific antibodies . from these data we concluded that the correct distribution of gfp.s, required for export, was warranted by interaction with co-expressed s chains. to determine whether the co-secreted gfp.s and sha proteins resembled authentic subviral hbv envelope particles, the culture supernatant of transfected cells was fractionated by isopycnic cscl gradient centrifugation and fractions were analyzed by an s-specific elisa. as shown in fig. 3a , the co-secreted proteins banded at a buoyant density of about 1.22 g/ml, typical for wild-type s lipoprotein particles (heermann and gerlich, 1991) . peak fractions were then subjected to specific western blot analysis that confirmed the presence of both the gfp.s fusion and the sha protein (fig. 3a ). in addition, when fig. 2 . gfp.s co-localizes with wild-type s at the er. shown is the intracellular distribution of gfp.s expressed either alone (squares a-c) or together with sha (squares d-i) in cos-7 cells. cells were fixed, permeabilized, and examined by fluorescence microscopy. (a, d, and g) gfp fluorescence (green); (b and e) immunostaining with a mouse antibody to pdi followed by alexafluor 494-conjugated goat anti-mouse igg (red); (h) immunostaining with a mouse anti-ha antibody followed by alexafluor 494-conjugated goat anti-mouse igg (red) to visualize sha. squares c, f, and i are the corresponding merged images so that overlapping red and green signals appear yellow. gfp.s + sha and wild-type sha particles were analyzed by sucrose gradient velocity centrifugation, their sedimentation profiles were nearly coincident with a buoyant density of about 1.130 g/ml in sucrose (fig. 3b) . together, these data demonstrated that gfp.s was successfully incorporated into subviral particles that closely resembled s spheres with respect to density and size. because the fluorescence of gfp depends on its native three-dimensional structure that might be altered within the chimeric context, we next examined whether purified gfp.s + sha particles were fluorescent. as imaged by fluorescence microscopy, a strong vesicular gfp staining was observed, indicating a native display of gfp within the particle (fig. 3c ). the intensity of fluorescence and hence the apparent size of the particles appeared slightly variable and brighter than expected, perhaps because of some particle aggregation or variation in the amount of gfp.s incorporation. the production of fluorescent subviral particles indicated that the individual constituents of the gfp.s chimeric protein were properly folded, at least when co-expressed with wild-type s chains. nonetheless, for further studies outlined below, it was important to know its precise topology, that is, the orientation of the gfp domain relative to the membrane. according to current models for the transmembrane structure of s, its n-terminus and hence the gfp fusion site are located to the lumenal side of intracellular membranes that is topologically equivalent to the virion surface ( fig. 4a ) (berting et al., 1995; stirk et al., 1992) . to analyze the topological features of gfp.s, expressed in the absence or presence of s molecules, we used proteinase k protection experiments of microsomes prepared from transfected cos-7 cells. both gfp.s and s proteins were synthesized in ha-tagged form to enable their simultaneous detection by immunoblotting. consistent with previous works , proteinase k did not cleave the p24/gp27 forms of sha unless the protecting microsomal membrane was disrupted by detergent ( fig. 4b , lanes 4-6). by contrast, gfp.sha synthesized in the absence of sha chains was accessible to cleavage with the protease in intact microsomes (fig. 4b , lanes 1-3). thereby, two ha-reactive fragments were generated that closely resembled the full-length p24 and gp27 forms of sha. therefore, cleavage of gfp.sha must have occurred at least at a very distal site within the nterminal fused gfp domain. in support of a fully cytoplasmic location of the gfp domain, we were unable to detect gfp-reactive proteolysis products by western blotting (data not shown). when expressed together with sha, the topology of gfp.sha surprisingly changed in such that a fraction of polypeptides was now protected from cleavage with proteinase k by the microsomal membrane (fig. 4b, lanes 4-6) . these results indicated that the presence of extra sha chains caused a partial topological change of the n-terminal gfp domain from cytosolic to lumenal, likely as a consequence of gfp.sha/sha interprotein subunit interaction. given the strict correlation between partial n-tail translocation and secretion competence and vice versa, we finally investigated the topological properties of gfp.sha complemented in trans with a secretion-defective s mutant protein. to this aim, we took advantage of sdtm1ha that lacks the first transmembrane domain and is blocked in the assembly and secretion of subviral particles (prange et al., 1992) . consistent with our previous results (prange et al., 1992) , the deletion mutant predominantly appeared in a glycosylated 24-kda form (gp24; fig. 4b, lane 7) . importantly, sdtm1ha failed to impart a partial lumenal orientation of gfp.sha (fig. 4b , lanes 7-9). together, these data indicated that the topological change of gfp.sha in the presence of extra s molecules was likely established during the formation and extracellular release of the gfp.sha/sha mosaic particles. if this notion was correct, we would expect the gfp tag located outside and inside of the secreted particle. for a formal proof of a surface display of the gfp domain, we tried to immunoprecipitate the particles under non-denaturing conditions using two monoclonal a-gfp antibodies directed against different epitopes. however, neither antibody brought down the particles in the absence of detergent (data not shown; and see below). although these results might argue against an exterior location of gfp, it seemed equally possible that the epitopes were not accessible in the bulky beta barrel of gfp on the intact particle. incorporation of gfp.s into envelopes allows viral particle formation next we examined whether gfp.s would also be incorporated into the viral envelope. for virus production, human hepatoma huh-7 cells were transiently transfected with a cloned over-length copy of the hbv genome. on cotransfection with the gfp.s expression plasmid at a 1:1 ratio, virus secretion into the cellular supernatant was reduced about 3-fold (data not shown), whereas no inhibitory effect of gfp.s on the virus secretion profile was observed if a 4-fold excess of the hbv encoding vector was used for co-transfection. because transfected huh-7 cells released viral and subviral particles into the culture medium, virions were separated from empty envelope particles by isopycnic cscl gradient centrifugation. a resolution of these particle types was verified by assaying of fractions for the presence of hbv dna by the endogenous polymerase reaction. as shown in fig. 5 (a and b) , dna-containing virions banded at a density near 1.24 g/ml around fractions 18-22, with the strongest signal in fraction 20, while empty envelopes were found at their typical density of 1.22 g/ml. importantly, when probed with a gfp-specific elisa, the virus-containing fractions were clearly reactive, thus giving first hints to an incorporation of the gfp-tagged s protein during virus assembly. as a final proof, supernatants of hbv + gfp.s co-transfected huh-7 cells were immunoprecipitated with a gfp-specific antibody prior to detection of the viral genome by dot-blot analysis. for reasons mentioned above, such a precipitation, however, required the presence of detergent (0.5% np-40) that decreased the limits of detection, likely because some nucleocapsids might leak from the disordered envelope network. nonetheless, despite these restraints, we could specifically detect the viral dna in a-gfp-precipitated supernatants (fig. 5c) . to investigate whether these fluorescent hbv virions supported attachment to hepatocytes, cell binding assays were performed. although most permanent cell lines are not permissive to hbv infection, the human hepatoblastoma hepg2 cell line is able to bind the virion (lu et al., 1996) . for binding, we applied the same assay conditions as validated previously for in vitro infection of primary human hepatocytes (gripon et al., 1993) . accordingly, recombinant hbv + s.gfp particles secreted from co-transfected huh-7 cells (see above) were precipitated with polyethylene glycol (peg) and reacted with hepg2 cells grown on glass bottom fig. 4 . gfp.s forms a mixed topology when co-expressed with wild-type s. (a) model of the transmembrane topology of s at the er membrane and the virion envelope. the predicted four membrane spanning segments of s project its n-and c-terminus into the er lumen that is topologically equivalent to the virus outside. the n-glycosylation site at asn-146 is indicated by w. (b.) proteinase k protection assay of gfp.s. cos-7 cells were transfected with ha-tagged gfp.s either alone (lanes 1-3) or together with ha-tagged s (lanes 4-6) or the secretion-defective sdtm1 mutant (lanes 7-9). two days after transfection, microsomes were prepared and either left untreated or digested with proteinase k (prot. k) in the absence (à) or presence (+) of np-40, as denoted above each lane. samples were analyzed by ha-specific immunoblotting. the gfp.s-and s-specific p and gp forms are indicated on the left; the corresponding forms of sdtm1ha are denoted on the right. dishes. after incubating cell cultures overnight, followed by several washes, a strong gfp fluorescence almost evenly distributed on the plasma membrane of the hepatocytes was observed (figs. 6a and b) . to test the specificity of adsorption, we analyzed the binding properties of gfp.s + s subviral particles that lacked the l envelope protein and thus the receptor binding site. as expected, these particles failed to attach to hepg2 cells thereby yielding only some diffuse background staining (figs. 6c and d) . however, because infected liver cells and hbv-producing cell lines are known to also produce subviral particles containing the l protein (nassal, 1996) , we could not totally exclude the possibility that the gfp fluorescence, shown in figs. 6a and b, might be also generated by binding of this particle type. in recent years, the adaptation of the aequorea victoria gfp for visualization of protein expression and protein localization in living organisms has provided a powerful new tool. here we applied this approach to hepatitis b virus and obtained fluorescent subviral and viral particles by incorporation of the viral s envelope protein, tagged with gfp, in trans, thereby preserving all the functions necessary for the viral life cycle. because hbv empty envelope particles, built from the s protein, provide a safe antigen delivery system, their use as a carrier for the presentation of various antigens is a long established practice. to date, chimeric particles have been produced in mammalian cells with inserts in the range of 10-80 amino acids (delpeyroux et al., 1986; michel et al., 1988; . for example, the n-terminal ectodomain of the s protein has been shown to tolerate 84 foreign residues while its c-terminus accommodated 42 residues without effects on particle assembly and secretion (michel et al., 1988; . in agreement and extension, the current study demonstrated that the nterminus of s is more permissive for insertions than the c-terminus and is even amenable for addition of the complete gfp protein. hence, proteins of at least up to 238 amino acids can be displayed on hbv envelope containing virion particles were further subjected to an envelope-specific immunoprecipitation and radioactive labeling of the viral genome by the endogenous viral polymerase. the migration of the hbv dna genome as visualized by agarose electrophoresis and phosphorimaging is indicated. (c) pooled extracellular viral particle fractions from hbv + gfp.s cotransfected cells were immunoprecipitated with either an envelope-specific (a-hbsag) or a gfp-specific (a-gfp) antibody prior to extraction of the viral genome and dna dot blot analysis. for control of nonspecific immunoprecipitation, native hbv particles (control) were reacted with the a-gfp mab (third panel from the left). in addition, the control material was probed with a-hbsag, as shown in the right panel. fig. 6 . gfp-tagged hbv binds to hepg2 cells. in squares a and b are shown the adsorption of gfp-tagged hbv particles (hbv + gfp.s) to live hepg2 cells, grown on glass bottom dishes, as detected by gfp autofluorescence (magnification, â1600). under the same assay conditions, gfp-tagged subviral particles (s + gfp.s) did not bind to cells (squares c and d). particles, raising intriguing perspectives for their future use as a carrier system. particle formation of the gfp.s chimeric protein, however, depended on the presence of the wild-type s protein, indicating that the fusion construct itself failed to fold into a functional conformation required for assembly. nonetheless, as judged by the autofluorescence of gfp.s in cells, at least the gfp-tag must have folded properly. thereby, gfp.s surprisingly appeared in sequestered speckles that overlapped with the er compartment. the formation of similar structures, termed concentric membranous er bodies, has been recently observed upon expression of a misfolded mutant of the cystic fibrosis transmembrane conductance regulator that accumulates in these bodies prior to erassociated degradation (okiyoneda et al., 2004) . accordingly, the s moiety of gfp.s might not attain its native transmembrane structure thereby tending to aggregate into discrete speckles. on co-expression with wild-type s, the intracellular distribution of gfp.s completely changed in such that it now yielded a typical membranous er staining pattern and a high degree of co-localization with s. this result indicates that the proper folding and localization of gfp.s were warranted by interprotein interactions with wildtype s chains. as a consequence thereof, highly fluorescent mosaic particles were formed and secreted, which closely resembled authentic spherical s lipoprotein particles according to density and size. furthermore, the gfp.s fusion protein could be also natively incorporated into the virion envelope. gfp, by virtue of its properties, has been successfully used to tag various viruses like, for example, herpes simplex virus type i, vesicular stomatitis virus, vaccinia virus, hiv, and mouse hepatitis coronavirus (bosch et al., 2004; dalton and rose, 2001; desai and person, 1998; stauber et al., 1999; ward and moss, 2001) . however, unlike these viruses, hbv is a very small particle with a diameter of 42 nm and a dense packed structure. even so, it can apparently accommodate multiple copies of gfp, indicating that there is substantial space available between the viral envelope and the nucleocapsid as well as sufficient flexibility allowing the incorporation of proteins in trans. although we were unable to unequivocally define the location of the gfp-tag within the secreted hbv envelope, our topological analysis of gfp.s suggested an interior (i.e., cytosolic side of microsomes) and exterior (i.e., lumenal side of microsomes) display of gfp. the formation of such a mixed topology is rare among membrane proteins but is a prominent feature of the large l envelope protein of hbv (bruss et al., 1994; ostapchuk et al., 1994; . in that case, it is established post-translationally at the er membrane and is uncoupled from envelope assembly (lambert and prange, 2001) . by contrast, the two differently orientated isoforms of gfp.s were only generated upon coexpression with secretion-competent wild-type s molecules, thus hinting to a link between envelope subunit interactions and topogenesis of gfp.s. while the underlying mechanism remains to be determined, it is tempting to speculate that the dual location of the gfp domain might be beneficial for the functionality of gfp-tagged hbv. by splitting the tag to both sides of the virion envelope, the local density of gfp might be lowered thereby limiting the risk of sterical hindrance of the envelope/nucleocapsid interactions at the interior and the envelope/receptor binding at the exterior. by using hepg2 cells, an established hepatocyte-derived cell line that is permissive for hbv attachment but nonpermissive to a productive infection (lu et al., 1996) , a specific binding of gfp-tagged viral particles was observed. hence, these particles must imitate the natural virus and viral attachment appeared to proceed through the authentic pathway. however, whether these particles even retained their infectivity remains to be addressed. in vitro hbv infection studies had been restricted to primary human hepatocytes that are not readily available. more recently, however, successful experimental infections of tree shrews were carried out that might present a new animal model (köck et al., 2001) . in addition, a human hepatoma cell line has been established that supports hbv infection (gripon et al., 2002) . therefore, the gfp-tagged hbv envelope might be useful for the investigation of the early events of virus penetration such as monitoring entry and uncoating in living infected cells and tracing the fate of the envelope structure. with gfp as a marker, it should also be possible to approach sites of viral assembly and to follow the intracellular trafficking and egress of (sub)viral particles during the late stages of an hbv infection. the mammalian expression vectors carrying the hbv s gene with or without a c-terminally tagged influenza virus hemagglutinin (ha) epitope under the control of the human metallothionein iia promoter had been described (pni2.-sha and pni2.s, respectively) (hartmann-stühler and . for construction of the gfp.s chimera, plasmid pegfp-c1 (bd biosciences clontech) was cut within the multiple cloning site by using ecori and smai. to introduce an ecori restriction site downstream of the start codon of the hbv s gene, site-directed mutagenesis with a recombinant m13mp19 bacteriophage carrying a 2.3kb bglii-bglii fragment (nucleotide [nts] 2839 to 1986, as referred of the hbv genome, subtype ayw) with the antisense oligonucleotide 5v-gaatcctgaattcatgttctc-3v (the ecori site is italic) was performed. the newly created ecori site, cutting between codon positions 4 and 5 (nts 170) of the s gene, together with the cognate filled-in acci site (nts 827) was then used to generate an s encoding fragment for in-frame insertion into pegfp-c1, giving rise to plasmid pegfp.s. in parallel, the ha-tagged version of the s gene was similarly cloned, giving rise to gfp.sha. a reciprocal chimera, containing the yellow-shifted yfp colinearly fused to the c-terminus of s (s.yfp), was created using plasmid peyfp-n1 (bd biosciences clontech), which was opened with xhoi and bamhi. the ha-tagged s gene was derived from pni2.sha by cleavage with xhoi (nts 127) and psii (cutting between the ha-tag and the translational stop codon) and inserted into peyfp-n1 after filling-in of its bamhi site. plasmid pni2.sdtm1ha carries an in-frame deletion of tm1 that was achieved by removal of a styi (nts 180)-xbai (nts 249) fragment from pni2.s. to produce viral envelope proteins, transient transfection of cos-7 cells by electroporation was used. unless otherwise indicated, 5 â 10 6 cells were transfected with 12 ag of plasmid dna, while in the case of co-transfections, 12 ag of each dna was used. three days posttransfection, cellular supernatants were harvested and clarified by low-speed centrifugation. cells were washed twice in tris-buffered saline (tbs, 50 mm tris-cl [ph 7.5], 150 mm nacl) and lysed with 1 ml of tbs-0.5% nonidet p-40 (np-40), supplemented with a protease inhibitor cocktail (roche), for 30 min on ice. after centrifugation for 5 min at 13 000 â g and 4 8c, proteins of lysates and cell supernatants were precipitated with 10% trichloroacetic acid (tca), washed twice with 5% tca, and once with acetone. tca precipitates were resolved by sds-page and western blotted to nitrocellulose membranes. enzymatic n-deglycosylation of proteins with pngase f (new england biolabs) was done according to the instructions of the supplier. immunoblots were incubated with a mouse monoclonal antibody (mab) against the ha epitope (babco), diluted 1:2000 in blotting buffer (pbs with 5% skim milk) or a mouse mab specific for gfp (jl-8; bd biosciences clontech) applied in 1:4000 dilution. peroxidase-labeled secondary antibodies (dianova) were diluted as instructed by the manufacturer, and the blots were developed with enhanced chemiluminescence detection reagents (amersham biosciences). in parallel, cell lysates and supernatants were analyzed by elisas. hbsag was measured using the auszyme ii diagnostic kit (abbott laboratories). for simultaneous detection of gfp and hbsag, a sandwich elisa with gfp-specific antibodies in the solid phase and s-specific antibodies in the detection phase was employed. briefly, the a-gfp-mab jl-8 was coated to microtiter plates (1:500 dilution in tbs) for 3 h at room temperature. nonspecific binding sites were blocked with tbs-1% bovine serum albumin for 18 h at 4 8c, followed by three washes with tbs-0.1% tween 20. samples were reacted for 2 h at 37 8c in the presence of 0.5% np-40, washed out, and incubated with peroxidase-labeled hbsag-specific mabs (auszyme ii) for 1 h at 37 8c. transfected cos-7 cells on glass cover-slips were fixed and permeabilized with ice-cold methanol containing 2 mm egta for 15 min at à20 8c. for staining of internal antigens, cells were incubated with the ha-specific mab (1:200 dilution in pbs) or a mouse mab specific for protein disulfide isomerase (spa-891, stressgen biotechnologies) (1:2000 dilution in pbs) prior to incubation with an alexafluor 594-conjugated goat anti-mouse immunoglobulin g (2 ag/ml in pbs; molecular probes). spontaneous gfp fluorescence and immunostaining were visualized with a fluorescence microscope (axiovert 200m, zeiss), and images obtained with a zeiss axiocam digital camera were processed using the zeiss axiovision software. to assess for fluorescent particles, cellular supernatants were concentrated 50-fold by ultracentrifugation (see below). a 10-al aliquot was spotted on glass bottom dishes (bd biosciences), briefly air dried, and imaged with a â100 objective. three days after transfection of cos-7 cells, microsomes were prepared essentially as described . briefly, cells were disrupted by dounce homogenization, and microsomes were recovered by ultracentrifugation prior to proteolysis with proteinase k (100 ag/ml) in the presence or absence of 0.5% np-40. after incubation on ice for 60 min, proteinase k was inactivated by the addition of 10 al/ml phenylmethylsulfonyl fluoride. each sample was then adjusted to 0.5% np-40 and solubilized for 20 min on ice. cleared samples were precipitated with 10% tca and subjected to western blot analysis. cesium chloride and sucrose gradient purification of particles subviral particles of cellular supernatants were pelleted through a 1-ml cushion of 20% sucrose in tne (10 mm tris-cl [ph 7.5], 150 mm nacl, 10 mm edta) using a sw 40 rotor (beckman) at 37 000 rpm for 4 h at 4 8c. the resuspended pellets were separated by isopycnic gradient centrifugation through 10-50% (wt/vol) cesium chloride in tne. after centrifugation at 35 000 rpm and 10 8c (sw 40 rotor), fractions were collected from the top and screened for chimeric particles by elisa, immunoblotting, and fluorescence analysis. for velocity sedimentation, pelleted particles were layered on top of a 12-ml linear sucrose gradient (10 to 50% [wt/wt] sucrose in tne buffer) with a 68% sucrose cushion, centrifuged at 35 000 rpm and 10 8c for 16 h (sw 40 rotor), and processed as above. for replication of hbv in the huh-7 liver cell line, plasmid phbv was used that carries a 1.1 mer of the hbv dna genome (radziwill et al., 1990) . this plasmid was cotransfected with pegfp.s by the calcium phosphate precipitation technique. four days after transfection, extracellular virions were separated from subviral empty envelopes and non-enveloped core particles by isopycnic cscl gradient centrifugation. to this aim, 3.8 g of cscl was dissolved in 10 ml cleared supernatant and 28 ml of this solution was spun for 20 h at 45 000 rpm at 10 8c in a beckman 50vti rotor. fractions were collected from the bottom, supplemented with the protease inhibitor cocktail, and were screened for hbsag and gfp by immunoassays. following dialysis of fractions against 10 mm tris-cl [ph 7.5], 150 mm nacl at 4 8c, virions were isolated by an envelope-specific immunoprecipitation, as described previously (löffler-mary et al., 2000) . for detection of gfp.s within the virion envelope, particles were immunoprecipitated with the a-gfp mab jl-8 that had been coated to a 10% (wt/vol) suspension of protein g-sepharose (10-al antibody was bound to 100 al sepharose solution in an overnight reaction at 4 8c). immunoprecipitation was performed in the presence of 0.5% np-40 and precipitates were then washed two times with ten buffer (50 mm tris-hcl [ph 7.5], 1 mm edta, 75 mm nh 4 cl). detection of the encapsidated viral progeny dna by radioactive labeling of the partially double-stranded genome with 10 aci [a 32 p]datp (amersham biosciences) by the endogenous polymerase, isolation, and separation of the dna by agarose gel electrophoresis have been described (löffler-mary et al., 2000) . the radioactive signal was detected by phosphorimager scanning. for detection of hbv dna on dot-blots, an ecorilinearized unit-length hbv genome was labeled with digoxygenin-dutp by random priming as instructed by the manufacturer (roche). after extraction of the dna genomes from the immunoprecipitated samples as described, they were denatured by boiling in 0.4 m naoh-10 mm edta and filtered with a dot-blot manifold (schleicher and schqll) onto nylon membranes. the membrane was baked at 120 8c for 30 min and hybridized with the labeled probe according to the roche dig dna labeling and detection kit. for binding analysis, human hepatoma hepg2 cells were cultivated in glass bottom dishes in dulbecco's modified eagle's medium with 10% fetal calf serum (fcs). hbv and subviral particles secreted from huh-7 (co)-transfected cells were precipitated from 10-ml culture supernatant in the presence of 6% polyethylene glycol 8000 (peg; sigma). the pellet was resuspended in 200-al phosphate-buffered saline (pbs) containing 25% fcs. hepg2 cells were incubated with 50 al of this concentrate, diluted in 1 ml culture medium with 4% peg, for 20 h at 37 8c. cells were washed three times with pbs and analyzed for gfp fluorescence by microscopy. computer-aided studies on the spatial structure of the small hepatitis b surface protein coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent carboxypeptidase d (gp180), a golgi-resident protein, functions in the attachment and entry of avian hepatitis b viruses the role of envelope proteins in hepatitis b virus assembly post-translational alterations in transmembrane topology of the hepatitis b virus large envelope protein functions of the large hepatitis b virus surface protein in viral particle morphogenesis expression of hepatitis b virus large envelope polypeptide inhibits hepatitis b surface antigen secretion in transgenic mice the earliest steps in hepatitis b virus infection vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles a poliovirus neutralization epitope expressed on hybrid hepatitis b surface antigen particles incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid reproducible high level infection of cultured adult human hepatocytes by hepatitis b virus: effect of polyethylene glycol on adsorption and penetration infection of a human hepatoma cell line by hepatitis b virus phindependent uptake of hepatitis b virus in primary human hepatocytes hepatitis b virus large envelope protein interacts with g2-adaptin, a clathrin adaptor-related protein surface proteins of hepatitis b viruses hepatitis b surface antigen assembles in a post-er, pre-golgi compartment efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis b virus dual topology of the hepatitis b virus large envelope protein: determinants influencing post-translational pre-s translocation infection process of the hepatitis b virus depends on the presence of a defined sequence in the pre-s1 domain hepatitis b virus assembly is sensitive to changes in the cytosolic s loop of the envelope proteins protease-induced infectivity of hepatitis b virus for human hepatoblastoma cell line induction of anti-human immunodeficiency virus (hiv) neutralizing antibodies in rabbits immunized with recombinant hiv-hepatitis b surface antigen particles hepatitis b virus morphogenesis. cmti identification and chemical synthesis of a host cell receptor binding site on hepatitis b virus df508 cftr pool in the endoplasmic reticulum is increased by calnexin overexpression a dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis b viral morphogenesis novel transmembrane topology of the hepatitis b virus envelope proteins deletions in the hepatitis b virus small envelope protein: effect on assembly and secretion of surface antigen particles properties of modified hepatitis b virus surface antigen particles carrying pres epitopes mutational analysis of the hepatitis b virus p gene product: domain structure and rnase h activity secreted hepatitis b surface antigen polypeptides are derived from a transmembrane precursor direct visualization of hiv-1 entry: mechanisms and role of cell surface receptors a topological model for hepatitis b surface antigen visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-b5r membrane protein chimera characterization of early hepatitis b virus surface protein oligomers we acknowledge the perfect technical assistance of tatjana dfring and thank rolf e. streeck, martin sapp, and hans-christoph selinka for helpful discussion. this work was supported by grants to r. p. from the deutsche forschungsgemeinschaft (sfb 490-a1, pr305/1-1). key: cord-332881-mkm4ygh6 authors: kirchhoff, jana; uhlenbruck, sabine; goris, katherina; keil, günther m; herrler, georg title: three viruses of the bovine respiratory disease complex apply different strategies to initiate infection date: 2014-02-18 journal: vet res doi: 10.1186/1297-9716-45-20 sha: doc_id: 332881 cord_uid: mkm4ygh6 bovine respiratory disease complex (brdc) is the major cause of serious respiratory tract infections in calves. the disease is multifactorial, with either stress or reduced immunity allowing several pathogens to emerge. we investigated the susceptibility of bovine airway epithelial cells (baec) to infection by the three major viruses associated with the brdc: bovine respiratory syncytial virus (brsv), bovine herpesvirus type 1 (bhv-1) and bovine parainfluenza virus type 3 (bpiv3). for this purpose, two culture systems for well-differentiated baec were used: the air-liquid interface (ali) system, where filter-grown baec differentiate into a pseudostratified respiratory epithelium and precision-cut lung slices (pcls) where baec are maintained in the original tissue organisation. comparative infection studies demonstrated that entry and release of bpiv3 occurred specifically via the apical membrane with ciliated cells being the major target cells. by contrast, airway epithelial cells were largely resistant to infection by bhv-1. when the epithelial barrier was abolished by opening tight junctions or by injuring the cell monolayer, bhv-1 infected mainly basal cells. respiratory epithelial cells were also refractory to infection by brsv. however, this virus infected neither differentiated epithelial cells nor basal cells when the integrity of the epithelial barrier was destroyed. in contrast to cells of the airway epithelium, subepithelial cells were susceptible to infection by brsv. altogether, these results indicate that the three viruses of the same disease complex follow different strategies to interact with the airway epithelium. possible entry mechanisms are discussed. virus infections of the respiratory tract are the most common cause of viral diseases worldwide ranging from common colds to life-threatening pneumonia [1] . a large number of both rna and dna viruses uses the respiratory tract to initiate host infection. infection may be restricted to or most evident in certain sections of the airway system such as trachea, bronchi or alveoli. for some viruses, the respiratory tract may just serve as a primary entry site from where infection spreads to other organs or tissues. all these viruses encounter the respiratory epithelium as a primary barrier against invading pathogens. this barrier is composed of differentiated epithelial cells that have different functions. an important defence strategy is the mucociliary clearance system. while some epithelial cells are specialized to produce and release mucins, other cells are equipped with cilia that enable them to contribute to the transport of the mucus out of the respiratory tract. viruses differ in their ability to infect the differentiated respiratory epithelial cells. productive infection by human influenza viruses has been reported to preferentially occur in mucus-producing cells [2] . other viruses, e.g. parainfluenza virus 3 (piv3), may have a preference for ciliated cells [3] . characteristic features of differentiated epithelial cells such as ciliary activity are not maintained in any of the available immortalized cell lines. analysis of such cells requires primary cell cultures. when tracheal or bronchial epithelial cells are grown on filter supports under air-liquid interface (ali) conditions, they differentiate and acquire properties of specialized cells, i.e. mucus production or ciliary activity [4, 5] . another culture system for differentiated respiratory epithelial cells are precision-cut lung slices (pcls), where the epithelial cells are maintained in their original setting. in addition to mucus production and ciliary activity, this culture system provides another characteristic feature of the airway, bronchoconstriction. as submucosal cells are also present in pcls, they can be included in the investigation [6] [7] [8] . bovine respiratory disease complex (brdc) is a leading cause of morbidity and mortality in feedlot cattle. the disease is considered as a multifactorial disorder caused by a combination of viral and bacterial pathogens together with environmental risk factors. the most important viral pathogens associated with brd are bovine respiratory syncytial virus (brsv), bovine parainfluenza virus 3 (bpiv3) and bovine herpesvirus 1 (bhv-1), a member of the subfamily alphaherpesvirinae [9] [10] [11] [12] . brsv and bpiv3 are paramyxoviruses that use the fusion protein f on the cell surface to mediate fusion of the viral envelope with the cellular membrane and thus to get the viral genome into the cell. prior to fusion, the virions attach to the cell surface. attachment of piv3 is accomplished by another surface protein, the hn protein which interacts with sialic acid-containing cellular glycoconjugates and thus binds to cells in a sialic acid-dependent manner [13, 14] . attachment of human rsv (hrsv) can be mediated by the surface protein g that binds to glycosaminoglycan structures [15] as well as by the fusion protein f that may bind to both glycosaminoglycans as well as to a specific protein receptor [16, 17] . nucleolin has recently been reported to promote infection though the authors did not exclude the possibility that another so far unknown protein may serve as a receptor for entry of hrsv [18] . no data on receptors for brsv have been reported yet. alphaherpesviruses like bhv-1, herpes simplex virus 1 (hsv-1) or pseudorabies virus use the surface glycoprotein gc for initial attachment to heparan sulphate structures. for entry, virions have to bind to a specific receptor (nectin-1 or herpesvirus entry mediator (hvem)) which is accomplished by the surface protein gd. the subsequent fusion reaction is mediated by the combined action of the surface proteins gb/gd/gh/gl [19, 20] . we have reported recently that well-differentiated respiratory epithelial cells are susceptible to infection by bpiv3 whereas they are rather resistant to infection by brsv [7] . here we have included another important virus of the brdc, bhv-1 and analysed virus entry into and release from differentiated respiratory epithelial cells in more detail. our results show that the three viruses have developed different strategies to infect the cells of the airway epithelium. the generation of recombinant brsv expressing green fluorescent protein (brsv-gfp) and preparation of viral stocks has been described in detail elsewhere (7; brsv atue51908 was kindly provided by karl-klaus conzelmann, max-von-pettenkofer-institut, munich). bpiv3 was provided by friedrich-loeffler-institut (insel riems, germany). bpiv3 stocks were prepared in kop-r cells [7] . bhv-1-gfp was generated from the strain bhv-1/aus12 as described previously [21] and propagated in kop-r cells. for this purpose, cells were grown in 75 cm 2 -flasks till 80% confluence and then infected with an moi of 0.1 for 2 h. after 1-3 days a cytopathic effect was observed and supernatants were collected and centrifuged by low speed centrifugation (2000 × g). all virus stocks were frozen in liquid nitrogen and stored at −80°c. mdbk (madin-darby bovine kidney cells; kindly provided by wolfgang garten, philipps-universität marburg, marburg, germany), vero cells (african green monkey cells; atcc, ccl-81) as well as kop-r cells (bovine oropharynx tissue, rie 244; friedrich-loeffler-institut, insel riems, germany) were cultured in dulbecco's modified eagle medium (dmem) supplemented with 5 or 10% fetal calf serum (biochrom ag, berlin). all cell lines were maintained in 75-cm 2 culture flasks at 37°c and 5% co 2 and passaged twice a week. primary baec (bovine airway epithelial cells) were derived from bovine bronchi of 6-8 months old calves and epithelial cells were prepared as described previously [5, 7] . briefly, cells were seeded on collagen coated, semipermeable membrane supports (greiner, 24 well, 0.4-μm pore size) at a density of 2.5 × 10 5 cells per filter support. after baec had reached confluence, the apical medium was removed and cells were cultured under ali conditions for at least 3 weeks to establish a polarized and differentiated epithelium. prior to infection, the transepithelial electrical resistance (teer) was determined to verify an intactness of the epithelial barrier. for this purpose, a millicell ers-2 volt-ohm meter (millipore corporation, billerica, ma, usa) was used. to obtain the actual resistance of the epithelial cell layer, the resistance value of a cell-free filter support was subtracted from the teer values determined across the filter-grown cultures. teer values were above 400 ω • cm 2 prior to infection. pcls were prepared from the lobus accessorius of 6-8 months old calves as described previously [6, 7] . lobes were filled with low-melting agarose (agarose lm gqt, gerbu, gaiberg, germany). after the agarose had solidified, cylindrical portions containing a section of an airway were stamped out. using a krumdieck tissue slicer (tse systems, model md4000-01) slices, 250 μm thick, were generated and incubated in 1 ml of rpmi 1640 medium (invitrogen/gibco, germany) in 24-well plates at 37°c and 5% co 2 . the viability of the slices was determined by screening for ciliary activity using a light microscope (zeiss axiovert 35). baec were washed extensively with phosphate-buffered saline (pbs) to remove secreted mucus. subsequently, the virus suspension, diluted in dmem, was added at 100 μl per filter to the cells for 2 h (bpiv3, bhv-1-gfp) or 3 h (brsv-gfp) at 37°c and 5% co 2 . for infection of the cells from the basolateral surface, filters were inverted for the duration of infection. after unbound virus had been removed by washing, the cells were further incubated at 37°c for 1 to 3 days. to open tight junctions, the cells were washed thrice with ca 2+ /mg 2+ -free pbs and then apically treated with ca 2+ /mg 2+ -free pbs containing 0.1 m egta (applichem, darmstadt) at 37°c. after 10 min, egta was removed and cells were infected with either bhv-1-gfp or brsv-gfp. teer was determined before and after egta treatment. pcls were washed three times with pbs to remove mucus and then infected with virus (10 5 ffu/ml) diluted in 500 μl of rpmi medium. after 2 h (bhv-1-gfp, bpiv3) or 3 h (brsv-gfp), the inoculum was removed and the slices were washed twice with pbs followed by the addition of 1 ml of rpmi medium. slices were incubated for 1-7 days in 5% co 2 at 37°c. to analyze the importance of sialic acids for bpiv3 infection, the slices were washed with pbs followed by incubation with 100 mu/pcls of neuraminidase (na) type v from clostridium perfringens (sigma/aldrich, munich) for 1 h at 37°c. after removal of na, the slices were rinsed in pbs prior to virus infection. to assess viral shedding, supernatants of apically infected ali cultures were taken post-infection over a period of 1 week. for this purpose, 100 μl aliquots of prewarmed emem were applied to the apical surface of the filter inserts and harvested after a 30 min incubation period at 37°c. for basolateral virus release, 500 μl of the basolateral medium was collected and replaced by fresh medium. supernatants were combined with 10 × viral stabilizing solution (1 m mgso 4 , 0.5 m hepes, ph 7.5), quick-frozen on dry ice and stored at −80°c. teer was determined during the course of the experiment. in the case of pcls, 100 μl of the supernatant was collected at each time point and replaced by the same amount of rpmi to avoid differences in virus concentration. for each virus, cultures from at least 3 animals were used and experiments were performed in duplicate. cells grown on membrane filters and pcls were fixed for 20 min at room-temperature with 3% paraformaldehyde in pbs. fixed cells were washed twice with 0.1 m glycine in pbs and then permeabilized with 0.2% triton x-100 diluted in pbs for 10 min. all antibodies were diluted in pbs containing 1% bovine serum albumin at room temperature. bpiv3-infected cells were stained with a polyclonal antiviral antiserum (vmrd, caprine origin) followed by a fitc-labeled secondary antibody (sigma-aldrich). cilia were stained by using a cy3-labeled monoclonal antibody against β-tubulin (sigma-aldrich). basal cells were visualized by a monoclonal mouse anti-human cd142 antibody (serotec) and a primary antibody specific for ttf-1 (clone bgx-397a mouse monoclonal, bio-genex) was used to stain pneumocytes type ii. nuclei were visualized with dapi (4′, 6′-diamidino-2-phenylindole) which was added to the apical surface of the cells and removed after incubation for 15 min (37°c) followed by three washing steps with pbs. after washing, cells were embedded in mowiol resin and photomicrographs were generated using a leica tcs sp5 aobs confocal laser scanning microscope. for image processing, adobe photoshop (version 10.0, adobe systems, usa) was used. one day prior to infection, mdbk (bpiv3, bhv-1-gfp) or vero cells (brsv-gfp) were grown to confluence in 96 well plates and then exposed to serial tenfold dilutions of virus suspensions in duplicate. for virus adsorption, cells were incubated at 37°c for 2 h (bpiv3, bhv-1-gfp) or 3 h (brsv-gfp). after inoculation, cells were overlaid with 3% methylcellulose and plaques were counted 2 (bhv-1-gfp, bpiv3) or 3 (brsv-gfp) days post-infection. in case of bpiv3, the cells were stained for virus antigen. the viral titer is indicated in foci-forming units per ml (ffu/ml). the three viruses associated with the bovine respiratory disease complex were compared for their efficiency to infect differentiated respiratory epithelial cells. for this purpose, baec were grown for about four weeks under ali conditions to build up a polarized and pseudostratified epithelium. cells were infected by bpiv3, brsv-gfp or bhv-1-gfp at an moi of 0.1 and analyzed two (bpiv3 and bhv-1-gfp) or three (brsv-gfp) days post infection (dpi) for the presence of infected cells. as described in earlier studies [7] , bpiv3 efficiently infected the respiratory epithelium. co-staining of β-tubulin indicated that ciliated cells were the target cells ( figure 1a ). on the other hand, infection by brsv-gfp was very inefficient ( figure 1b) ; only a few infected cells were detectable and those cells were identified as ciliated cells using an antibody directed against β-tubulin. only occasionally non-ciliated cells were infected by brsv-gfp. when bhv-1-gfp was applied to the apical surface, the epithelium was found to be refractory to infection ( figure 1c ). not a single infected cell was detectable at two dpi; if the incubation time was increased up to five days, only occasionally infected cells were found. the same result was obtained when the titer was enhanced tenfold and infection was monitored up to five days. only when an moi of 10 was applied, a few cells were infected and appearance of infected foci suggested that infection spread to adjacent cells (see additional file 1). infection of baec by the three bovine viruses was also evaluated by determining the kinetics of virus production ( figure 2 ). apical release of infectious bpiv3 was detected at 12 hours post infection (hpi) and increased exponentially by 24 hpi; the maximum infectivity titer of~7 × 10 5 ffu/ml was determined at 96 hpi. brsv-gfp was detectable in the supernatant only at 48 hpi and the maximum titer of 3 × 10 4 ffu/ml was reached after seven days. the apical release of infectious bhv-1-gfp was as slow as that of brsv-gfp but proceeded only to a maximum titer of 2.5 × 10 3 ffu/ml. basolateral release of all three viruses was below detection level. for any of the three viruses, we did not observe a general decrease in the transepithelial resistance values during the course of the experiment. despite some fluctuation of individual teer values post-infection and variability between individual trans-wells, apical infection of differentiated cultures did not affect the integrity of the epithelium (see additional file 2a). since brsv-gfp and bhv-1-gfp were not able to establish an efficient infection of baec when applied from the apical side, we analyzed whether they could initiate infection from the basolateral side of the cells. for this purpose, the device containing the membrane was turned upside down. under these conditions, bhv-1-gfp was able to infect baec as indicated by gfp expression after a 24 h incubation time. the efficiency of infection could be enhanced by increasing the moi tenfold ( figure 3a ). in contrast, brsv-gfp was not able to establish an infection after basolateral inoculation ( figure 3b ). the results presented above indicate that bhv-1 -but not brsv-gfp is able to infect baec from the basal side. to rule out that the pore-size of the filter membrane and the size and morphology of the virions prevented access to the basal side, we used an epithelium injury model to investigate infection. well-differentiated baec were mechanically injured with a sterile needle and subsequently infected by either bhv-1-gfp or brsv-gfp from the apical surface for two or three h. as shown in figure 4a , infection occurred exclusively within the region of injury and the underlying cells are the site of infection ( figure 4c ). further staining with anti-cd142 which is specifically expressed on bronchial epithelial basal cells [22] showed that bhv-1-gfp infected this cell type ( figure 4b ). in contrast, in case of brsv-gfp, hardly any cells at the injury site expressed gfp (data not shown). as shown above, a few infected cells were detected in the undamaged region; these cells were identified as ciliated cells by staining for β-tubulin. to further analyze bhv-1 entry into baec, egta was used to transiently open the tight junctions. cells were treated with 0.1 m egta for 10 min prior to infection with either bhv-1-gfp or brsv-gfp. in contrast to untreated cultures, teer values dropped to the baseline after egta treatment (see additional file 2b). as shown in figure 5a , pretreatment with egta enabled bhv-1-gfp entry from the apical side as indicated by gfp-positive foci distributed all over the filter-grown cells. by contrast, the disruption of the epithelial barrier had no effect on the efficiency of brsv-gfp infection or on the cell type infected by this virus. virus titration revealed that the yield of bhv-1-gfp released into the supernatant was pcls provide an alternative culture system for differentiated respiratory epithelial cells and have the advantage that they contain baec in the original setting. moreover, connective tissues and alveolar regions are present within these slices as well as immunomodulatory cells. to analyze the infection strategies of bpiv3, brsv-gfp as well as bhv-1-gfp, pcls were prepared from the lungs of 6-8 months old calves. for infection studies, only those slices were used, thatupon microscopic inspection -did not show any loss of the ciliary activity. pcls were infected with the same infectious dose (10 5 ffu/ml) of any of the three viruses. the patterns of infected cells were quite different. bpiv3 exclusively infected ciliated cells of the respiratory epithelium, whereas bhv-1-gfp-infected cells were located underneath the uppermost epithelial cell layer (figures 6 and 7a) . it is known that sialic acids serve as receptor determinants for entry of bpiv3. to determine the importance of sialic acids in the infection of differentiated respiratory epithelial cells by bpiv3, slices were treated with neuraminidase from c. perfringens prior to infection. as shown in figure 6 , desialylation almost completely abolished infection confirming that infection of differentiated respiratory epithelial cellslike that of immortalized cells (data not shown) -is sialic aciddependent. infection of the slices by bhv-1-gfp or brsv-gfp was not affected by neuraminidase treatment. staining for cd 142 revealed that basal cells, the progenitor cells of the differentiated respiratory epithelial cells, are primarily infected by bhv-1-gfp ( figure 7b ). when the incubation time was increased to one week, infection spread to the periphery of the slice ( figure 7c ). in contrast, basal cells were not targeted by brsv-gfp. the latter virus predominantly infected subepithelial cells within the peribronchiolar connective tissue at three dpi and only a low number of ciliated cells were gfp-positive ( figure 7d ). the morphologies of at least some infected cells in the submucosal figure 6 bpiv3, bhv-1-gfp and brsv-gfp infection of pcls before or after neuraminidase treatment. pcls were first incubated for 1 h with or without 100 mu/pcls neuraminidase type v and then infected with bpiv3 or bhv-1-gfp with 10 5 ffu/ml for 2 h or with brsv-gfp for 3 h. at 2 dpi, the slices were fixed and in case of bpiv3 stained for virus antigen (green). cilia were visualized by staining against β-tubulin (red). scale bar = 100 μm. area resembled those of dendritic cells ( figure 7e ). after one week of incubation, brsv-infected cells were also detected in the peripheral part of the slice comprising the alveoli ( figure 7f ). incubation with anti-ttf-1 (thyroid transcription factor −1), which stains nuclei of type ii pneumocytes revealed that both, ttf-1 positive and negative cells, were infected. stained cells were cuboidal in shape and found at the intersections of two or more alveoli whereas infected cells that were not stained by anti-ttf-1 had a flattened morphology (see additional file 3). the efficiency of infection was also determined with respect to infectious progeny virus released into the culture medium. for this purpose, supernatants were collected at different time points after infection. as shown in figure 8 , bpiv3 reached a viral titer of 2.3 × 10 3 ffu/ml after 48 h, which increased to 2 × 10 5 ffu/ml by day seven post infection (pi). infection of pcls by brsv-gfp resulted in titers in the supernatant that were hardly above the detection level. virus release of bhv-1-gfp was intermediate with titers that were 5 -to 20-fold lower than those determined for bpiv3. consistent with the differences in the amount of virus released into the supernatant, there was also a difference in the kinetics of virus release: bpiv3 was detectable in the supernatant at 12 hpi followed by bhv-1-gfp at 24 hpi; brsv-gfp was released into the supernatant not until 48 hpi. culture systems for differentiated respiratory epithelial cells are models of choice to investigate how respiratory viruses interact with the epithelial barrier when they enter the airways. they comprise cell types like ciliated cells that are important for airway function and cannot be cultured as immortalized cells. using these culture systems, it is possible to determine which of the different cell types is susceptible to infection. these results may be different from those obtained with immortalized cells. a striking example is brsv which infects almost all immortalized cells [18, 23] , whereas airway epithelial cells are rather refractory to infection ( [7] , this paper). membrane-grown respiratory epithelial cells cultured under air-liquid interface conditions undergo mucociliary differentiation to generate a pseudostratified epithelium including ciliated cells, secretory cells and progenitor cells. they allow targeted access of reagents or microorganisms to the apical and basal membrane. though we succeeded in establishing ali cultures from bovine airway epithelial cells, most studies were performed so far with human cells. establishing ali cultures for cells of different species is still challenging [24, 25] . the alternative culture system for differentiated respiratory epithelial cells, pcls, can be applied to lungs from different species [7, 8, 26, 27] . they allow analyzing infection of not only the epithelial cells but also of submucosal cells. brsv, bpiv3 as well as bhv-1 are important viral pathogens within the bovine respiratory disease complex [9, 12] . therefore, it was of interest to compare the entry strategies of these viruses. bpiv3 efficiently infected the airway epithelial cells using sialic acids on the apical surface as a receptor determinant for virus entry from the apical side. as virus egress also proceeds via the apical plasma membrane, bpiv3 -like human piv3 (hpiv3)has the characteristics of several other viruses that cause a localized infection of the respiratory tract, e.g. influenza viruses [3, 28, 29] . it remains to be further elucidated why bpiv3 preferentially targets the ciliated cells. this is a question that can be addressed in the future with culture systems for differentiated airway epithelial cells. bhv-1 showed a completely different infection pattern. when bhv-1-gfp was applied to the apical side of the well-differentiated airway epithelial cells, infected cells were detected only occasionally. recently, an explant culture system derived from the upper trachea of cows was used to analyze bhv-1 infection. single sites of infection were detected in the epithelium when the virus was applied at 10 7 tcid 50 /ml [30] . this infectious dose is about 100-fold higher than that required for efficient infection by bpiv3. thus, differentiated epithelial cells from both the proximal trachea and the bronchi are rather resistant to infection by bhv-1. successful infection required the access to the basal side of the epithelium either after opening of the tight junctions by calcium depletion or after mechanical injuring of the cell monolayer. infection studies with ali cultures indicated that the cells that are sensitive to bhv-1-gfp infection are not the differentiated epithelial cells but cells with the phenotype of basal cells, i.e. the progenitor cells of the differentiated epithelial cells. also in pcls, basal cells were found to be the primary site of infection. when the inoculation time was increased up to one week, virus was also found in the periphery of the slice, and the distribution pattern suggests direct cell-to-cell spread. this infection pattern could not be predicted from studies with polarized immortalized cells. our results most closely resemble those reported for polarized mdck cells where infection was not possible via the apical surface, but via the basolateral side after opening of the tight junctions or after injuring the cell monolayer [31, 32] . however, this cell system could not reveal the preference for the basal cell phenotype. other studies with polarized cells reported virus entry from both sides [33, 34] or preferentially via the apical membrane [35] . these conflicting reports underline the importance of infection studies with differentiated airway epithelial cells. from our results, the question arises how bhv-1 gets across the epithelial barrier. a model that has been proposed for hsv-1 suggests entry through small lesions within the epithelium [31, 32, 34, 36] . further possible explanations will be discussed below. a third infection pattern was observed when differentiated epithelial cells were infected with brsv-gfp. though brsv-gfp -like bpiv3 -preferentially infected ciliated cells, the overall efficiency of infection was much lower. whereas bpiv3 readily infected airway epithelial cells, only single brsv-infected cells were observed even when the virus was applied at an increased moi. the difference in infection efficiency was also evident from the amount of infectious virus released by the infected cultures. the titers of infectious progeny brsv-gfp were up to 600-fold lower when compared to bpiv3. in contrast to bhv-1-gfp infection, the relative resistance of the airway epithelium against brsv-gfp infection could not be overcome by opening the tight junctions or by injuring the cell monolayer. the infection pattern observed with brsv-gfp resembles that reported for hrsv. a previous study reporting the release of a substantial amount of progeny virus had applied an unusual high multiplicity of infection [37] . later on, it was reported also for hrsv that airway epithelial cells are infected with low efficiency [38, 39] . a striking feature of the brsv infection is the susceptibility of submucosal cells to infection. some of these cells have morphologies that resemble those of dendritic cells. others were located within the epithelium lining the alveolar lumen. replication of brsv within the alveolar epithelium in both type i and type ii pneumocytes has been reported in an ultrastructural analysis of brsv-infected calves [40] . staining of pcls for the presence of anti-ttf-1, which was identified as a marker for type ii pneumocytes in sheep and swine [41, 42] , demonstrated that type ii pneumocytes are susceptible to infection by brsv-gfp. alveolar cells that were negative for ttf-1 were also infected by brsv-gfp. these latter cells had a flattened morphology that is characteristic for type i pneumocytes. therefore, both type i and type ii pneumocytes appear to be susceptible to infection by brsv-gfp. unfortunately, the identity of the infected cells in the submucosal area could not be determined. immunological reagents available for bovine cells are limited and those few that were suitable for staining of pcls did not provide an unambiguous result. as differentiated airway epithelial cells are refractory to infection by brsv-gfp and bhv-1-gfp, it remains to be elucidated how these viruses get across the epithelial barrier. as mentioned above, passage through leaky sites within the epithelial monolayer is a possibility proposed for hsv-1. an alternative explanation has been suggested for measles virus. here, the major receptor, slam, is present on lymphoid cells but absent from epithelial cells. the recently identified measles receptor on epithelial cells, nectin 4, has a polarized surface distribution enabling infection via the basolateral side but not via the apical plasma membrane. therefore, it has been suggested that measles virus infection is initiated in alveolar macrophages or dendritic cells which are used as a ferry through the epithelial barrier [43, 44] . we cannot exclude that dendritic cells that uptake antigen from the airway lumen via their dendrites capture brsv and carry infection to the submucosal infection sites as well. invitro studies have demonstrated rsv infection and replication within dendritic cells [45] . also it has been suggested that the virus may persist in lymphoid or other tissues [12, 46, 47] . a further way to get across the epithelium without infecting the epithelial cells is transcytosis. this entry option has been shown for several viruses, e.g. for epstein-barr virus to get across the oral epithelium ( [48] , and references therein). there are no data available supporting transcytosis as an entry strategy for bhv-1 and brsv. an alternative explanation of the infection pattern of brsv and bhv-1 may be that the viruses enter differentiated bronchial epithelial cells without immediate initiation of replication. we do not have any evidence for this possibility, because brsv and bhv-1 infection of differentiated bronchial epithelial cells was not enhanced even when the infection time was extended to seven days. we would like to suggest another entry strategy that may apply to brsv and bhv-1. both viruses have been associated with the bovine respiratory disease complex, a multifactorial disease that involves the interplay of different factors such as stress and microbial co-infections. bacterial pathogens associated with brd are mannheimia haemolytica, pasteurella multocida or mycoplasma bovis [12, 49, 50] . infection of the epithelium by either of these microorganisms may induce a response reaction in the epithelial cells that make them susceptible to virus infection. for example, if the cells are responding by expressing suitable virus receptors that are otherwise absent from the cell surface, brsv or bhv-1 may initiate infection. thus, in the future it will be interesting to analyze the interaction of bacterial pathogens with differentiated airway epithelial cells and to determine whether bacterial infection predisposes the epithelium to virus infection. the analysis of the entry mechanism of the three bovine viruses is hampered by the fact that suitable antibodies against bovine nectin-1, the receptor for bhv-1, are not available commercially and the receptor for brsv has not been identified. however, when bacterial co-infection increases the susceptibility to infection by brsv, this approach may help to identify the receptor for brsv. also, our finding that pneumocytes are susceptible to brsvinfection, may be helpful in this respect. taken together, our data provide new insights in the airway infection by bovine respiratory viruses that may help not only to understand the pathogenicity of viruses involved in brdc but also to develop new intervention strategies. additional file 1: infection of well-differentiated baec by bhv-1-gfp at different mois. bhv-1-gfp was applied to the apical surface of ali cultures at an moi of 0.1, 1.0 or 10 for 2 h. cultures were fixed at 1 -5 dpi. virus-infected cells are shown in green. additional file 2: teer time course of infected ali cultures. cultured cells were apically infected with either of the three viruses or mock-infected and the transepithelial resistance was measured before infection, post infection and further at daily intervals (a). teer was also measured in egta-treated cultures before infection, directly after egta treatment, and at the indicated time points post-infection (b). values were corrected for the blank resistance. mean values and standard deviation of three independent cultures are shown. additional file 3: ttf-1 labeling of pcls. brsv-infected pcls were stained for ttf-1 followed by a mouse-cy3 second antibody (red). upper panels show a ttf-1 positive, infected cell typically located at the intersection of two or three alveoli. the middle panel represents an example of an infected cell with ttf-1 negative staining and a characteristic flattened shape. lower panels show ttf-1 positive but uninfected control samples. infected cells are shown in green. nuclei were stained by dapi. scale bar = 50 μm. the authors declare that they have no competing interests. epidemiology of community-acquired respiratory tract infections in adults. incidence, etiology, and impact human and avian influenza viruses target different cell types in cultures of human airway epithelium infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells isolation and air-liquid interface culture of human large airway and bronchiolar epithelial cells reactivity of equine airways -a study on precision-cut lung slices differential sensitivity of differentiated epithelial cells to respiratory viruses reveals different viral strategies of host infection differential sensitivity of well-differentiated avian respiratory epithelial cells to 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through apical surfaces of polarized epithelial cells in culture and in vivo access to nectin favors herpes simplex virus infection at the apical surface of polarized human epithelial cells disruption of adherens junctions liberates nectin-1 to serve as receptor for herpes simplex virus and pseudorabies virus entry respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology growth of respiratory syncytial virus in primary epithelial cells from the human respiratory tract in vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo ultrastructural features of alveolar lesions in induced respiratory syncytial virus pneumonia of calves sheep pox virus induces proliferation of type ii pneumocytes in the lungs immunohistochemical characterization of type ii pneumocyte proliferation after challenge with type i porcine reproductive and respiratory syndrome virus early target cells of measles virus after aerosol infection of non-human primates measles virus infection of alveolar macrophages and dendritic cells precedes spread to lymphatic organs in transgenic mice expressing human signaling lymphocytic activation molecule (slam, cd150) respiratory syncytial virus infection of monocyte-derived dendritic cells decreases their capacity to activate cd4 t cells quantitative investigation of population persistence and recurrent outbreaks of bovine respiratory syncytial virus on dairy farms persistent infection of b lymphocytes by bovine respiratory syncytial virus epstein-barr virus transcytosis through polarized oral epithelial cells immune evasion by pathogens of bovine respiratory disease complex mannheimia haemolytica and bovine respiratory disease three viruses of the bovine respiratory disease complex apply different strategies to initiate infection part of this work was performed by j.k. in partial fulfillment of the requirements for a phd from tieraerztliche hochschule hannover. we are grateful to prof. klaus conzelmann for providing recombinant brsv and dr wiebke köhl for generating brsv-gfp. we thank gyula balka for information about ttf-1 as a marker for pneumocytes. financial support was provided by a grant of deutsche forschungsgemeinschaft (sfb587, tpa1). authors' contributions jk and gh conceived and designed the experiments; jk, su and kg performed the experiments; jk, su, gk, kg and gh analyzed the data; gk contributed reagents/materials/analysis tools; jk and gh wrote the paper. all authors read and approved the final manuscript.submit your next manuscript to biomed central and take full advantage of: key: cord-001123-n2e4s7bu authors: lin, yue-zhi; yang, fei; zhang, shu-qin; sun, liu-ke; wang, xue-feng; du, cheng; zhou, jian-hua title: the soluble form of the eiav receptor encoded by an alternative splicing variant inhibits eiav infection of target cells date: 2013-11-22 journal: plos one doi: 10.1371/journal.pone.0079299 sha: doc_id: 1123 cord_uid: n2e4s7bu equine lentivirus receptor 1 (elr1) has been identified as the sole receptor for equine infectious anemia virus (eiav) and is a member of the tumor necrosis factor receptor (tnfr) superfamily. in addition to the previously described membrane-associated form of elr1, two other major alternative splicing variant mrnas were identified in equine monocyte-derived macrophages (emdms). one major spliced species (elr1-in) contained an insertion of 153 nt, which resulted in a premature stop codon situated 561 nt upstream of the predicted membrane spanning domain. the other major species (elr1-de) has a deletion of 109 nt that causes a shift of the open reading frame and generates a stop codon 312 nt downstream. because elr1-de presumably encodes a peptide of a mere 23 residues, only elr1-in was further analyzed. the expression of a soluble form of elr1 (selr1) by elr1-in was confirmed by western blot and immunofluorescence analyses. similar to elr1, the transcription level of elr1-in varied among individual horses and at different time points in the same individuals. the ratio of elr1-in mrna species to elr1 mrna was approximately 1∶2.5. pre-incubation of the recombinant selr1 with eiav significantly inhibited eiav infection in equine macrophages, the primary in vivo target cell of the virus. fetal equine dermal (fed) cells are susceptible to eiav in vitro, and the replication of eiav in fed cells transiently transfected with elr1-in was markedly reduced when compared with replication in cells transfected with the empty vector. finally, the expression levels of both forms of the eiav receptor were significantly regulated by infection with this virus. taken together, our data indicate that selr1 acts as a secreted cellular factor that inhibits eiav infection in host cells. for most retroviruses, the viral envelope binds to receptors in a ph-independent manner, suggesting that the virions can fuse directly to the cell membrane [1] . therefore, viral receptors on the cell membrane provide binding sites for the virus and are also involved in the structural modulation of viral envelopes, leading to the fusion of the cellular and viral membranes and virion entry, the first step in viral infection of target cells [2] . accordingly, studies of the role of viral receptors in the invasion of the virus are important to the development of antiviral reagents and vaccines. the equine infectious anemia virus (eiav) is a member of the genus lentivirus, family retroviridae, and its structure is the simplest out of all the known lentiviruses [3] . the receptor of eiav is equine lentivirus receptor 1 (elr1), which was identified by zhang et al. in 2005 using a functional cloning approach [4] . in contrast to most other lentiviruses, such as human immunodeficiency virus (hiv)-1, simian immunodeficiency virus (siv) and feline immunodeficiency virus (fiv), which require co-receptors for successful infection, eiav appears to depend only on a functional elr1 for the invasion of target cells. based on its sequence and structural characteristics, elr1 belongs to the tnf receptor (tnfr) superfamily [4, 5] , and many receptors of this superfamily, such as the growth factor receptor, leptin receptor and fas, also have soluble forms. soluble forms have also been identified for some immunoglobulins and chemokine receptors [6] [7] [8] . soluble receptors can be processed posttranscriptionally or posttranslationally. the release of membrane-associated forms from the cell surface contributes significantly to the formation of soluble receptors at the posttranslational level; this process is usually catalyzed by enzymes and highly is regulated. in addition, the alterative splicing of mrnas during the maturation of eukaryotic pro-mrna is another mechanism for the formation of soluble receptors. the translation of receptor mrna can be prematurely terminated due to alterative splicing, which produces receptors that lack the transmembrane and cytoplasmic domains [6, 7, 9] . much evidence has demonstrated that soluble viral receptors are functionally important for viral infections [10] [11] [12] [13] . the soluble receptors for hiv-1, ebv (epstein-barr virus) and rhinovirus are reportedly able to inhibit infection by the corresponding viruses [11, 14, 15] . another study found that the soluble form of the avian sarcoma leukosis virus subgroup a (aslv-a) receptor tva (stva) inhibited the infectivity of this virus by 90% at a low concentration (25 pm) but mediated aslv-a infection in cells lacking the receptor at a high concentration (5 nm) [16] . brindley et al. demonstrated that preincubation of eiav with the soluble ectodomain of elr1 dramatically reduced the viral infectivity on the target cells [17] . these data further implicate soluble viral receptors in the interaction between viruses and their host cells. as mentioned above, the investigation of the alternative splicing isoforms of a given receptor facilitates a better understanding of its functions. to the best of our knowledge, there are no reports on naturally expressed soluble eiav receptors. therefore, the present study had three objectives: 1) to identify alternative splicing variants for elr1, 2) to determine whether any of these variants encode soluble elr1 and 3) to characterize the roles of any soluble forms of the receptor in eiav infection. horses and related experimental protocols used in this study were approved by the institutional animal care and use committee (iacuc) of the harbin veterinary research institute (hvri), chinese academy of agricultural sciences. there were no animals scarified specifically for this study. fetal equine dermal (fed) cells and 293t cells were proliferated from the stock of the cell bank of epidemic disease examination and service center of hvri. equine monocyte-derived macrophages (mdms) were prepared from peripheral blood mononuclear cells (pbmc) that were isolated from 200-300 ml horse peripheral blood taken from the jugular vein by veterinarians. horses were infected with eiav in a previous study [19] , and the animals infected with pathogenic eiav strains were euthanized at the end of experiment or when demonstrated severe disease-associated symptoms resulting in distress by intravenous injection of pelltobarbitalum natricum (100 mg/kg of body weight, dissolved in saline) in the jugular vein by veterinarians according to protocols approved by iacuc of hvri. fetal equine dermal (fed) cells, equine monocyte-derived macrophages (mdms) and human 293t cells were used for the transfections and the expression of recombinant proteins. the cells were maintained in high-glucose dulbecco's modified eagle's medium (dmem, gibco, usa) supplemented with penicillin and streptomycin. the medium was supplemented with 10% fetal calf serum (fcs) for the fed and 293t cells. equine mdms (emdms) were enriched from whole blood as described previously [18] . eiav dlv34 (dlv34) is a cell culture-adapted eiav pathogenic strain, which was derived by 33 passages of the virulent eiav ln40 strain in donkey mdms. similar to eiav ln40 , inoculation with 10 4 tcid50 of dlv34 resulted in acute eia in all the experimentally infected horses [19] . the bdna is a sandwich nucleic acid hybridization assay, in which target mrna molecules are captured through cooperative hybridization of multiple probes. unlike pcr, in which a region of the intended target is exponentially amplified in order to generate detectable signal, in bdna assays only signals are amplified. bdna assays are consequently not susceptible to contamination risks associated with pcr-based assays [20, 21] . equine mdms were cultured and elr1-in mrna was extracted and quantified using bdna assay as described previously [18] . samples were read by a luminex 200 (molecular devices, usa) and all data were analyzed using the luminex is2.3 program. an acquisition gate between 5000 and 20,000 was set to exclude any doublet events and ensure that only single microspheres were measured. the construction of vectors for the expression of ha-and gfp-tagged elr1 and elr1-in the elr1 cdna was cloned by reverse transcription pcr (rt-pcr) using equine mdm with primers based on the published sequence of equine elr1 [4] . the green fluorescent protein (gfp) gene was subcloned from the pgfp-n3 vector (clontech, usa), and the gfp-elr1 fusion gene fragment was amplified by overlapping pcr. a human influenza hemagglutinin (ha) fragment was linked to the 39 of the elr1 cdna for the detection of the expressed recombinant elr1. the elr1-ha fusion fragment was amplified using the primers 59-gcgaattc-ttagcacagggacgcgtagtccgggacgtcgtatggg-taggcctggcagctct-39 for ha and 59-gcgaattctt-agcacagggacgcgt agtccgggacgtcgtatgggt-aggtctgaaggac-39 for elr1. both of these pcr products were digested with ecori and hindiii and inserted into the pcdna3.1 (+) vector (invitrogen, usa) at the same cloning sites. vectors for the expression of gfp-and ha-tagged elr1-in were constructed using the same procedures. plasmids containing only the gfp or ha gene fragment were used as the controls for gfp and ha expression, respectively. the translation of selr1, i.e., the protein predicted to be encoded by elr1-in, was confirmed using western blot and confocal imaging. briefly, 293t cells were transfected with plasmids expressing ha-tagged elr1 and elr1-in using polyjet transfection reagents (roche, usa). the expression of membraneassociated or soluble elr1 was examined by western blot using a monoclonal antibody against the ha tag (sigma, usa) fused to the receptor at the c-terminus. protein bands reacting with the ha antibody were visualized using a horseradish peroxidase (hrp)-conjugated goat anti-mouse antibody (sigma, usa). to perform confocal imaging, 293t cells seeded on coverslips were transfected with the ha-tagged selr1 or elr1 expression vectors. after 72 h of incubation, transfected cells were fixed with 4% paraformaldehyde in pbs for 30 min and permeabilized with 0.1% triton x-100 for 15 min. cells were blocked with pbs/ fish gelatin (5%) for 2 h, reacted with anti-ha monoclonal ab for 1 h and then incubated with an anti-mouse igg (whole molecule)-fluorescein isothiocyanate (fitc)-labeled antibody (f2012, sigma). cells were washed with pbs for three times and then stained with 4,6-diamidino-2-phenylindole (dapi, sigma) for 30 min and examined using a leica tcs sp5 confocal system (leica microsystems, germany). appropriate igg isotype controls were included to rule out non-specific binding. in addition, a 3d live cell imaging system (ultraview vox, perkinelmer, usa) was applied to observe the dynamically distribution of the selr1 and elr1 that fused to gfp at the n-terminus after being transiently expressed in 293t cells. an in vitro eiav infection inhibition assay was applied to examine the ability of selr1 to interact with eiav. specifically, selr1 was transiently expressed in 293t cells by transfection of the expression plasmid pcdna3.1-elr1-in. the culture medium that contained the secreted selr1 was collected at 48 h after transfection and incubated with 10 3 tcid50 of the dlv34 strain at 4uc for 30 min. the viruses that were pre-incubated with selr1 were then added to emdms in 6-well plates. cell-free culture medium was collected at 1, 3, 5 and 7 days post-infection (dpi) and measured for viral reverse transcriptase (rt) activity using a reverse transcriptase assay colorimetric kit (roche, switzerland). the dlv34 strain incubated with the culture medium of cells transfected with the empty pcdna3.1 vector was used as the control for non-specific bindings. the effect of selr1 overexpression on the eiav infection of target cells selr1 was transiently overexpressed in fed cells by transfection with pcdna3.1-elr1-in using polyjet transfection reagents. the fed cells were infected with 100 ml of appropriately diluted dlv34, 24 h after the transfection. the virion release into the medium was quantified at 2, 4, 6 and 8 dpi by measuring the viral rt activity. the mrna levels of elr1 and elr1-in in equine mdms infected with eiav were determined using quantitative real-time pcr. cultivated emdms were infected with 0.1 plaque-forming units (pfu) of dlv34 and collected at 4, 8 and 12 hours and 3 and 5 dpi. mrna samples were prepared from these cells and reverse transcribed into cdna using m-mlv reverse transcriptase (invitrogen, usa), and the expression levels of elr and elr1-in were then quantified using real-time pcr with a sybr green amplification and detection kit (qiagen, germany). the expression levels of the elr1 transcripts were normalized to that of the b-actin mrna. plasmids containing either the elr1 or elr1-in cdna were constructed to establish standard curves for copy number determination. the elr1 and elr1-in fragments were amplified by pcr, cloned into a pmd18-t vector and verified by dna sequencing. the standard plasmid was serially diluted from 1610 8 to 1610 2 copies/ml and then measured using real-time pcr. for each reaction, 5 ml of standard plasmid or cdna sample was added to a 20 ml reaction mixture containing 12.5 ml sybr green pcr master mix and 3 mm of each primer. the amplification protocol was as follows: 95uc for 10 min, followed by 35 cycles at 95uc for 30 s, 60uc for 30 s and 65uc for 30 s; the final extension was at 68uc for 10 min. all the samples were measured twice. the elr1 and elr1-in mrna levels were calculated by dividing the receptor copy number by the b-actin copy number. statistical analysis and data presentation were performed using student's t-test (two-tailed, confidence intervals of 95%). and graphpad prism version 4.0 (graphpad software, usa) programs, respectively. a probability value less than 0.05 was considered statistically significant. the identification of alternative splicing variants for elr1 the elr1 cdna was amplified from mrna templates extracted from emdms by reverse-transcription pcr using primers designed based on the sequence published in genbank. polyacrylamide gel electrophoresis revealed three bands migrating at approximately 1000 bp (fig. 1a) , and subcloning of the amplified fragments further confirmed the existence of three elr1 cdna fragments with different molecular masses (fig. 1b) . nucleotide sequencing revealed two other species of elr1: one containing an insertion of a 153 nucleotide (nt) fragment of intron 6 (elr1-in) and another with a deletion of 109 nt (elr1-de). to avoid the contamination of introns of chromosomal dna, the elr1-in mrna samples were pre-treated with dnase to remove any contaminating chromosomal dna and then pcr amplified with a pair of primers, one of which targeted the insert. the 125 bp elr1 cdna fragment was amplified from both mrna samples with or without dnase pre-treatment, indicating that elr1-in originated from the mrna. as a control, we confirmed that a 501 bp fragment from intron 3 of the equine mhc-i gene was amplified from the dna preparation without dnase treatment but not a preparation that was pre-incubated with the nuclease (data not shown). in addition, to exclude the possible artificial effect resulting in the elr1-in molecule during reverse transcription and pcr amplification, the presence of elr1-in mrna was further measured by a bdna assay, which detects specific mrna species directly from total rna preparations by hybridizing with signalamplified probes specific for the inserted sequence without being revers transcribed and amplified. as shown in fig. 1c , the average light unit of elr1-in mrna was 1,594 while that of the blank control was 53.5. to further investigate the status of the elr1 transcript variants in vivo, the variants were examined by cloning and sequencing the pcr-amplified specific cdna fragments from the pbmcs of three horses collected at different time points. all three major variants of elr1 mrna were found in all the horses and at all the time points examined. however, the ratios of each isoform were varied among the different horses and at the different sampling times. among the total 126 clones analyzed, the elr1 species was predominant (83/126), followed by elr1-in (31/126) and elr1-de (12/126) ( fig. 2a) . furthermore, additional isoforms with insertions or deletions were identified. among the 31 clones of elr1-in, 29 contained a fragment of intron 6, and the other two contained a fragment of intron 2 or intron 7. in addition, five types of deletions at different sites of the coding region were detected (fig. 2b) . all the insertions and deletions resulted in a shift of the open reading frame (orf). for the major form of elr1-in, the inclusion of a 153 nt fragment of intron 6 created an isoform with five different amino acid residues and then a premature stop codon 18 residues upstream of the predicted transmembrane domain (fig. 2c) . the cdna of elr1-de, which lacked exon 3, contained two orfs; the first encoded a peptide of only 27 residues, and the second encoded a receptor lacking the eiavbinding crd1 (fig. 2c) . because both of these predicted proteins were considered not to function as eiav receptors, they were not further evaluated in the present study. the receptor encoded by elr1-in was predicted to be a truncated form lacking the transmembrane domain and the cytoplasmic domain and, therefore, was presumed to be a soluble form of the receptor. the expression of soluble elr1 (selr1) was examined by western blot, flow cytometry and confocal microscopy. an selr1-ha fusion protein was overexpressed in 293t cells, and the 26 kda selr1-ha protein was detected in both the cell lysate and the culture medium by western blot using an anti-ha antibody. as a control, membrane-associated elr1-ha was detected only in the cell lysate (fig. 3a) . in addition, both selr1 and elr1 were transiently overexpressed in 293t cells with green fluorescence protein (gfp) fused at the n-terminus. flow cytometry analysis showed that, although the transfection efficiency was similar for both of the transfected cell populations, the fluorescence intensity of the gfp-selr1-expressing cells was markedly weaker than that of the gfp-elr1expressing cells, indicating a dramatic reduction in the intracellular accumulation of gfp-selr1 relative to gfp-elr1 (fig. s1 ). the aforementioned experiment suggested that the eiav receptor encoded by elr1-in was expressed as a soluble form of the receptor. to further examine the dynamic cellular distribution of selr1 and compare with that of elr1, a live cell imaging of the dynamic expression and distribution of the gfp-elr1 and gfp-selr1 was taken. as shown in fig. 3b , the membrane-associated gfp-elr1 was accumulated as a few large spots in the cell plasma at the early phase post transfection (fig. 3b) . the fluorescence extended and covered to the whole cell surface and plasma thereafter (intermediate phase). at the late phase of cell life, the intracellular fluorescence was seen as bands and islands, which was considered the remaining membrane-bound elr1 after the damage of plasma membrane. on the other hand, the transmembrane domain-free gfp-selr1 was observed as diffused fluorescence, distributed like donuts at the early phase, evenly covered to the whole cell at the intermediate phase and gradually attenuated to weak and faint images at the late phase. at certain time points, the leak of fluorescent content from some cells could even be observed (see the video of 3d live cell image in movie s1, e.g. at the recombinant selr1 was expressed as a fusion protein with an ha tag at the c-terminus in 293t cells, and its presence in the cell lysate or in the culture medium was examined by western blot using an anti-ha antibody. b-actin was applied as the internal control protein. (b) live cell imaging analysis of elr1 and selr1 expression in hek 293t cells. these two forms of receptor were expressed as proteins with a gfp fused at the nterminus of each. the three images of selr1 were enhanced at a higher level than elr1. (c) cellular location of elr1 and selr-1. ha-tagged elr1 and selr1 were expressed in 293t cells for 72 h. cells were then examined by confocal microscopy after being fixed, stained with dapi for the nucleus (blue) and dii for the cell membrane (red) and reacted with an anti-ha antibody and a fitc-labeled anti-mouse igg (green). appropriate igg isotype controls were included to rule out non-specific binding. doi:10.1371/journal.pone.0079299.g003 05:00:00). these images vividly demonstrated the behaviors of gfp-selr1 as a soluble protein. the cellular location of elr1 and selr1 was also detected by confocal microscopy. these two forms of eiav receptor were expressed as ha-tagged proteins in 293t cells and were detected by an ha monoclonal antibody and visualized by a fitc-labeled anti-mouse antibody. as displayed in fig. 3c , the elr1-ha was mostly detected on the cell membrane, so as the selr1-ha, but at a much lower level. combined with all the results submitted in fig. 3 and supplementary materials, it indicates that selr1 is expressed as a soluble protein and can be secreted out of the cell. however, some molecules of this soluble form of receptor are associated with the cell surface membrane. the ability of selr1 to inhibit eiav infection on target cells is the most important characteristic of the soluble receptor studied here. the recombinant selr1 was overexpressed in 293t cells, and eiav preparations pre-incubated with either the culture medium of selr1-expressing cells or the culture medium of cells transfected with the empty pcdna3.1 vector were used to infect emdms. the replication of the infected eiav was then examined by measuring the viral rt activity. as shown in fig. 4 , eiav incubated with the medium of pcdna3.1-transfected cells and the untreated virus replicated at similar rates. in contrast, the virus that was pre-incubated with medium from selr1-expressing cells showed a dramatically retarded replication that was characterized by delayed detectable rt activity and low rates of increase. this result strongly indicated that overexpressed recombinant selr1 was able to inhibit eiav infection in vitro, probably by competing with the membrane-associated receptor for the binding of virions on target cells. this result also suggests that the receptor encoded by elr1-in was a secreted protein. because selr1 interacted with eiav in culture medium, significantly blocking the subsequent infection of target cells by the selr1-preincubated virus, it was logical to examine whether the expression of the soluble receptor by the target cells interferes with viral infection. thus, the soluble form of elr1, tagged with ha, was overexpressed in fed cells, a major target of eiav in vitro, by transfection with the selr1 expression vector pcdna3.1-elr1-in. the cells were then infected with eiav 24 h after the transfection. the results revealed that eiav replicated at a significantly slower rate in the fed cells transfected with the selr1 expression vector than in the cells transfected with the empty vector at 6 and 8 dpi (p,0.05, see fig. 5a ), indicating the inhibitory effect of selr1 expressed in the target cells upon eiav infection. in addition, the presence of recombinant selr1 in the culture medium was confirmed by western blot using an anti-ha antibody. there was no b-actin detected in the medium, which ruled out the possibility that these soluble receptor molecules were leaked from broken cells (fig. 5b ). as shown in fig. 2 , the level of elr1 expression varied in different hosts and at different detection times. thus, we tested the effect of eiav infection on the expression of the membraneassociated and soluble forms of elr1. the elr1 and selr1 mrnas were quantified in emdms infected with eiav for 4 hours to 5 days using sybr green real-time pcr with b-actin as the internal control. the fold changes of elr1 and selr1 mrna (elr1-in) after eiav infection at each time point are shown in fig. 6 , with the fold changes of the mrna levels in mock-infected cells as the baseline (indicated as 1). both the elr1 and elr1-in levels showed no apparent change or a slight decrease during the 4 to 12 h post-infection. however, a notable increase (approximately 1.5-fold, p,0.05) was observed for the elr1 and selr1 mrnas in cells infected with eiav for 3 and 5 d when compared with the untreated controls. alternative splicing is an important posttranscriptional regulation mechanism in eukaryotic cells. most genes in eukaryotes are alternatively spliced into several forms, and the percentage of these genes can be as high as 93% in humans [22] . through such a mechanism, multiple proteins with different functions can be produced from a single gene, thus providing an opportunity for adaptation to different environments [23] . multiple alternative splicing variants of elr were identified in this study for the first time. the predominant isoform that produces a functional elr1 is a species of 942 bp containing exons 1-9. the other two major isoforms include elr1-in, which contains an insertion of a fragment of intron 6, and elr-de, in which exon 3 is deleted following splicing. these three major forms of spliced mrna exhibit distinct expression levels and ratios in different individuals and at different time points. the inserted fragment in elr1-in was determined to be a fragment of an intron, and intron retention is considered to occur in unprocessed or incompletely spliced pre-mrna [24, 25] . it is known that abnormal transcript variants, such as intron retentions, are occasionally generated in cells but normally do not exist long enough to have a functional impact. in addition, it has been reported that, although intron retention events are common in human genes, their frequency relative to the dominant transcript is the culture medium, containing secreted selr1, was incubated with an eiav strain dlv34 at 4uc for 30 min. virus that had been pre-incubated with selr1 was used to infect emdms in 6-well plates. dlv34 pre-incubated with medium from cells that had been transfected with the empty vector pcdna3.1 or untransfected cells were also used to infect emdms as controls. eiav replication was monitored by measuring viral rt activity at 1, 3, 5 and 7 days post infection (dpi). the data shown represent the means and standard errors of the means from three separate experiments. *p,0.05, compared with empty vector transfected cells. doi:10.1371/journal.pone.0079299.g004 generally low [22, 25] . our results demonstrated that elr1-in differed from a typical intron retention transcript: it contained a fragment of an intron and appeared with a high frequency compared with the predominant transcript of elr1 (approximately 25%). elr1-in was consistently detected in different individuals at different times, and it was translated into a functional soluble elr1 when the recombinant molecule was expressed. in addition, we did not find the typical ''gt/ag'' and ''gc/ag'' splicing sites in these spliced elr1 variants. however, due to the absence of a sufficient equine alternative splicing database, the splicing model of elr1-in remains unclear [24] . the protein encoded by elr1-in was confirmed to be a soluble receptor by several different approaches, including western blot, live cell imaging, confocal microscopy and flow cytometry, which demonstrated that most of the receptor molecules were secreted out of the cell after being expressed. however, 3d live cell imaging and confocal microscopy showed that a small portion of selr1 associated with the cell surface, although the fluorescent signal was much weaker than that of elr1, the membrane-associated form of eiav receptor. because the predicted hydrophobic transmembrane domain is absent in the selr1, the association of this soluble protein on cell membrane is mediated by the glycoside chains, like that observed for the eiav gp90 surface protein [4, 5] . because soluble viral receptors containing virus-binding motif(s) can be used as valuable tools for evaluating viral entrance into target cells, these molecules have played important roles in studies of virus-host interactions [26, 27] . it was reported that the coincubation of soluble cd4 with hiv-1 envelope protein resulted in the dissociation of the gp120 surface protein and gp45 transmembrane protein [6, 28] . cd4 is also the cellular receptor of hiv-1, hiv-2 and simian immunodeficiency virus sivmac and sivagm. the soluble form of cd4 (scd4) can block hiv-1 and hiv-2 infection in human lymphoma cell lines, but it enhances sivagm infection in these cell lines by 10 to 100-fold [29] . a similar augmenting effect of soluble viral receptors on the invasion of viruses was also observed in studies on herpes simplex virus (hsv) [30, 31] . in addition, a study on jaagsiekte retrovirus (jsrv) found that although the soluble form of its receptor, hyal2 (shyal2), did not mediate the entrance of a jsrv-pseudotype retroviral vector into cells lacking the integrated hyal2, this purified soluble protein significantly inhibited the infection of the pseudotyped vector in the target cells of jsrv [13] . moreover, some soluble receptors in the plasma can be considered markers of disease progression, such as soluble tnf receptor (stnfr) ii and soluble il-2 and il-6 receptors in hiv-1-infected patients [10, 14, 32] . therefore, soluble receptors play important roles in the interactions between viruses and hosts [33] [34] [35] [36] [37] . studies on the mechanisms regulating the formation of soluble viral receptors should lead to a better understanding of the cellular responses to invading viruses. in this study, the soluble form of the eiav receptor selr1 markedly inhibited eiav infection of its target cells when examined by in vitro competition studies, and this inhibitory effect appeared to increase with increasing infection time. additionally, the expression levels of elr1 and its alternative splicing variants were regulated by eiav infection. in a separate study on the restriction of eiav superinfection, we found that the up-regulation of selr1 induced by the initially infected viral strain was correlated with protection against subsequent viral infection (unpublished data). at present, it remains unclear whether the inhibitory effect of selr1 on eiav infection is based on the competition of the binding of the virus to membraneassociated elr1 or on the polymerization with eir1, which may trigger or may reduce signal transduction of membrane-associated receptors, therefore interfering with the expression of downstream cellular proteins [6] . however, our data indicate that selr1 is not merely a truncated product of a spliced elr1 transcript but is a functional protein with important biological roles. elr1 is a member of the tnfr superfamily, which is known to have important roles in regulating immune responses and inducing pathological outcomes [4, 38] . previous studies have shown that plasma tnfa concentration is positively correlated with the clinical symptoms of eiav-infected horses [18] . it was also reported that stnfr was significantly elevated in patients with hiv-1 infection [10] . because elr1 acts as both the receptor of eiav and a member of tnfr family, the finding that selr1 is regulatable and can compete its membrane-associated prototype form implicates the involvement of this soluble protein in both of the viral infectivity and the host response, especially that tnfa is closely related with eia symptoms. the possible role of selr1 in elr1 signaling is an interesting question to be explored. the results of this study indicate that the ability to inhibit eiav infection of target cells in vitro may be related to reduced viral entry into host cells in vivo. the regulated expression levels of both the soluble and membrane-associated forms of elr1 by eiav reveal the possible role of selr1 in the interaction between virus and host. therefore, selr1 may be considered a secreted cellular factor that regulates susceptibility to eiav and the pathological and immunological responses of the host. furthermore, the identification of elr1-in and other alternative splicing variants of elr1 suggests additional approaches for further studies on the pathogenesis and immunogenicity of eiav infection. figure s1 recombinant selr1 was expressed in 293t cells as a fusion protein with a gfp tag linked at the nterminus. the percentage of gfp-expressing cells and the fluorescence intensity of selr1-gfp in cells were examined by flow cytometry. the gfp-tagged elr1 was also expressed and analyzed in parallel in the experiments shown in this figure. all the experiments were performed for three times, and a representative result is shown. movie s1 a 3d live cell imaging of the dynamic expression and distribution of the gfp-elr1 and gfp-selr1 was taken to examine the dynamic cellular distribution of selr1 and compare with that of elr1. the images of selr1 were enhanced at a higher level than elr1. 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processing and its regulation: a genomic perspective alternative splicing in human transcriptome: functional and structural influence on proteins selecting for functional alternative splices in ests an upstream ag determines whether a downstream ag is selected during catalytic step ii of splicing widespread production of novel soluble protein isoforms by alternative splicing removal of transmembrane anchoring domains identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants soluble receptor-induced retroviral infection of receptor-deficient cells soluble cd4 enhances simian immunodeficiency virus sivagm infection enhanced resistance to herpes simplex virus type 1 infection in transgenic mice expressing a soluble form of herpesvirus entry mediator novel, soluble isoform of the herpes simplex virus (hsv) receptor nectin1 (or prr1-higr-hvec) modulates positively and negatively susceptibility to hsv infection soluble il-2 receptor serum levels-a marker for disease progression in patients with hiv-1 infection soluble interleukin-6 receptor induces motor stereotypies and co-localizes with gp130 in regions linked to cortico-striato-thalamo-cortical circuits soluble il-2ralpha (scd25) exacerbates autoimmunity and enhances the development of th17 responses in mice splicing fidelity, enhancers, and disease levels of soluble endothelial protein c receptor are associated with cd4+ changes in maraviroc-treated hiv-infected patients synergistic effects of soluble pd-1 and il-21 on antitumor immunity against h22 murine hepatocellular carcinoma tnf receptor (tnfr)-associated factor (traf) 3 serves as an inhibitor of traf2/5-mediated activation of the noncanonical nf-kappab pathway by traf-binding tnfrs key: cord-338811-2bi2edcw authors: lennemann, nicholas j.; evans, azia s.; coyne, carolyn b. title: imaging-based reporter systems to define cvb-induced membrane remodeling in living cells date: 2020-09-25 journal: viruses doi: 10.3390/v12101074 sha: doc_id: 338811 cord_uid: 2bi2edcw enteroviruses manipulate host membranes to form replication organelles, which concentrate viral and host factors to allow for efficient replication. however, this process has not been well-studied in living cells throughout the course of infection. to define the dynamic process of enterovirus membrane remodeling of major secretory pathway organelles, we have developed plasmid-based reporter systems that utilize viral protease-dependent release of a nuclear-localized fluorescent protein from the endoplasmic reticulum (er) membrane during infection, while retaining organelle-specific fluorescent protein markers such as the er and golgi. this system thus allows for the monitoring of organelle-specific changes induced by infection in real-time. using long-term time-lapse imaging of living cells infected with coxsackievirus b3 (cvb), we detected reporter translocation to the nucleus beginning ~4 h post-infection, which correlated with a loss of golgi integrity and a collapse of the peripheral er. lastly, we applied our system to study the effects of a calcium channel inhibitor, 2apb, on virus-induced manipulation of host membranes. we found that 2apb treatment had no effect on the kinetics of infection or the percentage of infected cells. however, we observed aberrant er structures in cvb-infected cells treated with 2apb and a significant decrease in viral-dependent cell lysis, which corresponded with a decrease in extracellular virus titers. thus, our system provides a tractable platform to monitor the effects of inhibitors, gene silencing, and/or gene editing on viral manipulation of host membranes, which can help determine the mechanism of action for antivirals. positive-strand rna viruses represent a large group of viruses that are responsible for the development of severe disease manifestations worldwide. enteroviruses, including coxsackievirus b3 (cvb) and enterovirus 71 (ev71), are small, non-enveloped, positive-strand rna viruses. infection by these viruses can lead to the development of severe disease, including acute flaccid paralysis, meningitis, and encephalitis [1] [2] [3] [4] [5] [6] . currently, there are no antivirals and vaccines are only available for enterovirus 71 and poliovirus. thus, a better understanding of the interactions of these viruses with the host cell can aid in the development of anti-enterovirus small molecule therapeutics. all positive-strand rna viruses manipulate host cell membranes to form membranous structures, termed replication organelles (ros), that concentrate viral and host factors to allow for efficient genome replication [7] [8] [9] [10] . enteroviruses induce extensive remodeling of the host secretory pathway during infection, which results in the accumulation of large clusters of vesicular structures in the cytoplasm that harbor viral replication proteins [11] [12] [13] [14] . previous studies have shown that at early stages of enterovirus infection, ros are observed in contact with the endoplasmic reticulum (er) membrane [10, 15] . as infection progresses, the accumulation of viral nonstructural proteins 3a and 2b leads to an inhibition of secretory trafficking, resulting in the dispersal of the golgi complex [14, [16] [17] [18] [19] [20] . the infection eventually leads to the release of progeny virion through disruption of the plasma membrane or non-lytic release [21] . while these events have been well-studied using traditional imaging techniques on fixed samples, the single-cell kinetics have not been well defined. imaging of virus-infected cells typically involves taking still images of fluorescently labeled fixed samples. while these methods are widely used and highly practical, there are a number of limitations. the choice of fixation and permeabilization reagents can have a significant impact on the outcome of immunofluorescence staining [22] [23] [24] . additionally, imaging of fixed samples only allows for 'snapshots' of different cells at various time points, but infection is a dynamic process. thus, a large sample size is needed to accurately confirm observed phenotypes. to overcome these limitations, live-cell imaging has been used in combination with recombinant viruses encoding fluorescent reporter proteins, such as gfp. these recombinant viruses are highly beneficial for many assays, including high-throughput screens. however, the insertion of a fluorescent protein open reading frame in small positive-strand rna viruses, including enteroviruses and flaviviruses, can dramatically attenuate viral progeny [14, 25, 26] , thereby limiting the scope of assays for which these viruses can provide accurate information. previous reports have overcome these limitations by developing plasmid-based reporters to detect hepaci-and flavivirus infections [27] [28] [29] . these reporters rely on viral protease-dependent cleavage to allow for the translocation of a fluorescent protein to the nucleus from the mitochondria [27] or the er [28, 29] . however, these reporters only detect virus infection and do not allow for the characterization of virus-induced remodeling of intracellular host cell structures without the introduction of multiple fluorescent protein marker expression vectors. thus, we sought to develop plasmid-based multipartite reporters for live-cell imaging that allow for the visualization of host cell organelles and enterovirus infection. in this report, we describe the characterization of enterovirus reporter constructs that allow for the visualization of the er and golgi complex throughout infection. using long-term time-lapse imaging over the complete course of infection, we define the real-time remodeling of host secretory pathway organelles during cvb infection. hela cells were cultured in modified eagle's medium supplemented with 5% fetal bovine serum, 1× non-essential amino acids, and 1% penicillin/streptomycin. u2os osteosarcoma cells were cultured in dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. u2os cells were transfected using xtremegenehp (roche, mannheim, germany), and selected and maintained in 15 µg/ml and 2.5 µg/ml blasticidin s hcl (research products international corp, mount prospect, il, usa), respectively. heterogeneous populations of stable cells were propagated and used for experiments. all enteroviruses were propagated in hela cells and stocks were prepared and titrated by plaque assay, as previously described [30] . plasmids expressing myc-tagged wild-type and catalytically inactivated cvb 3c pro have previously been described [31, 32] . all primer sequences for cloning are listed in supplementary materials table s1 . reper: a pcr fragment corresponding to amino acids 280-368 of the poliovirus receptor, which includes the type i transmembrane region, was amplified with flanking kpni and ecori restriction sites from cdna and cloned into pcdna3.1-v5-his topo ta vector. the er-localized mcherry-kdel (a gift from gia voeltz, university of colorado), which includes the 5 bip signal sequence and a 3 flavivirus octapeptide host signal peptidase cleavage sequence (lvnslvta), was amplified and inserted upstream of the pvr fragment at hindiii and kpni sites. next, an enterovirus 3c pro cleavage sequence (leaefq↓gppk) flanked by ecori and bamhi sites was inserted with an amplified gfp with the c-myc nuclear localization sequence (paakrvkld) flanked by bamhi and xhoi sites into the vector. lastly, the neomycin resistance gene was replaced with a blasticidin resistance gene using gibson assembly (neb, ipswich, ma, usa). to generate the reper mutant that is unable to be cleaved by 3c pro , the protease recognition-gfp-nls fragment was amplified and ligated into reper at ecori-xbai sites. repor: the gfp-nls sequence in reper was replaced with an ebfp2-nls sequence, ebfp2-nucleus-7 was a gift from michael davidson (addgene plasmid # 55249). a fluorescent golgi marker, memerald-golgi-7 (a gift from michael davidson, addgene plasmid # 54108), flanked by hindiii and ecori was produced by pcr. next, the emcv ires sequence flanked by ecori and noti was amplified. the new reper cassette containing ebfp2-nls flanked by noti and xbai was amplified and ligated with memerald-golgi-7 and emcv-ires amplicons into pcdna3.1 reper plasmid at the hindiii and xhoi. rabbit polyclonal antibodies against c-myc (a-14) and gfp (fl) were purchased from santa cruz biotechnology. rabbit polyclonal antibodies against gapdh were purchased from proteintech. mouse monoclonal antibodies against vinculin, clone hvin-1, were purchased from sigma aldrich (st. louis, mo, usa). mouse monoclonal antibodies against enterovirus vp1 (ncl-entero) were purchased from novocastra (buffalo grove, il, usa). xtremegene hp plasmid transfection reagent was purchased from sigma aldrich. brefeldin a (invitrogen, carlsbad, ca, usa) was dissolved in dmso and used at a final concentration of 5 µg/ml. also, 2-aminoethoxydiphenylborane (2apb, sigma aldrich) was diluted in dmso and used at a final concentration of 100 µm. cvb growth kinetics were determined using u2os cells transfected with the indicated plasmids. u2os cells were bound with cvb (100 pfu/cell) for 1 h at 4 • c, washed ×3 in pbs, and placed at 37 • c. at the indicated time, samples were collected from infected cells and used for plaque assays to determine the titer of the extracellular virus. inhibition of infection using 2apb was performed in u2os cells. cells were treated with 2apb (100 µm) for 1 h at 37 • c, cvb (50 pfu/cell) was absorbed for 1 h in the presence of 2apb at room temperature with gentle rocking, washed three times, and replaced with growth media containing 2apb for 10 h. supernatants were collected to determine the extracellular virus titer by plaque assay. u2os cells or hela cells were transfected with the indicated plasmids using xtremegenehp (roche). when specified, hela and u2os cells were infected with the indicated virus for 7 h. cell lysates were prepared on ice in cold ripa buffer (millipore, 50 mm tris-hcl, ph7.4, 1% np-40, 0.25% sodium deoxycholate, 150 mm nacl, 1 mm edta) supplemented with 1× pierce protease inhibitor cocktail (aebsf, aprotinin, bestatin, e64, leupeptin, pepstatin a). lysates were clarified by centrifugation at 13,000× g for 15 min at 4 • c. lysates (10-30 µg) were separated on 4-20% tgx pre-cast gels (bio-rad laboratories, hercules, ca, usa) and transferred to nitrocellulose membranes. membranes were blocked in pbs + 10% nonfat milk for 30 min prior to probing with primary and ir-dye conjugated secondary antibodies (li-cor biosciences, lincoln, ne, usa) in pbs-t + 5% nonfat milk. immunoblots were visualized using an odyssey clx infrared imaging system (li-cor biosciences). quantification was performed using imagestudio (li-cor biosciences). u2os cells were transfected with reper plasmid using xtremegenehp (roche). cells were cultured in chamber slides (millipore, burlington, ma, usa) and infected with cvb (100 pfu/cell) for 7 h. cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% triton x-100. monolayers were washed, incubated with anti-vp1 primary antibody, washed, incubated with secondary antibody conjugated to alexa fluor-633 (invitrogen), washed, and mounted with vectashield containing 4 ,6-diamidino-2-phenylindole (dapi; vector laboratories, burlingame, ca, usa). when indicated, samples were incubated with primary antibodies for 1 h at room temperature, washed, incubated with alexa fluor conjugated secondary antibodies for 30 min, and mounted using vectashield (vector laboratories). images were captured using a zeiss lsm 710 confocal microscope (zeiss, oberkochen, germany). u2os cells stably expressing reper or repor were plated into 35 mm dishes with a 14 mm glass no. 1.5 coverslip (mattek corporation, ashland, ma, usa). live-cell imaging was performed as previously described [33, 34] . briefly, dishes were placed in a 37 • c, co 2 -controlled incubator positioned over a motorized, inverted fluorescent microscope to allow for long-term time-lapse imaging (vivaview fl; olympus, tokyo, japan). between 5 and 10 stage positions were selected for each sample. cvb (300 pfu/cell) was added to cells and images were captured every 15-20 min for 16-18 h. image series were cropped using imagej software (nih, bethesda, ma, usa) and multi-panel movies were rendered using photoshop cc 2017 (adobe, san jose, ca, usa). the plot profile tool in imagej was used to perform line plot analyses on the red, green, and blue channels of confocal images. data was plotted and the area under the curve was calculated for the area corresponding to the nucleus using prism 8 software (graphpad, san diego, ca, usa). imagej was used to determine the fluorescence intensity of the whole cell and nuclear, gfp and mcherry signals for individual cells. the cytoplasmic fluorescence intensity was calculated by subtracting the gfp value from the whole cell value. due to variable fluorescence intensity between cells, we normalized nuclear fluorescence intensity to the cytoplasmic intensity. the ratio of gfp to mcherry was then calculated for each cell to determine if infection changed nuclear gfp fluorescence compared to mcherry. in order to account for changes in fluorescent signal throughout live-cell imaging, due to debris moving into the field of view, the fluorescent signal was normalized to an internal control. imagej was used to measure the mean signal intensities of a circular area within the nucleus and in er-dense microtubule-organizing center region for individual cells from every image series frame. infection was then calculated as the ratio between the nuclear and perinuclear signals. nonlinear regression curve analysis in prism 8 (graphpad) was used to determine the time at which 50% of the maximum infection signal was observed for individual infected cells. peripheral er integrity was determined by quantifying the ratio of peripheral er signal to dense er sheet-like signal. images were thresholded to exclude the fine er structures in the cell periphery. the area of the nucleus was subtracted from the area of the thresholded region to calculate the er sheet area. next, a boundary was drawn around the perimeter of the er network and the area was measured. the areas of the nucleus and er sheets were subtracted from this value to calculate the peripheral er area. the ratio of peripheral er to er sheet area was used to quantify peripheral er integrity. these measurements were performed on individual cells from every image series frame using the same thresholding values. golgi dispersal was measured by quantifying golgi area, which was performed similarly to previously described methods for imaging flow cytometry [35] . the first frame of an image series was thresholded to include the high-intensity cluster of memerald signal, indicative of the golgi, for a single cell, and this area was measured. this measurement was taken for the same cell for every frame of an image series using the same thresholding value. this was performed for individual cells from every image series frame. image series were used to determine the percentage of infected cells at 8 hpi by observing infection reporter translocation. infected cells with aberrant er structures at 8 hpi in the image series were manually counted using imagej (nih). image series were used to calculate the percentage of lysed cells, which was determined by counting the number of cells showing infection at 8 hpi, then tracking these cells up to 16 hpi and looking for the loss of the cytoplasmic reporter signal due to loss of plasma membrane integrity. student's t-tests and one-way anova with bonferroni multiple comparisons tests were performed using prism 8 software (graphpad). to monitor enterovirus infection in real-time, we adapted cell-based reporter methodologies previously used for flaviviruses and hepatitis c virus that rely on viral protease cleavage-dependent translocation of a membrane-anchored cytoplasmic fluorescent proteins to the nucleus [27] [28] [29] . however, given that these systems do not allow for the visualization of virus-induced manipulation of host cell organelles, we sought to design reporter plasmids expressing a fluorescent protein to indicate infection in addition to fluorescent protein-targeted organelle markers. as a proof of principle, we developed a dual reporter construct (reper) that expresses an er lumen localized mcherry containing an er retention signal (kdel) and signal peptidase cleavage site fused to a type i transmembrane domain followed by an enterovirus 3c protease (3c pro ) target sequence and a cytoplasmic gfp containing a nuclear localization signal (gfp-nls) ( figure 1a ). upon infection, the expression of 3c pro is predicted to release the er-tethered gfp-nls, allowing for translocation to the nucleus ( figure 1a) . importantly, given that the mcherry-kdel remains localized in the er, we can monitor er dynamics in parallel. we first validated the cleavage of the reporter by expressing myc-tagged cvb 3c pro and 3c mut , a catalytically inactive protease mutant, in u2os cells. immunoblot analysis of cell lysates indicated that expression of cvb 3c pro led to the efficient release of gfp-nls from the full reper protein, whereas cleavage was absent in 3c mut expressing cells (figure 1b ). to ensure that cleavage of reper is dependent on viral protease cleavage during infection, we generated a construct containing a double alanine mutation (aa) of the glutamine-glycine (qg) 3c pro cleavage site. we found that mutation of the viral protease cleavage site blocked cvb-induced cleavage of reper ( figure 1c ). next, we sought to determine if reper was cleaved by a panel of enteroviruses during infection. we found that infection of reper-expressing hela cells with cvb, poliovirus, echovirus 11, and enterovirus 71 resulted in cleavage of reper to varying degrees ( figure 1d ) and efficiency of cleavage correlated with moi ( figure 1e ). lastly, using immunofluorescence microscopy, we verified that cvb infection resulted in translocation of gfp-nls to the nucleus, whereas mcherry was retained in the er (figure 1f ). notably, we did not observe complete gfp-nls translocation, which is consistent with previous reports demonstrating that enterovirus infection restricts nuclear transport [36] ; additionally, there may be uncleaved reporter present on the er, which can account for some of the cytoplasmic gfp fluorescence. intensity profile analysis of confocal images showed that mock-infected cells had lower gfp-nls reporter and mcherry-er signal intensities in the nuclear region, defined by peaks of dapi signal (figure 1g) . importantly, cvb infection led to an increase in gfp-nls reporter signal intensity in the nuclear region, while mcherry-er maintained a lower signal intensity in this region. upon quantification of confocal images, we observed a significant increase in the ratio of gfp to mcherry signals in the nucleus relative to the cytoplasmic during cvb infection compared to mock (figure 1h ). together, these results indicate that the reper reporter can be used to efficiently detect infection by a broad range of enteroviruses and monitor virus-induced changes of the er. viruses 2020, 12, x for peer review 6 of 15 cytoplasmic gfp fluorescence. intensity profile analysis of confocal images showed that mockinfected cells had lower gfp-nls reporter and mcherry-er signal intensities in the nuclear region, defined by peaks of dapi signal (figure 1g ). importantly, cvb infection led to an increase in gfp-nls reporter signal intensity in the nuclear region, while mcherry-er maintained a lower signal intensity in this region. upon quantification of confocal images, we observed a significant increase in the ratio of gfp to mcherry signals in the nucleus relative to the cytoplasmic during cvb infection compared to mock (figure 1h ). together, these results indicate that the reper reporter can be used to efficiently detect infection by a broad range of enteroviruses and monitor virus-induced changes of the er. we next utilized long-term time-lapse imaging to determine the kinetics of gfp-nls translocation during virus infection. we used u2os cells, which are highly conducive to live-cell imaging. cvb infection of reper expressing u2os cells resulted in a clear translocation of gfp-nls reporter to the nucleus and eventual dispersal due to cell lysis, while the mcherry-kdel was maintained in the membranous structure of the er (video s1 and figure 2a) . image series analysis revealed that infected cells exhibited a linear increase in nuclear translocation of gfp-nls starting around~4 hpi on average (figure 2b) with 50% of the maximum infection signal occurring~5.5 hpi (figure 2c) , whereas mock-infected cells showed no increase in nuclear gfp signal. we next utilized long-term time-lapse imaging to determine the kinetics of gfp-nls translocation during virus infection. we used u2os cells, which are highly conducive to live-cell imaging. cvb infection of reper expressing u2os cells resulted in a clear translocation of gfp-nls reporter to the nucleus and eventual dispersal due to cell lysis, while the mcherry-kdel was maintained in the membranous structure of the er (video s1 and figure 2a) . image series analysis revealed that infected cells exhibited a linear increase in nuclear translocation of gfp-nls starting around ~4 hpi on average (figure 2b ) with 50% of the maximum infection signal occurring ~5.5 hpi (figure 2c ), whereas mock-infected cells showed no increase in nuclear gfp signal. the er has recently been shown to be involved in the early stages of cvb ro formation [15] . thus, we sought to determine virus-induced changes to the er throughout infection using long-term time-lapse imaging. we observed a dramatic loss of the structural integrity of the er, as shown by the collapse of the peripheral er network in cells infected with cvb (video s1, video s2, and figure 2a ). analysis of image series from individual cells indicated a decrease in the ratio of peripheral er to sheet areas, a measurement of peripheral er integrity, that coincided with an increase in signal for infection ( figure 2d) . furthermore, analysis of multiple image series showed a reverse correlation between the infection signal and the peripheral er integrity (figure 2e ). interestingly, we found that the collapse of the peripheral er network preceded cell lysis by ~7 h (videos s1 and s2). the er has recently been shown to be involved in the early stages of cvb ro formation [15] . thus, we sought to determine virus-induced changes to the er throughout infection using long-term time-lapse imaging. we observed a dramatic loss of the structural integrity of the er, as shown by the collapse of the peripheral er network in cells infected with cvb (video s1, video s2, and figure 2a ). analysis of image series from individual cells indicated a decrease in the ratio of peripheral er to sheet areas, a measurement of peripheral er integrity, that coincided with an increase in signal for infection ( figure 2d) . furthermore, analysis of multiple image series showed a reverse correlation between the infection signal and the peripheral er integrity (figure 2e) . interestingly, we found that the collapse of the peripheral er network preceded cell lysis by~7 h (videos s1 and s2). enterovirus infection has been previously shown to restrict secretory trafficking, resulting in golgi dispersal [14, [16] [17] [18] [19] [20] . thus, we developed a bicistronic, triple reporter to visualize the er and golgi as well as monitoring virus infection (repor). the repor construct encodes an memerald-golgi marker followed by an ires-driven reper cassette that has been modified to include a bfp-nls reporter protein (figure 3a) . time-lapse imaging of cells expressing repor showed correct localization of organelle reporters that were maintained throughout time-lapse imaging (video s3 and figure 3b ). to initially validate this construct, we performed time-lapse imaging of repor cells treated with brefeldin a (bfa), an inhibitor of er to golgi protein transport [37] . as expected, bfa treatment resulted in a clear change in the localization of the golgi marker from a tight cluster to a dispersed signal that colocalized with the er marker, which indicated a disruption of golgi integrity and a block in protein transport out of the er (video s4 and figure 3b ). next, we sought to monitor virus-induced changes to the golgi during infection of repor expressing cells. to do this, we performed time-lapse imaging of cells infected with cvb. consistent with previous reports [14, [16] [17] [18] [19] [20] 38 ], cvb infection led to a clear dispersal of the golgi marker, as shown by a decrease in the high-intensity golgi signal area and presence of a diffuse low-intensity signal, indicating a disruption of golgi integrity (video s5 and figure 3b ). analysis of this image series indicated that dispersal of the golgi coincided with an increase in the signal for infection (figure 3c ) and analysis of multiple image series showed a reverse correlation between infection signal and golgi area (figure 3d ). together, these results indicated the repor construct can be used to monitor pharmacological and viral manipulation of the secretory pathway. previous studies have utilized recombinant enteroviruses that encode fluorescent protein reporters to visualize infection in real-time. however, these viruses display dramatic defects in replication kinetics [14, 25, 26] . thus, we sought to determine if reporter plasmid expression attenuates virus replication. we performed single-step growth curves for cvb in u2os cells transfected with each reporter plasmid. our results indicated there were no differences in growth of cvb in u2os cells transfected with empty, reper, or repor plasmids (figure 4) . these results suggest our reporter plasmids can be used to accurately monitor viral replication kinetics and virus-induced manipulation of host cells in real-time. previous studies have utilized recombinant enteroviruses that encode fluorescent protein reporters to visualize infection in real-time. however, these viruses display dramatic defects in replication kinetics [14, 25, 26] . thus, we sought to determine if reporter plasmid expression attenuates virus replication. we performed single-step growth curves for cvb in u2os cells transfected with each reporter plasmid. our results indicated there were no differences in growth of cvb in u2os cells transfected with empty, reper, or repor plasmids (figure 4) . these results suggest our reporter plasmids can be used to accurately monitor viral replication kinetics and virus-induced manipulation of host cells in real-time. lastly, we sought to demonstrate the utility of our reporter system by visualizing the effect of a pharmacological inhibitor on virus infection. we have previously reported that inhibition of inositol 1,4,5-triphosphate receptors (ip3r), which function as calcium channels in the er, restricts the release of an infectious virus through modulating cell death pathways [39] . long-term time-lapse imaging of cvb infected repor cells treated with dmso produced results similar to those described above (video s6, video s7, and figure 5a ). time-lapse imaging of repor cells in the presence of 2aminoethoxydiphenylborane (2apb), an ip3r inhibitor, did not prevent the nuclear translocation of the virus reporter or dispersal of the golgi (video s8, video s9, and figure 5a ). furthermore, image series analysis showed no defect in the kinetics of nuclear translocation of the reporter, indicating this drug does not affect viral protein production and replication (figure 5b) . additionally, we did not observe a difference in the percentage of cells infected at 8 hpi, indicated by reporter translocation to the nucleus (figure 5c ). interestingly, 2apb treatment of cells led to the formation of aberrant er structures that coincided with reporter translocation to the nucleus (video s8 and video s9). these structures resembled large membrane aggregates and inflated regions of er (figure 5a , asterisks and arrowheads, respectively), which were rarely observed in infected dmso-treated cells (figure 5c ) and not observed in uninfected cells. furthermore, treatment with 2apb led to a significant decrease in the number of infected cells that progressed to lysis (figure 5c ), which is consistent with a significant decrease in the titer of extracellular virus (figure 5d ). together, these results indicate that our plasmid-based reporters can be used to monitor virus-induced changes to host membranes upon inhibition of specific cellular functions, which can further our understanding of inhibitor mechanisms of action during infection. lastly, we sought to demonstrate the utility of our reporter system by visualizing the effect of a pharmacological inhibitor on virus infection. we have previously reported that inhibition of inositol 1,4,5-triphosphate receptors (ip 3 r), which function as calcium channels in the er, restricts the release of an infectious virus through modulating cell death pathways [39] . long-term time-lapse imaging of cvb infected repor cells treated with dmso produced results similar to those described above (video s6, video s7, and figure 5a ). time-lapse imaging of repor cells in the presence of 2-aminoethoxydiphenylborane (2apb), an ip 3 r inhibitor, did not prevent the nuclear translocation of the virus reporter or dispersal of the golgi (video s8, video s9, and figure 5a ). furthermore, image series analysis showed no defect in the kinetics of nuclear translocation of the reporter, indicating this drug does not affect viral protein production and replication ( figure 5b ). additionally, we did not observe a difference in the percentage of cells infected at 8 hpi, indicated by reporter translocation to the nucleus (figure 5c ). interestingly, 2apb treatment of cells led to the formation of aberrant er structures that coincided with reporter translocation to the nucleus (video s8 and video s9). these structures resembled large membrane aggregates and inflated regions of er (figure 5a , asterisks and arrowheads, respectively), which were rarely observed in infected dmso-treated cells (figure 5c ) and not observed in uninfected cells. furthermore, treatment with 2apb led to a significant decrease in the number of infected cells that progressed to lysis (figure 5c ), which is consistent with a significant decrease in the titer of extracellular virus (figure 5d ). together, these results indicate that our plasmid-based reporters can be used to monitor virus-induced changes to host membranes upon inhibition of specific cellular functions, which can further our understanding of inhibitor mechanisms of action during infection. the data was collected from two independent experiments, with four separate fields/experiment. significance was determined by one-way anova, *** p < 0.0005. (d) titer of extracellular virus from cvb infected u2os cells treated with dmso or 2apb (100 µm) at 10 hpi. the data is shown as the average ± sd titer (pfu/ml) from three independent experiments. significance was determined by student's t-test, ** p < 0.005. live-cell imaging is a powerful tool to study virus-host interactions in real-time. to facilitate this, we developed and characterized multipartite fluorescent-reporters to monitor enterovirusinduced remodeling of organelles in the host secretory pathway. these reporters rely on enterovirus 3c pro activity to release an nls-tagged fluorescent protein, which translocates to the nucleus while retaining er and golgi localized fluorescent markers. live-cell imaging of cvb infected cells the data was collected from two independent experiments, with four separate fields/experiment. significance was determined by one-way anova, *** p < 0.0005. (d) titer of extracellular virus from cvb infected u2os cells treated with dmso or 2apb (100 µm) at 10 hpi. the data is shown as the average ± sd titer (pfu/ml) from three independent experiments. significance was determined by student's t-test, ** p < 0.005. live-cell imaging is a powerful tool to study virus-host interactions in real-time. to facilitate this, we developed and characterized multipartite fluorescent-reporters to monitor enterovirus-induced remodeling of organelles in the host secretory pathway. these reporters rely on enterovirus 3c pro activity to release an nls-tagged fluorescent protein, which translocates to the nucleus while retaining er and golgi localized fluorescent markers. live-cell imaging of cvb infected cells expressing these reporters allowed for the real-time visualization of virus-induced changes to the host cell, including the collapse of the peripheral er network and loss of golgi integrity. furthermore, we used live-cell imaging with repor to show that a calcium channel inhibitor, 2apb, resulted in cvb-induced formation of aberrant er structures and decreased the lytic effects of the virus. overall, we show that our reporters present innovative opportunities for studying enterovirus-induced remodeling of host cell membranes during infection in real-time. previous studies have utilized plasmid-based reporters for live-cell imaging of other small positive-strand rna virus infections [27] [28] [29] . plasmid-based reporters for dengue virus and zika virus utilized viral nonstructural protein 4b (ns4b), a multi-pass er membrane-anchored protein, fused to gfp-nls [28, 29] . however, expression of flavivirus ns4b has previously been associated with the induction of autophagy and remodeling of the er, which can complicate interpretation of results and restrict the applications in which these reporters can be used [40, 41] . additionally, these reporters are limited to the detection of virus infection. to monitor virus-induced changes to the host-cell they would need to be used in combination with other fluorescent-marker expression vectors or stable cells. thus, we expanded on these reporters to develop multipartite fluorescent reporters that anchor the virus reporter via a single-pass host-derived transmembrane domain. this design eliminates the need for transfection of multiple constructs to visualize host structures and virus infection and significantly simplifies the generation of stable cells or transgenic animals. whereas the flavivirus reporters are virus-specific [28] , the reper and repor reporters can be used for a broad array of enteroviruses. while all enterovirus infections tested resulted in reporter cleavage, there were differences in efficiency with cvb infection consistently resulting in the highest reporter cleavage efficiency. one explanation is the engineered cleavage site may not be optimal for all enteroviruses and may need to be modified for specific viruses. alternatively, there may be slight differences in replication kinetics between these viruses, which results in lower levels of cleavage by the time point tested. thus, these reporters can be used to compare virus-specific manipulation of host membranes in a wide variety of target cells. all positive-strand rna viruses remodel host membranes to form ros that facilitate the sequestration of viral and host factors to promote efficient replication [7] [8] [9] [10] . recently, a recombinant cvb was developed to show in real-time that the accumulation of the viral 3a coincides with golgi disruption [14] . this recombinant virus was also used to identify the er as an early source for ro membranes [15] . however, the manipulation of the cvb genome to introduce fluorescent-based reporters resulted in dramatic attenuation, which is common in the development of most recombinant small positive-strand rna viruses [14, 25, 26] . an important benefit of our reporters is their expression did not restrict cvb infection. thus, we utilized the reporters to evaluate the kinetics of cvb-induced membrane remodeling at a single-cell level. using live-cell imaging and image series analysis, we were able to demonstrate that golgi dispersal and loss of peripheral er integrity coincided with an increase in cvb infection. interestingly, the diffuse reporter signal in the cytoplasm indicated that the loss of the peripheral er network did not coincide with cell rounding, shrinkage, or result in a sudden progression to cell death, which occurred~7 h post er collapse. interestingly, cmv infection leads to the collapse of the er due to the expression of the viral pul37×1 protein [42] . thus, future studies are warranted to determine if interactions between viral and host proteins are responsible for cvb-induced remodeling of the er. we applied our reporter systems to better understand the effects of calcium channel inhibition on infection, which we have previously shown decreases the release of infectious virus [39, 43] . we found that 2apb inhibition of calcium channels did not block virus-induced nuclear translocation of the virus reporter, golgi dispersal, or er collapse. however, we did find that 2apb treated cells showing virus reporter translocation to the nucleus contained aberrant er structures and prevented cell lysis. previous studies have shown similar structures represent er whorls or stacked er, which have been proposed to regulate cargo exit from the er [44] [45] [46] . in yeast, these structures have also been associated with induction of er stress and the unfolded protein response [47, 48] , which could explain their presence in cvb infected cells that are unable to regulate ca 2+ release. thus, our results demonstrate an important application for our reporters in understanding the real-time effects of pharmacological inhibitors on virus-induced manipulation of the host cell. overall, we have shown the benefits of using multipartite plasmid-based reporters to visualize virus-induced remodeling of the host cell using live-cell imaging. our system can be readily modified to include combinations of other fluorescent markers of host cell structures or viral host factors. additionally, these constructs can be modified to include recognition sites for proteases encoded by other positive-strand rna viruses, including flaviviruses, alphaviruses, and coronaviruses. furthermore, these reporters can be used for other applications, including diagnostics and high-throughput screens. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/10/1074/s1, table s1 : primer and oligonucleotide sequences, video s1: enterovirus infections of the central nervous system neonatal enterovirus infections reported to the national enterovirus surveillance system in the united states centers for disease control and prevention. enterovirus surveillance-united states pediatric group b coxsackievirus infections neurologic complications in children with enterovirus 71 infection pathogenesis of enterovirus 71 brainstem encephalitis in pediatric patients: roles of cytokines and cellular immune activation in patients with pulmonary edema architecture and biogenesis of plus-strand rna virus replication factories the transformation of enterovirus replication structures: a three-dimensional study of single and double-membrane compartments building viral replication organelles: close encounters of the membrane types remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles association of polioviral proteins of the p2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography electron microscopic study of the formation of poliovirus cellular origin and ultrastructure of membranes induced during poliovirus infection illuminating the sites of enterovirus replication in living cells by using a split-gfp-tagged viral protein origins of enterovirus replication organelles established by whole-cell electron microscopy viral reorganization of the secretory pathway generates distinct organelles for rna replication hijacking components of the cellular secretory pathway for replication of poliovirus rna inhibition of cellular protein secretion by poliovirus proteins 2b and 3a poliovirus infection blocks ergic-to-golgi trafficking and induces microtubule-dependent disruption of the golgi complex enterovirus protein 2b po(u)res out the calcium: a viral strategy to survive? nonlytic viral spread enhanced by autophagy components tissue preparation for immunocytochemistry pfa fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins immunolabeling artifacts and the need for live-cell imaging dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro development and characterization of a stable egfp enterovirus 71 for antiviral screening real-time imaging of hepatitis c virus infection using a fluorescent cell-based reporter system a fluorescent cell-based system for imaging zika virus infection in real-time a plasmid-based reporter system for live cell imaging of dengue virus infected cells comparative rnai screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers the coxsackievirus b 3c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling rip3 regulates autophagy and promotes coxsackievirus b3 infection of intestinal epithelial cells adap2 is an interferon stimulated gene that restricts rna virus entry bpifb3 regulates autophagy and coxsackievirus b replication through a noncanonical pathway independent of the core initiation machinery effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells calcium signals and calpain-dependent necrosis are essential for release of coxsackievirus b from polarized intestinal epithelial cells induction of endoplasmic reticulum-derived replication-competent membrane structures by west nile virus non-structural protein 4b zika virus ns4a and ns4b proteins deregulate akt-mtor signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy human cytomegalovirus pul37x1 induces the release of endoplasmic reticulum calcium stores release of intracellular calcium stores facilitates coxsackievirus entry into polarized endothelial cells mouse stbd1 is n-myristoylated and affects er-mitochondria association and mitochondrial morphology yip1a structures the mammalian endoplasmic reticulum membrane biogenesis during b cell differentiation: most endoplasmic reticulum proteins are expressed coordinately er-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response we thank kaman fan (tsinghua university school of medicine) for technical assistance and members of the coyne laboratory for discussion of the data presented in the manuscript. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-272018-txdc0c3j authors: laing, eric d.; sterling, spencer l.; weir, dawn l.; beauregard, chelsi r.; smith, ina l.; larsen, sasha e.; wang, lin-fa; snow, andrew l.; schaefer, brian c.; broder, christopher c. title: enhanced autophagy contributes to reduced viral infection in black flying fox cells date: 2019-03-14 journal: viruses doi: 10.3390/v11030260 sha: doc_id: 272018 cord_uid: txdc0c3j bats are increasingly implicated as hosts of highly pathogenic viruses. the underlying virus–host interactions and cellular mechanisms that promote co-existence remain ill-defined, but physiological traits such as flight and longevity are proposed to drive these adaptations. autophagy is a cellular homeostatic process that regulates ageing, metabolism, and intrinsic immune defense. we quantified basal and stimulated autophagic responses in black flying fox cells, and demonstrated that although black flying fox cells are susceptible to australian bat lyssavirus (ablv) infection, viral replication is dampened in these bat cells. black flying fox cells tolerated prolonged ablv infection with less cell death relative to comparable human cells, suggesting post-entry mechanisms interference with virus replication. an elevated basal autophagic level was observed and autophagy was induced in response to high virus doses. pharmacological stimulation of the autophagy pathway reduced virus replication, indicating autophagy acts as an anti-viral mechanism. enhancement of basal and virus-induced autophagy in bat cells connects related reports that long-lived species possess homeostatic processes that dampen oxidative stress and macromolecule damage. exemplifying the potential that evolved cellular homeostatic adaptations like autophagy may secondarily act as anti-viral mechanisms, enabling bats to serve as natural hosts to an assortment of pathogenic viruses. furthermore, our data suggest autophagy-inducing drugs may provide a novel therapeutic strategy for combating lyssavirus infection. bats, order chiroptera, host a significantly greater richness of virus species compared to other mammalian taxa [1] . viruses hosted by bats include several that are highly pathogenic in human hosts such as henipaviruses (e.g., nipah virus (niv) and hendra virus (hev)) [2] , filoviruses (e.g., marburg virus (marv)) [3, 4] , coronaviruses (e.g., sars-like coronavirus) [5] , and lyssaviruses (e.g., rabies virus (rabv)) [6] . bats experimentally infected with niv and hev [2, 7, 8] , ebola virus in a dose-dependent manner, in both black flying fox-and human-derived cell lines, which we confirmed in primary black flying fox brain cells. activation of autophagy through pharmacological methods decreased ablv replication in both black flying fox and human cells, which suggested (1) that autophagy functions as an anti-viral defense during ablv infection, and (2) that activation of autophagy might be an effective treatment against neurotropic viruses such as ablv or related lyssaviruses. lastly, we observed that in contrast to human cells, black flying fox brain-derived cells withstood a high dose of ablv over a long incubation period and experienced significantly less cell death. our findings provide an initial in vitro exploration for future studies that may illuminate the importance of autophagy as an enhanced post-transcriptional anti-viral pathway in bats. black flying fox brain (pabrh) and kidney (pakit) tissue-derived cell lines and primary brain (pabr) cells have been previously described [44] . pabrh and pakit cells were maintained in dmem (dulbecco's modified eagle medium (gibco laboratories; gaithersburg, md, usa), 10% hyclone ™ cosmic calf ™ serum (ccs) (thermo fisher scientific; waltham, ma, usa), and 1% l-glutamine (thermo fisher scientific)) complete cell culture media (dmem-10). primary pabr cells were maintained in dmem/nutrient f-12 ham media (sigma-aldrich; st. louis, mo, usa) with 10% fetal bovine serum (gibco) and 1% antibiotic-antimycotic (gibco). a human neuroblastoma cell line (nbf-l) was obtained from dr. aviva symes (uniformed services university, bethesda, md, usa) and maintained in dmem supplemented with 10% cosmic calf ™ serum (thermo fisher scientific), 5% fetal bovine serum (gibco), and 1% glutamax ™ (thermo fisher scientific). human embryonic kidney (hek) 293t (atcc ® crl-3216 ™ ) and mouse neuro-2a (atcc ® ccl-131 ™ ) cells were obtained from the american type culture collection (atcc; manassas, va, usa) and maintained in dmem-10 complete media. a recombinant australian bat lyssavirus (rablv), human isolate [52] , anti-genome plasmid was used to generate a reporter virus through reverse genetics and a wild-type ablv (wt-ablv), pteropus isolate [40] , was also used for infection studies (ncbi genbank accession number: af418014). the open reading frame of turbo green fluorescent protein (gfp; evrogen; moscow, russian federation) was cloned into the rablv anti-genome plasmid between the ablv glycoprotein (g) and large rna polymerase (l) genes. to rescue rablv-gfp, hek293t cells were seeded 2 × 10 5 cells/well in 6-well plates and transfected with 2 µg full-length rablv-gfp anti-genome plasmid and 1 µg nucleoprotein (n), 0.5 µg phosphoprotein (p), and 0.5 µg l protein helper plasmids. the total recombinant plasmid dna (4 µg) was mixed with 10 µl lipofectamine ltx ® (thermo fischer scientific) in opti-mem (gibco), following standard transfection protocol. hek293t cells were maintained in dmem-10 complete media. cell culture supernatants were replaced 24 hours post-transfection with dmem, 6% ccs and 1% l-glutamine. three days post-transfection, fluorescent microscopy was used to examine gfp expression. wells that contained gfp-positive cells were passaged and co-cultured with mouse neuro-2a cells. cells were continuously monitored for gfp expression and cell culture supernatant was passaged to fresh hek293t cells. to purify rablv-gfp, cell culture supernatant containing rablv-gfp was collected and ultracentrifuged (27,500 rpm, 2 h) with a 20% sucrose cushion. pabrh, pakit, hek293t, nbf-l, or primary pabr cells (2.5 × 10 5 cells/well, 6-well cell culture plates) were infected with rablv-gfp or wt-ablv at the indicated multiplicities of infection (moi) (e.g., 0, 1.0, 10). to compare replication kinetics between pabrh, pakit, hek293t, and nbf-l, cell lines were incubated with 1 ml dmem-10 containing rablv-gfp (moi 1) for 2 h. after 2 h, virus/dmem-10 was removed and replaced with fresh dmem-10. cell culture supernatants were collected and centrifuged to remove cell debris (2500 rpm, 10 min) then serially diluted and incubated with hek293t cells to determine ablv titers. gfp expression from rablv-gfp was used to count the number of fluorescent virus plaques, whereas fitc-conjugated anti-rabies monoclonal globulin (fujirebio diagnostics, inc.; malvern, pa, usa) was used to quantify wt-ablv titers. to quantify gfp expression by flow cytometry (lsr ii; bd biosciences; san jose, ca, usa), cell culture supernatants were collected and centrifuged to collect floating cells, then mixed with the collected adherent cells, and fixed with 4% paraformaldehyde. gfp median fluorescence intensity (mfi) and the percentage of gfp positive cells were then analyzed. bafilomycin a1 (bafa1; 400 nm; invivogen; san diego, ca, usa) was used to inhibit fusion of autophagosomes and autolysosomes, i.e., autophagic flux. to block autophagy, cells were incubated for two hours at the end of the infection time course with bafa1 prior to whole cell lysate processing. whole cell protein lysates were collected at indicated hours post-infection (hpi) and processed following protocol guidelines for autophagic quantification by lc3b western blots [53] . cell culture protein lysates were normalized to 15 µg, and loaded into 4-12% bis-tris protein gel (invitrogen; carlsbad, ca, usa); protein gels were transferred to pvdf membranes (bio-rad; hercules, ca, usa; 350 ma, 2 h) for standard western blotting techniques. antibodies used for immunoblots included: anti-lc3b polyclonal antibody (sigma-aldrich), anti-p62 polyclonal antibody (sigma-aldrich), anti-ndp52 polyclonal antibody (aviva systems biology; san diego, ca, usa), anti-β-actin polyclonal antibody (abcam; cambridge, uk), anti-gapdh polyclonal antibody (abcam), and anti-turbo gfp polyclonal antibody (evrogen). an anti-rabies virus n protein polyclonal rabbit serum was provided by dr. ina smith and anti-rabies virus p protein polyclonal antibody was purchased (mybiosource; san diego, ca, usa). protein levels were quantified using image j software (nih; bethesda, md, usa). pabrh and nbf-l cell lines at a density of 2.5 × 10 5 cells/well (6-well plate) were infected with rablv-gfp. cell culture supernatant was not removed after the addition of rablv-gfp until the conclusion of the post-infection time course. cell culture supernatant and cells were collected, centrifuged for 5 min, 2500× g, resuspended in 1 ml pbs, and dyed with live/dead™ fixable violet dead cell stain kit, for 405 nm excitation (thermo fisher scientific) for 30 min following the manufacturer's guidelines. cells were fixed with 4% paraformaldehyde and incubated at 37 • c for 20 min. cells were then pelted and washed with pbs, this process was repeated twice. resuspended stained cells were analyzed with an lsr ii flow cytometer (bd biosciences) for ablv gfp expression and live/dead violet signal. pabrh, pakit, and nbf-l cells at a density of 2.5 × 10 5 cells/well (6-well plate) were infected with rablv-gfp (moi 1.0) for 48 h. at 24 hpi, rapamycin (rapa; sigma-aldrich), small molecule enhancer of autophagy-28 (smer; tocris; bristol, uk), or nvp bez235 (bez; selleck chemicals; houston, tx, usa) were added to the wells and incubated with the ablv-infected cell culture for 24 h. at 48 hpi, cell culture supernatants and whole cell lysates were collected. whole cell lysates were processed as described above. supernatants were centrifuged for 10 min at 2500 rpm to remove cell debris, then serially diluted and incubated with hek293t cells at 5 × 10 4 cells/well (96-well plate) to determine rablv-gfp titers; fitc anti-rabies monoclonal globulin (fujirebio diagnostics) was used to quantify wt-ablv titers after bez treatments. we attempted to monitor autophagy levels by flow cytometry after rapa and smer induction using a cyto-id ® autophagy detection kit (enzo life sciences, inc.; famingdale, ny, usa), which stains autophagosomes with a green fluorescent dye; a positive stain was confirmed using the pakit cell line. ablv, like all lyssaviruses, has a 3'-5'(-)ssrna genome that encodes five proteins: nucleoprotein (n), phosphoprotein (p), matrix protein (m), envelope glycoprotein (g), and large rna polymerase (l). we used reverse genetic techniques to generate a recombinant ablv reporter virus expressing a green fluorescent protein (rablv-gfp) ( figure 1a ). the ablv anti-genome plasmid was modified with the insertion of a gfp open reading frame between the g and l genes. first, we compared ablv replication in black flying fox and human cell lines. to conduct these experiments, we utilized brain (pabrh) and kidney (pakit) tissue-derived black flying fox cell lines [44] . the pabrh cell line is morphologically fibroblast-like in appearance so we chose to compare ablv replication with a human neuroblastoma cell line (nbf-l) that also had fibroblast-like morphology [54] . a human embryonic kidney cell line, that was similarly immortalized with a sv40 t antigen (hek293t), was included for comparative purposes with the pakit cell line. plasmid was modified with the insertion of a gfp open reading frame between the g and l genes. first, we compared ablv replication in black flying fox and human cell lines. to conduct these experiments, we utilized brain (pabrh) and kidney (pakit) tissue-derived black flying fox cell lines [44] . the pabrh cell line is morphologically fibroblast-like in appearance so we chose to compare ablv replication with a human neuroblastoma cell line (nbf-l) that also had fibroblast-like morphology [54] . a human embryonic kidney cell line, that was similarly immortalized with a sv40 t antigen (hek293t), was included for comparative purposes with the pakit cell line. lower rablv-gfp titers were quantified from both infected black flying cell lines compared to human cell lines at equivalent hpi ( figure 1b ). nearly 10-fold higher titers were achieved in both human compared to black flying fox cell lines at 48 and 72 hpi. quantification of gfp positive cells after rablv-gfp infection revealed that nearly double the number of human cells were infected compared to black flying fox cell lines at 48 and 72 hpi ( figure 1c ). investigation of ablv protein levels-including nucleoprotein (n), phosphoprotein (p), and the virus-expressed gfp (vgfp) ( figure 1d )-revealed lower amounts of structural and non-structural ablv proteins in black flying fox cells. gfp expression in black flying fox cells and human cells indicated that all four cell lines were susceptible to rablv-gfp infection, although multiple mechanisms of cellular entry, replication, or post-entry might contribute to the reduced level of replication in black flying fox cells relative to human cells. to avoid issues with inefficient ablv entry into black flying fox cells, in the following experiment the conditions of rablv-gfp infection were modified to promote infection of close to 100% of the cells. infection, although multiple mechanisms of cellular entry, replication, or post-entry might contribute to the reduced level of replication in black flying fox cells relative to human cells. to avoid issues with inefficient ablv entry into black flying fox cells, in the following experiment the conditions of rablv-gfp infection were modified to promote infection of close to 100% of the cells. we next sought to further investigate the ablv replication differences between the cells and determine whether black flying fox cells tolerated high doses of ablv infection. notably, ablv infection increased cell death in human and black flying fox cells in a dose-dependent manner ( figure 2a ). black flying fox (pabrh) cells experienced an increase in cell death at 96 hpi (moi 10), although a relatively lesser extent compared to human nbf-l cells, which was significantly higher than pabrh cells at this hpi ( figure 2b ). the percentage of gfp positive black flying fox cells was also dose-dependent. unlike in figure 1 , rablv-gfp was not washed out after two hpi, which would lessen the effects of inefficient virus entry of black flying fox cells. gfp positive percentages between human and black flying fox cells were comparable with moi 10 at 48 (~85 vs. 74%) and 96 hpi (~86 vs. 85%), whereas the percentage of gfp positive black flying fox cells was nearly half compared to human cells (~80 vs. 41%) 48 hpi, moi 1.0 ( figure 2c ). more strikingly, a significantly lower vgfp median fluorescence intensity (mfi), which is the quantification of vgfp expression and dependent on post-entry virus replication, was quantified in black flying fox cells compared to a human cell line 96 hpi at both moi 1 and 10 ( figure 1d ). thus, despite a similar percentage of infected cells at 48 and 96 hpi (moi 10) in both nbf-l and pabrh cells ( figure 2c ), lower vgfp in pabrh cells suggested that an intrinsic mechanism, not inefficient entry, restricted production of ablv encoded proteins (e.g., gfp) or replication. viruses 2019, 11, x for peer review 6 of 18 we next sought to further investigate the ablv replication differences between the cells and determine whether black flying fox cells tolerated high doses of ablv infection. notably, ablv infection increased cell death in human and black flying fox cells in a dose-dependent manner ( figure 2a ). black flying fox (pabrh) cells experienced an increase in cell death at 96 hpi (moi 10), although a relatively lesser extent compared to human nbf-l cells, which was significantly higher than pabrh cells at this hpi ( figure 2b ). the percentage of gfp positive black flying fox cells was also dosedependent. unlike in figure 1 , rablv-gfp was not washed out after two hpi, which would lessen the effects of inefficient virus entry of black flying fox cells. gfp positive percentages between human and black flying fox cells were comparable with moi 10 at 48 (~85 vs. 74%) and 96 hpi (~86 vs. 85%), whereas the percentage of gfp positive black flying fox cells was nearly half compared to human cells (~80 vs. 41%) 48 hpi, moi 1.0 ( figure 2c ). more strikingly, a significantly lower vgfp median fluorescence intensity (mfi), which is the quantification of vgfp expression and dependent on postentry virus replication, was quantified in black flying fox cells compared to a human cell line 96 hpi at both moi 1 and 10 ( figure 1d ). thus, despite a similar percentage of infected cells at 48 and 96 hpi (moi 10) in both nbf-l and pabrh cells ( figure 2c ), lower vgfp in pabrh cells suggested that an intrinsic mechanism, not inefficient entry, restricted production of ablv encoded proteins (e.g., gfp) or replication. to determine whether an intrinsic defense mechanism was constraining ablv replication, we next focused on the anti-viral potential of the autophagy pathway. autophagy functions as an antiviral mechanism during vesicular stomatitis virus (vsv) infection [55, 56] . vsv is a member of family to determine whether an intrinsic defense mechanism was constraining ablv replication, we next focused on the anti-viral potential of the autophagy pathway. autophagy functions as an anti-viral mechanism during vesicular stomatitis virus (vsv) infection [55, 56] . vsv is a member of family rhabdoviridae, which includes lyssaviruses such as rabies virus (rabv) and rabies virus-related viruses (e.g., ablv). wild-type rabv infection of a human neuroblastoma cell line was shown to activate the autophagy pathway [57] . a hallmark feature of autophagy induction is the cleavage and lipidation of the cytosolic microtubule-associated protein 1a/1b light chain 3b (lc3b-i) to autophagosomal-associated lc3b-phosphatidylethanolamine (lc3b-ii). lc3b-ii is incorporated into the developing autophagophore and retained in the autophagosome double membrane vesicle such that increased cellular levels of lc3b-ii are used as a marker for the induction of autophagy [58] . functional human and black flying fox lc3b proteins are identical in amino acid sequence after post-translational cleavage. therefore, we compared quantities of lc3b-ii between black flying fox and human cell lines by immunoblotting, following inhibition and stimulation of autophagy. bafilomycin-a1 (bafa1) inhibits autophagic flux by blocking the fusion of autophagosomes and autolysosomes [59, 60] , causing a buildup of autophagosomes and lc3b-ii. we treated pabrh, pakit, and nbf-l cells with bafa1 for two hours to inhibit autophagic flux and create a snapshot of basal autophagy levels in all three cell lines. as expected, bafa1 treatment significantly increased levels of lc3b-ii in all three cell lines ( figure 3a,b) . interestingly, in the bafa1 treated cells, levels of lc3b-ii were significantly higher in both pabrh and pakit compared to nbf-l ( figure 3a,b) , suggesting that these black flying fox cell lines maintained a higher basal level of autophagic flux. to determine whether autophagy was activated in bat versus human cells after ablv infection, we again monitored levels of lc3b-i and lc3b-ii. pabrh cells showed a significant percentage of lc3b-ii conversion when infected with a moi 10 at 24 and 48 hpi ( figure 3c,d) . the nbf-l cells had a significant percentage of lc3b-ii when infected for 48 hpi (moi 1.0 and 10) ( figure 3e ,f). we confirmed these results in primary black flying fox brain (pabr) cells. a significant increase in lc3b-ii was again dependent on a high virus dose, moi 10 ( figure 4a,b) . additionally, treatment with bafa1 increased lc3b-ii, however the levels of lc3b-ii remained statistically equivalent regardless of ablv moi. as there was not an additive effect of ablv infection plus bafa1 treatment, it is possible that the basal level of autophagic processes are so rapid that external stimulation (a) has little observable effect by comparison or (b) additional stimulation via ablv infection increased autophagic flux such that autophagosome and lc3b-ii turnover occurs more rapidly. these results suggested that although ablv induced autophagy in both black flying fox and human cells, further enhancement of autophagic flux in black flying fox cells required a high virus dose. this observation highlights the need for additional studies of autophagic kinetics, both basal and induced, in black flying fox cells. viruses 2019, 11, x for peer review 8 of 18 to further characterize the increase in lc3b-ii after ablv incubation, we monitored cytosolic levels of autophagy cargo receptor proteins, p62 and ndp52, which are associated with selective autophagy and used to monitor autophagic flux [61] [62] [63] [64] . virus proteins, such as hiv nef protein and influenza m2, inhibit autophagic flux by blocking fusion of autophagosomes with autolysosomes [65, 66] . inhibition of autophagic flux and the resulting accumulation of autophagosomes and their contents such as lc3b-ii could be misinterpreted as virus activation of autophagy. if autophagic flux is unobstructed by ablv infection, p62 and ndp52 will be degraded, whereas inhibition of autophagic flux will lead to an increase in these cargo proteins. we examined levels of p62 and ndp52 in pabrh cells after ablv infection, and in combination with bafa1 treatment. infection with ablv alone resulted in less p62 ( figure 5a ,b) and ndp52 ( figure 5a ,c) detected in pabrh cells at 48 hpi. in nbf-l cells, a mean fold decrease in p62 was observed at both 48 and 72 hpi ( figure 5d ,e). treatment with bafa1 alone or in combination with ablv infection at all time points resulted in a significant increase of p62 and ndp52 in pabrh cells ( figure 5a-c) and nbf-l cell ( figure 5d ,e), however p62 and ndp52 fold-changes between ablv alone and ablv plus bafa1 were insignificant. since additive effects of bafa1 inhibition of autophagic flux were not observed during ablv infection and we observed a fold decrease of p62 and ndp52 48 hpi, we concluded that ablv alone was not inhibiting autophagic flux. together, increased lc3b-ii (figures 3 and 4) and decreased p62/ndp52 protein levels ( figure 5 ) in black flying fox cells further demonstrated that ablv infection induced the autophagy pathway. viruses 2019, 11, x for peer review 9 of 18 (a). all data are representative of three independent experiments, mean ± sem and * p < 0.05 anova (two-way), and tukey's multiple comparison test (48 hpi moi 0 vs. moi 10). to further characterize the increase in lc3b-ii after ablv incubation, we monitored cytosolic levels of autophagy cargo receptor proteins, p62 and ndp52, which are associated with selective autophagy and used to monitor autophagic flux [61] [62] [63] [64] . virus proteins, such as hiv nef protein and influenza m2, inhibit autophagic flux by blocking fusion of autophagosomes with autolysosomes [65, 66] . inhibition of autophagic flux and the resulting accumulation of autophagosomes and their contents such as lc3b-ii could be misinterpreted as virus activation of autophagy. if autophagic flux is unobstructed by ablv infection, p62 and ndp52 will be degraded, whereas inhibition of autophagic flux will lead to an increase in these cargo proteins. we examined levels of p62 and ndp52 in pabrh cells after ablv infection, and in combination with bafa1 treatment. infection with ablv alone resulted in less p62 ( figure 5a ,b) and ndp52 ( figure 5a ,c) detected in pabrh cells at 48 hpi. in nbf-l cells, a mean fold decrease in p62 was observed at both 48 and 72 hpi ( figure 5d ,e). treatment with bafa1 alone or in combination with ablv infection at all time points resulted in a significant increase of p62 and ndp52 in pabrh cells ( figure 5a-c) and nbf-l cell ( figure 5d,e) , however p62 and ndp52 fold-changes between ablv alone and ablv plus bafa1 were insignificant. since additive effects of bafa1 inhibition of autophagic flux were not observed during ablv infection and we observed a fold decrease of p62 and ndp52 48 hpi, we concluded that ablv alone was not inhibiting autophagic flux. together, increased lc3b-ii (figures 3 and 4) and decreased p62/ndp52 protein levels ( figure 5 ) in black flying fox cells further demonstrated that ablv infection induced the autophagy pathway. to understand whether the autophagy pathway had an anti-viral role during ablv infection, we examined the effects of pharmacological activation of autophagy on ablv replication. the mammalian target of rapamycin (mtor) is a key modulator of autophagy [67] . we therefore tested the effects of both mtor-dependent autophagy activation using the mtor inhibitor rapamycin (rapa), as well as mtor-independent autophagy activation with small molecule enhancer of autophagy-28 (smer) stimulation [68, 69] . rablv-gfp infected black flying fox cell lines, pabrh and pakit, were treated with rapa or smer, respectively, during ablv infection to elucidate whether activation of autophagy had anti-viral effects; human nbf-l cells were treated the same in comparison. rapa and smer activation of autophagy was monitored by cyto-id ® (enzo) staining of autophagosomes in pakit cells. treatment with either rapa or smer induced autophagy in pakit cells, with rapa showing a stronger effect ( figure 6a ). chloroquine (chq), an inhibitor of autophagic flux, was included as a positive control for autophagosome staining [60] . treatment of black flying fox and human cells with rapa after ablv infection resulted in significant reductions in ablv replication ( figure 6b) . a significant reduction in ablv titers after smer treatment was only observed in human nbf-l cells, and a decreasing titer trend was observed in smer treated black flying fox cells. as a potential mechanism underlying the reduction in ablv, we examined whether treatment with rapa or smer resulted in degradation of virus proteins. cell lysates collected after rapa and smer treatment of pabrh and pakit cells, followed by analysis via western blot ( figure 6c -f). neither rapa or smer treatment had any significant effects on n or p protein levels in pabr cells, however, n protein fold change had a decreasing trend in rapa treated cells ( figure 6c,d) . ablv protein levels in bat pakit cells, infected and treated with rapa and smer, were examined by western blot (figure 6e,f) . as seen in the pabrh cells, n protein fold change had a decreasing trend in pakit cells when treated with rapa, but not smer. p protein levels were quantified for two independent experiments, and also had a trending decease in both rapa-and smer-treated cells. although, western blot quantification of vgfp can be difficult because of low replication and vgfp expression levels in bat cells, we successfully quantified vgfp from two of the four independent experiments with pakit cells. the noticeable vgfp fold decrease caused by rapa and smer stands in contrast to fold changes in n and p proteins. human nbf-l cells had a significant fold decrease of vgfp levels in both rapa-or smer-treated cells, but neither n nor p levels had any significant reductions ( figure 6g,h) . these results indicated that autophagy acts as an anti-viral response in both black flying fox cells and human cells during ablv infection. given our limited on-hand reagents (e.g., ablv n and p reactive antisera), we were only able assess the effects of autophagy activation on n and p protein levels. it is possible that other ablv proteins such as m protein or l rna polymerase, to which we lack reagents, are degraded by autophagy, and that degradation of these proteins leads to decreased ablv replication as assessed by lower ablv titers ( figure 6b ) and ablv vgfp expression ( figure 6f,h) . while conducting this study we observed that autophagy activation through pharmacological modulators (e.g., rapa) reduced ablv replication and protein expression in a human neuroblastoma cell line (nbf-l). infected neurons cannot rely on the anti-viral effects of ifn-induced cell death to control viral infection and risk potential damage to the central nervous system [70] . given the neurotropism of lyssaviruses such as ablv and rabv, we asked whether potent autophagy induction with dactolisib (a.k.a. nvp bez235 or bez), a dual inhibitor pan-class pi3k and mtor inhibitor evaluated in clinical trials, could also restrict ablv replication in nbf-cells. treatment of nbf-l cells with bez increased the amount of autophagosomal lc3b-ii indicative of autophagy activation ( figure 7a ) without causing significant cytotoxicity ( figure 7b ). to examine the anti-viral effect of bez treatment on ablv replication, nbf-l cells were infected with wt-ablv for 24 h then treated with bez. treatment with bez resulted in significant dose-dependent decreases of wt-ablv titers ( figure 7c ), in addition to n and p protein levels ( figure 7d,e) . although bez advanced to phase i/ii clinical trials as a potential anti-cancer therapy [71] [72] [73] , its use was discontinued due to adverse toxicities [74] . nevertheless, our results suggest that with improved selectivity or dosing, potent inducers like bez may be promising additional therapeutics to prevent lethality following lyssavirus exposure. fold change in ablv n, p, and vgfp proteins (β-actin-normalized) collected from nbf-l cell lysates. (c-h) whole cell lysates were collected 48 hpi (rablv-gfp moi 1.0). fold change is a representation of at least three independent experiments, mean ± sem and ** p < 0.001, * p < 0.05 anova (one-way) analysis, dunnett's multiple comparison test with treatments compared to dmso mock treated cells. while conducting this study we observed that autophagy activation through pharmacological modulators (e.g., rapa) reduced ablv replication and protein expression in a human neuroblastoma cell line (nbf-l). infected neurons cannot rely on the anti-viral effects of ifn-induced cell death to control viral infection and risk potential damage to the central nervous system [70] . given the neurotropism of lyssaviruses such as ablv and rabv, we asked whether potent autophagy induction with dactolisib (a.k.a. nvp bez235 or bez), a dual inhibitor pan-class pi3k and mtor inhibitor evaluated in clinical trials, could also restrict ablv replication in nbf-cells. treatment of nbf-l cells with bez increased the amount of autophagosomal lc3b-ii indicative of autophagy activation ( figure 7a ) without causing significant cytotoxicity ( figure 7b ). to examine the anti-viral effect of bez treatment on ablv replication, nbf-l cells were infected with wt-ablv for 24 h then treated with bez. treatment with bez resulted in significant dose-dependent decreases of wt-ablv titers ( figure 7c ), in addition to n and p protein levels ( figure 7d,e) . although bez advanced to phase i/ii clinical trials as a potential anti-cancer therapy [71] [72] [73] , its use was discontinued due to adverse toxicities [74] . nevertheless, our results suggest that with improved selectivity or dosing, potent inducers like bez may be promising additional therapeutics to prevent lethality following lyssavirus exposure. although bats are reservoir hosts for a richness of viruses, a comprehensive understanding of how their immune response can sense, respond to, and control viral infection is needed. the type i ifn pathway is well-conserved among vertebrates as an intrinsic first defense during virus infection, with unique anti-viral expression patterns noted in bat cells [75] . in black flying fox cells, ifn-α was constitutively expressed regardless of virus infection, whereas ifn-β was not induced [50] , consistent with another report that ifn-β was antagonized during hev infection [45] . regardless, the ifn pathway is a constant target of virus antagonism and evasion. here, we focused on another well-conserved, intrinsic immune response and present a first experimental attempt to study the autophagy pathway during a virus infection in cell lines derived from a bat species. collectively, our findings demonstrated that ablv infection increased autophagosome generation and autophagy in black flying fox and human cells, and that autophagy activation in black flying fox cells was dependent on a high ablv moi. yet, how ablv rna or proteins are sensed by host sensory machinery during ablv replication remain unanswered. a related rhabdovirus, vsv, induces autophagy through g protein engagement independent of virus replication [56] and rabv m protein may also contribute to autophagy activation [57] . autophagy has been implicated in either anti-and pro-viral processes for distinct viruses [33, 76] . for example, a protective role for autophagy was identified during vsv infection [55, 56] . in our study, pharmacological activation of autophagy decreased ablv replication in both black flying fox and human cell lines, suggesting autophagy antagonizes lyssavirus infection. host-virus adaptations that promote tolerance to virus infection may contribute to the ability of bats to act as primary animal hosts without developing pathogenic disease [77] . we found that the black flying fox brain cell line did not undergo significant cell death after what could be considered a pathogenic ablv infection (moi 10; 96 hpi). in contrast, ablv induced substantial cell death in human neuroblastoma cells, similar to that observed previously with rabv infection [57] . crosstalk and interplay between autophagic and apoptotic processes have long been recognized. dependent on the environment or stimulation, autophagy can promote survival under stressful conditions (e.g., nutrient deprivation) or itself act as a mechanism of programmed cell death if left unchecked [78, 79] . recently, we rescued a recombinant cedar virus [80] , a non-pathogenic henipavirus species also isolated from pteropus species [81] . such tools will facilitate opportunities to ask if autophagic response to ablv can be generalized to other bat-borne viruses, and better define how the balance between autophagy and cell death is calibrated in bat cells. the interactions between natural reservoirs hosts and viruses require that (a) the host is readily infected and (b) the virus needs to persist in the host long enough to be transmitted to another susceptible host, before it is either cleared by the host immune response or kills the host [82, 83] . epidemiology models suggest a possibility that latently infected individual bats contribute to episodic shedding and natural persistence of bat-borne viruses [84] . although horizontal transmission within a population is still regarded as the primary source of transmission [85] , hev and niv recrudescence in pteropus hosts have been reported [86] [87] [88] , yet the precise mechanism of latency or persistence is unclear. autophagy functions as a pro-survival defense by removing toxic virus n protein aggregates during sindbis virus infection, without having an effect on virus replication [89, 90] . indeed, a pro-survival autophagic response that removes toxic protein aggregates and dampens virus replication might be critical for promoting viral persistence at the cellular level in natural hosts, maintaining the potential to shed virus with minimal disease presentation. more investigation is needed to translate in vitro bat immunity studies into a models of virus persistence and shedding within individuals and populations. in conclusion, our working hypothesis is that black flying fox cells maintain a higher basal rate of autophagy that serves as both an anti-viral and pro-survival mechanism when further stimulated by virus infection. similarly, brook and dobson, 2015, postulated that autophagic removal of damaged mitochondria (i.e., mitophagy), a result of enhanced oxidative stress from the demands of flight, evolved a secondary anti-viral role for autophagy in bats [91] . new cell lines have recently been derived from additional bat species facilitating a greater comparative study across the bat taxa [92] . furthermore, comparisons with rodent cell lines will strengthen whether in vitro intrinsic responses to virus infection are unique to bats or shared co-adaptations among other natural host species. experimental challenges have limited our efforts to genetically silence or pharmacologically inhibit the autophagy pathways in black flying fox cells. although we cannot yet definitively associate autophagy function with host tolerance to ablv infection in black flying fox cells, ongoing experiments will refine our mechanistic understanding of enhanced basal autophagy and virus-induced autophagy in bat cells, and further characterize the pro-survival role of autophagy during virus infection. host and viral traits predict zoonotic spillover from mammals pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission isolation of genetically diverse marburg viruses from egyptian fruit bats oral shedding of marburg virus in experimentally infected egyptian fruit bats (rousettus aegyptiacus) bats are natural reservoirs of sars-like coronaviruses bats and lyssaviruses experimental hendra virus infectionin pregnant guinea-pigs and fruit bats (pteropus poliocephalus) experimental nipah virus infection in pteropid bats (pteropus poliocephalus) experimental 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ecology and transmission characterization of nipah virus from naturally infected pteropus vampyrus bats, malaysia evidence for nipah virus recrudescence and serological patterns of captive pteropus vampyrus recrudescent infection supports hendra virus persistence in australian flying-fox populations autophagy protects against sindbis virus infection of the central nervous system selective autophagy and viruses bats as 'special' reservoirs for emerging zoonotic pathogens tools to study pathogen-host interactions in bats we thank k. lund, for assistance with flow cytometry and c. olsen for statistical consultation. additionally, we thank a.j. symes for kindly providing human neuroblastoma cell lines. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-001270-l8aa9cl3 authors: wongsrikeao, pimprapar; saenz, dyana; rinkoski, tommy; otoi, takeshige; poeschla, eric title: antiviral restriction factor transgenesis in the domestic cat date: 2011-09-11 journal: nat methods doi: 10.1038/nmeth.1703 sha: doc_id: 1270 cord_uid: l8aa9cl3 studies of the domestic cat have contributed to many scientific advances, including the present understanding of the mammalian cerebral cortex. a practical capability for cat transgenesis is needed to realize the distinctive potential of research on this neurobehaviorally complex, accessible species for advancing human and feline health. for example, humans and cats are afflicted with pandemic aids lentiviruses that are susceptible to species-specific restriction factors. here we introduced genes encoding such a factor, rhesus macaque trimcyp, and egfp, into the cat germline. the method establishes gamete-targeted transgenesis for the first time in a carnivore. we observed uniformly transgenic outcomes, widespread expression, no mosaicism and no f1 silencing. trimcyp transgenic cat lymphocytes resisted feline immunodeficiency virus replication. this capability to experimentally manipulate the genome of an aids-susceptible species can be used to test the potential of restriction factors for hiv gene therapy and to build models of other infectious and noninfectious diseases. supplementary information: the online version of this article (doi:10.1038/nmeth.1703) contains supplementary material, which is available to authorized users. felis catus has been domesticated for over 9,000 years and presently numbers 0.5-1.0 billion worldwide. medical surveillance of this most common companion animal is extensive, and over 250 hereditary pathologies common to both cats and humans are known 1 . the f. catus genome was recently sequenced at light (1.9×) coverage and a 10× assembly is imminent 2 . over 90% of identified cat genes have a human homolog, and compared with the mouse there are fewer genomic rearrangements. intermediate size, prolific breeding capacity, similarity of systems to humans, abundance, modest costs and the neurobehavioral complexity of a carnivoran make the cat of value in experimental settings ranging from neurobiology to diverse genetic, ophthalmologic and infectious diseases. these include conditions in which mice or rats are not useful on the basis of disease susceptibility, organ size or other factors 1 . cat transgenesis is thus of interest for both human and cat health research and potentially for developing ways to confer protection from epidemic pathogens to free-ranging feline species, all 36 of which now face the threat of extinction 3 . the world has two aids pandemics, one in domestic cats and the other in humans. the causative lentiviruses, feline immunodeficiency virus (fiv) and hiv-1, are highly similar in genome structure, disease manifestations and host cell dependency factor use 4, 5 . the differences between these lentiviruses are also informative and potentially exploitable. for example, species-specific lentiviral restriction factors such as trim and apobec3 proteins 6 restrict fiv and hiv-1 with distinctive patterns [7] [8] [9] [10] . these genes have not been studied in a controlled manner at the systemic and species levels by introduction into the genome of an aids virus-susceptible species (old world primates or felids). given the challenges inherent to macaque transgenesis, the aids virus-susceptible cat would be singularly positioned for such studies if it can be accessed by genetic approaches used in mice. in contrast to primates, feline species lack antiviral trim5α genes 11 but have potently restrictive apobec3 proteins 9, 10 , which sets up intriguing possibilities for testing such genes at the whole-animal level, for conferring gene-based immunity with them or engineered variants 12, 13 , and potentially for hiv-1 disease model development 10 . to realize the potential of the species for virology and nonvirology models, a means for practical cat genome modification is needed. somatic cell nuclear transfer (scnt) was recently used to generate cats that express fluorescent proteins 14, 15 . however, the efficiency of animal cloning is extremely low 16 , and scnt results in faulty epigenetic reprogramming in most embryos 17 . cloned mammals with apparently normal gross anatomy can have many abnormalities resulting from failure to erase and reprogram epigenetic memory completely 17 . the two key approaches for generating transgenic mice are dna injection into fertilized embryo pronuclei and injection of genetically modified embryonic stem cell (esc) lines into blastocysts. however, in nonrodent mammals, pronuclear injection is very inefficient, and the second method is blocked by the lack of germline-competent escs. transgenesis with germline transmission has been achieved in some mammals by microinjecting lentiviral vectors into oocytes or single-cell zygotes 18 . this has not been achieved in any carnivore species. here we performed oocyte-targeted lentiviral transgenesis in the domestic cat. results multi-transgenic, nonmosaic cat embryo generation we optimized reagents, gamete collection, microinjection parameters, embryo culture and recipient queen preparation to establish an optimal cat transgenesis protocol (fig. 1a) . we obtained gametes from both sexes without additional animal procedures by microdissecting gonads discarded after spaying or neutering. in experiments summarized in supplementary table 1 , we subjected 195 in vitro-matured grade i and ii domestic cat oocytes to perivitelline space microinjection (pvsmi) with lentiviral vector tsing 5 ; we performed injection 10-12 h before or 10-12 h after in vitro fertilization (ivf) (supplementary fig. 1 ). then we cultured these embryos until blastocyst stage (day 7). comparisons of embryo development rates (supplementary tables 1 and 2) and enhanced gfp (referred to as gfp throughout) expression ( fig. 1b) showed that transgenesis rates were high (>75%) and the process was well tolerated, as cleavage and blastocyst formation rates did not differ substantially between pvsmi and control embryos (supplementary table 1 ). there were no differences in morphology or total cell number and no preference for vector injection timing before or after ivf (supplementary table 1) . however, mosaicism scored by nonuniform fluorescent protein expression in the blastocyst was negligible when we injected vectors before ivf but was substantial with injection after ivf (supplementary table 1) . to investigate whether more than one transgene could be expressed in cat embryos in a single step by pvsmi, we microinjected 418 oocytes with single-or dual-transgene lentiviral vectors. transgene assemblages were genes encoding gfp, gfp plus rfp, or gfp plus rhesus macaque trimcyp ( supplementary fig. 1) . the latter combination was expressed from either a dual promoter or as a single 2a peptide-linked preprotein. after microinjection we performed ivf with cat sperm 10 h later. we consistently observed embryo-pervasive, abundant expression of both proteins encoded by dual gene vectors in cat blastocysts when we injected lentiviral vector before ivf ( fig. 1b and supplementary table 2 ). we observed no detrimental effects of dual expression on embryo development or gfp expression irrespective of transgene combination (supplementary table 2 ). in addition, the 2a peptide or the dual promoter were each effective for simultaneous expression. the process from oocyte collection to fallopian tube transfer took 3-4 d (fig. 1a) . we randomly selected embryos for implantation from cleaved oocytes that had been subjected to ivf and transferred them into surgically exposed fallopian tubes at 48-72 h after lentiviral vector transduction. we carried out no preselection for transgene expression after microinjection (embryos were in any case not reliably fluorescent by the time of transfer). we performed transfers into hormonally synchronized queens prepared by a 14-10 h light-dark environment. we administered to queens pregnant mare serum gonadotropin on day -4 and human chorionic gonadrotropin on day -1 with respect to lentiviral vector transduction, and mated them ad lib from the day of human chorionic gonadrotropin injection until the day before embryo transfer with a vasectomized, azoospermia-verified tomcat to induce ovulation and corpus luteum formation. during surgery we punctured follicles with a needle if not naturally ovulated. twenty-two embryo-transfer procedures resulted in five pregnancies (labeled a-e), five births and three live kittens ( table 1) . we achieved a high rate of transgenesis, with 10 of 11 testable live-born or fetal offspring found to be transgenic (a twelfth, spontaneously miscarried 10 d preterm, was consumed by the surrogate mother and could not be tested). three male and two female transgenic cats, named tgcat1-5, were born by spontaneous vaginal deliveries at term and all five were transgenic (fig. 2 , table 1 and supplementary fig. 2 ). tgcat1 (male), tgcat2 (male) and tgcat3 (female) survived, whereas the fourth and fifth cats died perinatally from obstetrical complications ( table 1) . tgcats1-3 were vigorous from birth, fed, played, developed and socialized normally and were healthy, with the exception that tgcat2 is unilaterally cryptorchid. he also has intermittent pruritic dermatitis, which may be due to a food allergy. in the first year he developed a ventral abdominal hernia and a lower eyelid irritation (entropion), both of which we cured surgically. although we cannot exclude vector-insertion genotoxicity in tgcat2, the conditions do not constitute a recognizable syndrome. southern blotting on restriction enzyme-digested genomic dna from the three living transgenic kittens, from tgcat4 and from four miscarried fetuses showed that all eight were transgenic, with 6-12 insertions per cat (fig. 2b) . pcr assays on genomic dna confirmed the high level of genomic transduction (fig. 2c) . southern blot hybridization bands were specific, as all were (i) absent from control cat dna, (ii) different from cat to cat and (iii) of greater than the predicted minimum size determined by the distance from restriction site to end of the vector provirus ( fig. 2b) . sequencing of proviral genomic dna junctions (n = 4) from two cats was performed and each was a bona fide retroviral integration junction, with the genomic sequences mapping to the cat genome (supplementary table 3 ). tgcat3, in which transgene expression was driven by the standard (0.52-kilobase) human cytomegalovirus (hcmv) promoter of vector tsint2ag, was brightly and stably green fluorescent in integument and oropharyngeal mucosa surfaces (fig. 2a) , but surface tissue expression was less bright for tgcat1 and tgcat2 (vector tbdmgpt). for the live kittens, we collected cells for protein analyses by oral mucosa scrapings (which showed gfp-expressing squamous epithelial cells), and blood and semen collection. both transgenes were expressed in activated peripheral blood mononuclear cells (pbmcs) but with notable variation (fig. 3a,b) . percentages of gfp-positive cells as determined by facs were 15-80% in tgcat1, tgcat2 and tgcat3 and increased gradually as the kittens aged (fig. 3b,c) . tgcat2 had the most gfppositive cells in the pbmc compartment, being about 65% gfppositive early in life and then over 70-75% later (fig. 3a-c and supplementary fig. 3) . several specific aspects here are interesting for developing models that will depend on lymphocyte or monocyte lineage expression. first, irrespective of promoter used, facs and immunoblot detection of gfp and rhtrimcyp in pbmcs in living cats required activation by phytohemagglutin-e (pha-e) and interleukin 2 (il-2), and gfp expression increased steadily with time in culture (fig. 3c) . fluorescence intensity was variable ( fig. 3b and supplementary fig. 3a) . second, driving gfp expression from a minimal cmv (mcmv) promoter element adjacent to the pgk promoter was effective in tgcat2, but we observed only low gfp expression with the same vector in tgcat1, although even in this cat gfp expression increased steadily from rare positives to 14.8% by 20 months (fig. 3b,c) . third, all three cats expressed hemagglutinin epitope (ha)-tagged rhtrimcyp in the bulk pbmc population as detected by immunoblotting (fig. 3a) . tgcat1 consistently expressed more rhtrimcyp than the other two living cats by quantitative western blot analysis. however, this protein was more difficult to detect than gfp, and was clearly visualized by immunofluorescence, using an antibody to the ha tag, in only a fraction of the cells (supplementary fig. 3) . even so, rhtrimcyp transgenic cat pbmcs displayed resistance to fiv replication, with the greatest resistance to replication seen in cells from the cat that expressed the most rhtrimcyp (tgcat1; fig. 3d) . the resistance to fiv replication was partial, as predicted for cell populations that express such a restriction factor partially 19 . washed swim-up purified sperm from the two males had normal motility and strongly expressed the transgene as determined by pcr (fig. 4) . consistent with this result and with the lack of embryo mosaicism when ivf was done after vector microinjection (supplementary table 1 ) germline transmission was readily achieved by direct mating, with all progeny being transgenic. therefore, the transgenesis procedure preserves fertility, and the germline is transduced. transgene expression persisted in the f1 offspring of transgenic f0 parents, indicating that silencing did not occur (fig. 4) . matings of tgcat1 with three nontransgenic queens produced five additional kittens from three pregnancies. similar to the sire, they were less surface green-fluorescent but were strongly 'pcr-and southern blot-positive' (data not shown); of these one died perinatally owing to dystocia associated with a hypocontractile uterus. thus, all f1 cats were transgenic; 8 of 9 were alive and healthy. tgcat4 was born after an uncomplicated singleton pregnancy at a normal gestation time (65 d). it was morphologically normal but died during or shortly after parturition from an apparent obstetrical accident involving aspiration, although a precise cause could not be determined at autopsy. this cat provided the opportunity to study all tissues (fig. 5a) . detailed organ examination and histology did not identify abnormalities. tgcat4 is the product of an oocyte transduced by the tsing vector, in which gfp was driven by the hcmv promoter, and had ~10 vector insertions (fig. 5) . as was tgcat3, the kitten was brightly green fluorescent in fur and skin, and immunoblotting revealed abundant gfp expression in all tissues tested: brain, spinal cord, heart, spleen, skin, muscle, liver, kidney, small intestine and stomach ( fig. 5a and supplementary fig. 4) . solid viscera were visibly green fluorescent at the gross level, as were adipose tissues (for example, all omental and pericardial fat) and antibody labeling of fixed tissue showed uniform expression in all cells (fig. 5c) . when fresh tissue was sectioned and imaged directly for gfp by epifluorescence microscopy, pervasive expression was similarly evident (fig. 5d) . a fourth pregnancy (c; table 1 ), for which we identified five well-formed, appropriately sized fetal skeletons by x-ray analysis at day 45 of gestation ( supplementary fig. 2d) , ended in serial miscarriages between days 51 and 53 (~10 d before term). we recovered four of these preterm cats (named tgpre1-4; table 1 ) for gross and molecular autopsy. dissection did not identify birth defects. as for tgcat1-4, southern blotting showed that tgpre1-4 were each amply transgenic, with 10-13 genomic tsing vector insertions (fig. 5b) and gfp expression was similarly found in all tissues tested (fig. 5a,c) . we also probed rhtrimcyp expression (supplementary fig. 5 ) using organs from a cat that was stillborn after a placental abruption (table 1 ; tgcat5), and observed that rhtrimcyp expression was similarly widespread, including in the main lymphoid organs (lymph node, thymus and spleen). consistent with the immunoblotting data, tissues of individual organs were green fluorescent at the gross level. facs of fetal pbmcs from tgpre1-4 showed that 74-100% were gfp-positive (fig. 5e) . southern blots of genomic dna from the products of non-singleton pregnancies (figs. 2b and 5b) , showed also that each was the genetically unique product of a different transduced oocyte, and none were a product of twinning after transduction. our results indicate that transgenic cats may be used as experimental animals for biomedical research. the approach enables transgenesis by germ cell genetic modification for the first time in this species and in any carnivore. notably, we achieved uniformly transgenic outcomes, which reduce screening cost and time. a second implication of the high efficiency and the copy numbers achieved is that it should be possible to titrate vector dose down or to microinject a mix of vectors into one oocyte to produce complex multi-transgenics. the approach is accessible: feline oocytes competent for efficient transgenesis are readily obtained noninvasively and without added animal procedures from ovaries discarded during routine spaying (laparoscopic or ultrasound-guided percutaneous oocyte retrieval is also feasible). in vitro blastocyst development rates were higher than had been seen previously with scnt-developed transgenic embryos (19-20% versus 3%) 14 . we prevented mosaicism by microinjection before ivf and observed germline transmission. the persistence of transgene expression in f1 cats is encouraging for establishing useful transgenic lines. the lack of multiple inbred strains of cat, a current limitation, could be addressed in a focused breeding project. introducing a lentiviral restriction factor(s) into the genome of the cat has specific potential because this species is naturally susceptible to lentiviral infection (and aids) whereas mice, unmodified or transgenic, are not. several questions can therefore now be addressed. first, it is unknown whether introducing a single active restriction factor into the genome of an aids virus-susceptible species can protect it, and if so, at which of three broadly considered levels: transmission, establishment of sustained viremia and disease development. when antiviral genes are interrogated at the whole animal level by transgenesis in a natural host, results can be surprising and informative. for example, a recent transgenic intervention against influenza in chickens prevented secondary virus transmission to transgenic and nontransgenic contacts, but it had no effect on mortality after primary virus challenge 20 . because species-specific lentiviral restriction factors have not been tested by controlled experimental introduction into an animal, the most fundamental question directly answerable with the approach is whether restriction factor transgenesis can mimic natural experiments that normally take place over large expanses of evolutionary time, with selection by viral culling, and render a species genetically immune to its own lentivirus. it is not possible to make clear predictions. for example, there are natural macaque and sootey mangabey trim5 alleles that do not block simian immunodeficiency virus transmission to animals that carry them but appear to constrain extent of replication in vivo and to exert selection pressure on the capsid 21 . when breeding expansion is completed with our present restriction factor transgenic cats, fiv challenges can be done. whether or not more than one restriction factor will be needed to achieve antiviral protection, the concept of using them for this purpose in gene therapy has stimulated efforts to devise non-immunogenic human and feline versions 12, 13 . both of these recently bioengineered trimcyps restrict fiv and can be tested in our system. indeed, fiv is unique among lentiviruses in being restricted by both old and new world monkey trimcyps. we speculate that feline transgenesis with host defense molecules could also confer protection from viral pathogens to wild feline species, all of which face accelerating extinction threats and which are among the most charismatic, ecosystem-iconic taxa in the carnivora. cat transgenesis could have additional impact. as we recently proposed, the domestic cat may have potential for modeling hiv-1 disease itself because, except for entry receptors, the cat genome can supply the dependency factors needed for hiv-1 replication 10 . this is a fundamental difference compared to the mouse 22 . gene knockdowns and targeting are foreseeable by combining our approach with current technologies. furthermore, transgenesis in this accessible, abundant species with intermediate size and complex neurobehavioral repertoire will permit other human-relevant models in areas such as neurobiology, where the cat is already a paramount model. studies in the cat have revealed much of the present knowledge on organization of the mammalian brain, in particular the visual cortex [23] [24] [25] [26] [27] ; work in this area has been critical to unraveling the neural mechanisms of vision. although transgenesis in this species will not be as common as in rodents, the creation of a small number of lines with genetic tools could build on the large knowledge base in the species to dramatically alter capability for understanding the cerebral cortex. transgenic mice have many advantages, but fundamental differences with human physiology limit their utility in many ways. many diseases cannot be modeled in mice or rats, with size alone being sometimes intrinsically limiting. transgenesis has been performed in marmosets 28 , and, so far without demonstrated germline transmission, in macaques 29 . these two primate models have clear promise, but limitations arise from scarcity, expense, longer gestation times and, for macaques, prolonged time to sexual maturity (4-8 years) and the requirement to shield handlers from casually transmitted cercopithecine herpesvirus 1. for the purpose of aids-relevant work, new world monkeys such as marmosets are not susceptible to any lentivirus. even with a generic viral promoter we observed transgene expression in 16 of 16 cat organs tested. we observed rhtrimcyp expression in the main aids-relevant lymphoid tissues (lymph node, spleen and thymus). mature circulating hematopoietic lineages have notoriously specialized transcriptional environments, but 15-80% of pbmcs in the living cats were gfp-positive in culture. variation may reflect genome positional effects. whereas tissue-specific or alternative promoter or enhancer elements can be used, cats with partial pbmc expression profiles also provide a good experimental opportunity because they allow the question of virus-mediated cell lineage selection in vivo 30 to be addressed, modeling a realistic cell-based therapy situation, for example, gene therapy for hiv-1 disease. one important issue is whether fiv infection will result in long-term selection of a virus-refractory lymphocyte population as has been observed in nonobese diabetic severe combined immune deficiency (nod-scid) il2rγ null mice transplanted with ccr5 −/− human cd34 cells 30 . conversely, if systemic viral replication occurs, we can determine whether escape mutations arise. methods and any associated references are available in the online version of the paper at http://www.nature.com/naturemethods/. funding from us national institutes of health grants ai47536 and ey14411 assisted prior key technology developments. we thank the helen c. levitt foundation for initial pilot funding and a. keller for coordinating it, members of our laboratory for helpful discussions and assistance, h. fadel for assisting with site-directed mutagenesis, members of our transgenic mouse core for sharing microinjection equipment, g. towers (university college london) for a rhtrimcyp cdna, and mayo clinic veterinary staff for advice and surgical assistance. the domestic cat, felis catus, as a model of hereditary and infectious disease state of cat genomics feline leukemia virus and other pathogens as important threats to the survival of the critically endangered iberian lynx (lynx pardinus) shared usage of the chemokine receptor cxcr4 by the feline and human immunodeficiency viruses an essential role for ledgf/p75 in hiv integration restriction of retroviral replication by apobec3g/f and trim5alpha restriction of feline immunodeficiency virus by ref1, lv1 and primate trim5a proteins independent evolution of an antiviral trimcyp in rhesus macaques functions, structure, and read-through alternative splicing of feline apobec3 genes productive replication of vif-chimeric hiv-1 in feline cells truncation of trim5 in feliformia explains the absence of retroviral restriction in cells of the domestic cat potent lentiviral restriction by a synthetic feline trim5 cyclophilin a fusion potent inhibition of hiv-1 by trim5-cyclophilin fusion proteins engineered from human components generation of cloned transgenic cats expressing red fluorescence protein generation of domestic transgenic cloned kittens using lentivirus vectors mammalian nuclear transfer nuclear reprogramming to a pluripotent state by three approaches lentiviral transgenesis-a versatile tool for basic research and gene therapy mode of transmission affects the sensitivity of human immunodeficiency virus type 1 to restriction by rhesus trim5alpha suppression of avian influenza transmission in genetically modified chickens trim5alpha modulates immunodeficiency virus control in rhesus monkeys multiple blocks to human immunodeficiency virus type 1 replication in rodent cells receptive fields, binocular interaction and functional architecture in the cat's visual cortex the period of susceptibility to the physiological effects of unilateral eye closure in kittens innate and environmental factors in the development of the kitten's visual cortex development of cat visual cortex following rotation of one eye neuronal circuits of the neocortex generation of transgenic non-human primates with germline transmission toward a transgenic model of huntington's disease in a non-human primate human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to ccr5 control hiv-1 in vivo the entire rhesus trimcyp transgene (1.4 kb) was amplified using 500 nm each sense primer 5′-atgtacccatacgatgttcc-3′ and antisense primer 5′-gccgcttattcgagttgcc-3′. the program included an initial denaturation step at 98 °c for 30 s. pcr amplification was performed as follows; 98 °c for 7 s, 60 °c for 20 s, 72 °c for 30 s. a final extension step at 72 °c for 7 min concludes the program reproductive patterns of cats. in mcdonald's veterinary endocrinology and reproduction effect of light manipulation on ovarian activity and melatonin and prolactin secretion in the domestic cat effect of the quality of the cumulus-oocyte complex in the domestic cat on the ability of oocytes to mature, fertilize and develop into blastocysts in vitro lentiviral vectors production and use of feline immunodeficiency virus (fiv)-based lentiviral vectors intensive rnai with lentiviral vectors in mammalian cells development of 2a peptide-based strategies in the design of multicistronic vectors coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters nucleotide sequence and genomic organization of feline immunodeficiency virus efficient transduction of nondividing cells by feline immunodeficiency virus lentiviral vectors all authors designed experiments, analyzed data and critiqued the manuscript. e.p. conceived the project and recruited p.w. and t.o. e.p. and t.o. oversaw the project. p.w. and d.s. produced vector and retrieved gametes; p.w. microinjected vector and did embryo cultures. p.w. transfered embryos with assistance from t.r. and e.p. with surgery. p.w., d.s and t.r. monitored cats, did cell and tissue assays and virology. p.w., d.s. and e.p. wrote the manuscript. the authors declare no competing financial interests.published online at http://www.nature.com/naturemethods/. reprints and permissions information is available online at http://www.nature. com/reprints/index.html. gametes used for embryo formation were obtained from gonads discarded after routine elective sterilization. oocyte-cumulus complexes (cocs) were recovered within 6 h by repeated fine slicing of ovarian tissue in modified phosphate-buffered saline (mpbs) supplemented with 4 mg ml −1 bovine serum albumin (bsa) and 50 µg ml −1 l gentamicin. only grade i and ii oocytes 33 were used. selected cocs were washed and matured in modified tcm-199 (gibco) containing 1 iu ml −1 human chorionic gonadrotropin (hcg), 0.5 iu ml −1 pregnant mare serum gonadotropin (pmsg), 10 µg ml −1 epidermal growth factor (egf), and 4 mg ml −1 bsa in a humidified atmosphere of 5% co 2 in air at 38 °c for 28 h.in vitro fertilization and in vitro culture. twenty-eight hours after ivm, cooled spermatozoa were washed twice in brackett-oliphant medium supplemented with 137 µg ml −1 sodium pyruvate, 4 mg ml −1 bsa, and 50 µg ml −1 gentamicin by centrifugation at 1800 rpm for 5 min. the supernatant was removed and sperm pellet was diluted in 500 µl fertilization medium (g-ivf plus, vitrolife), and placed in the incubator to allow sperm swim-up for 30 min. the spermatozoa concentration was adjusted to 2 × 10 6 ml −1 . ten oocytes were transferred into each of 100 µl sperm microdroplets under mineral oil and co-cultured for 12 h, after which presumptive zygotes were removed from sperm with a small-bore pipette, washed, and cultured in a modified earl's balanced salt solution (mk-1) supplemented with 4 mg ml −1 bsa and 50 µg ml −1 gentamicin for 3 d. three days after sperm exposure, cleaved embryos were selected for transfer or subsequently cultured in mk-1 medium supplemented with 5% (v/v) fbs (fbs, hyclone laboratories) and 50 µg ml −1 gentamicin for a further 4 days to evaluate developmental capacities to morula and blastocyst stages.transgenic embryo production. before to lentiviral vector microinjection, cumulus cells were mechanically removed from the oocytes 18-20 h after incubation in maturation medium (pre-ivf injection group) or from presumptive zygotes at 12-14 h post-incubation in fertilization medium (post-ivf injection group). a volume of ~100 pl vector was injected directly into the oocyte perivitelline space 12 h before or 12 h after ivf using a finely pulled glass capillary (femtotips, eppendorf) connected to a microinjector (eppendorf femtojet) adjusted for injection and compensation pressure with an injection time of 12 s. after microinjection, the oocytes were washed and returned to culture in ivm medium until hour 28 of maturation when they were used for ivf. for post-ivf injection, zygotes were washed and subsequently cultured in mk-1 medium. with the conditions developed, oocytes were modified at high rates without apparent toxicity to the zygotes early development and timing microinjection before fertilization created reliably non-mosaic embryos.embryo transfer, pregnancy detection, parturition and photo graphy. healthy 2-3-year-old spf queens were the recipients for embryo transfer. they were induced with 150 iu pmsg injected intramuscularly at 96-120 h before ivf, followed by injection of 100 iu of hcg 72 h after the pmsg. in addition, ad lib mating with a vasectomized male was done from the day of hcg injection until the day before embryo transfer.the females were anesthetized on the day of transfer with ketamine (5 mg kg −1 ), medetomidine (0.03 mg/kg) and buprenorphine (0.01 mg kg −1 ) administered intramuscular and maintained with 1-3% isoflurane gas. prior to abdominal incision the medetomidine was reversed with an intramuscular injection of atipamezole to minimize any effects the alpha-2 agonist may have on transfer success. an approximately 2 cm ventral midline incision was made and ovaries and fallopian tube exteriorized. each ovary was examined for evidence of ovulation. if no corpus hemorrhagicum or corpus luteum was visualized, follicles were punctured with a needle to artificially induce ovulation. then, a transmural puncture of the fallopian tube was performed with a 28 gauge needle and this was replaced with a fine hand-pulled glass transfer pipette, through which fifteen to twenty-five pre-loaded embryos (transduced, cleaved, >4 cell stage) in 10-20 µl mk-1 medium were transferred per fallopian tube under microscopic visualization using gentle positive mouth-controlled pressure. the pipette was withdrawn and the incision was closed in three layers.pregnancy status was determined with a canine relaxin kit (synbiotics) on day 30 after transfer and by film radiography on day 45. pregnant recipients were monitored daily until delivery of term kittens which occurred by un-assisted spontaneous vaginal birth at term. all control and transgenic animal photographs were taken with a nikon camera at the same time using identical lighting, filter, and camera settings, with gfp imaged under blue light illumination with a long pass filter. supplementary figure 2 contains additional images.immunofluorescence microscopy and immunohistochemistry. blastocysts (fig. 1c) were attached to a slide with bd-cell tak, cell and tissue adhesive, fixed and permeabilized for 15 min at room temperature in pbs supplemented with 4% (w/v) paraformaldehyde and 1% (v/v) triton x-100 and blocked with 1% bsa in pbs for 15 min. transduced and control blastocysts and activated pbmcs were imaged by confocal microscopy with gfp fluroescence imaged directly and ha-tagged rhtrimcyp detected using primary anti-ha (high affinity anti-ha rat monoclonal, roche, used at 1:1000 dilution), with incubation for 1 h at rt, washed, followed by incubation with cy3-conjugated goat anti-rat igg secondary (1:500 dilution, chemicon international) for 1 h. controls with each protein alone verified no signal cross-reception nature methods between channels and blastocysts derived from untransduced ova were negative as shown. following three 5 min washing steps in pbs and mounting with addition of prolong gold anti-fade reagent with dapi (invitrogen) for nuclear dna staining, the embryos were analyzed by laser confocal microscopy (axiovert 100m; carl zeiss microimaging).animal tissues were fixed with 4% paraformaldehyde and paraffin-embedded. serial 10 µm sections were made. immunohistochemistry was performed using a dako envision plus kit. sections were dewaxed in xylene and rehydrated in alcohol. endogenous peroxidase activity was blocked with 0.03% hydrogen peroxide. sections were incubated with a 1:200 diluted primary mouse monoclonal antibody (clontech, jl8, 1:5000) for 2 h. dako envision anti-mouse secondary antibody (1:200) was then applied for 30 min. the sections were mounted using prolong gold anti-fading reagent and observed by light microscopy.vectors and fiv infections. all vectors and vector sequences are available from the authors upon request. lentiviral vectors were hiv-1-based to permit pcr-based tracking of infectious fiv in future experiments. gfp is the enhanced version (egfp). tsin series lentiviral vectors were previously described 5 , and were prepared using 293t transfection in nunc cell factories and concentrated by ultracentrifugation using established methods [34] [35] [36] . the transfer vectors have cppt-cts and wpre elements and are u3-deleted. dual gene vectors with rhesus (macaca mulatta) trimcyp 8 and egfp utilize either a porcine teschovirus 2a peptide 37 expressing a single pro-protein (human cytomegalovirus immediate early gene (hcmv)-promoted rhtrimcyp-p2a-gfp) or a bi-directional promoter kindly provided by amendola et al. 38 with tandemly arranged phosophglycerate kinase (pgk) and minimal cmv (mcmv, 0.16 kb) promoter elements driving rhtrimcyp and gfp respectively on opposite strands. vsv-g-pseudotyped vectors were produced in two-chamber cell factories (cf2) and concentrated by ultracentrifugation over a sucrose cushion as described 5, 36 . vectors were titrated on feline kidney cell line (crfk) cells using flow cytometry for gfp expression. reverse transcriptase activities were used to normalize preparations 36 . pbmcs were cultured in rpmi with 10% fcs, rhil-2 and antibiotics and were activated with 10 µg ml −1 pha-e. for fiv infection of pbmcs, 50,000 feline pbmcs were infected with 2 × 10 6 rt activity units (10 µl) of fiv 34tf10 39 generated by 293t cell transfection of pct5 orfarep , a version of pct5 40 in which we repaired the premature orf-a stop codon by overlap extension pcr to enable pbmc replication. supernatants were collected approximately every 2 d thereafter and assayed for reverse transcriptase activity as described above.immunoblotting. transfected cell lysate or minced tissue samples were homogenized in ripa (150 mm nacl, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 1% np-40, 150 mm tris-hcl, ph 8.0) supplemented with protease inhibitors (complete-mini, boehringer). fractions and lysates were boiled in laemmli supplemented with β-mercaptoethanol for 10 min, separated by gel electrophoresis, transferred onto pvdf membranes (immobilon-p, millipore), and blocked in mpbs containing 2 mg ml −1 bsa and 1% tween 20 for 1 h at room temperature (22-25 °c) . blots were treated with primary antibodies against: gfp (jl8, 1:5000, clontech), α-tubulin (mouse monoclonal antibody 1:8,000, sigma), ha (high affinity anti-ha antibody, rat monoclonal, 1:1,000, roche, cat # 11867423001) for 1 h at room temperature. after washing, secondary antibodies were applied: alkaline phosphataseconjugated goat anti-mouse igg (calbiochem) diluted 1:10,000, and alkaline phosphatase-conjugated goat anti-rat igg (santa cruz biotechnology, inc., santa cruz, ca) diluted 1:1000. membranes were then incubated with ecl reagent (thermo scientific) and exposed to film.sperm collection and storage. epidydymi were separated by dissection within 6 h and repeatedly finely sliced in mpbs supplemented with 4 mg ml −1 bsa and 50 µg ml −1 gentamicin to release spermatozoa. the medium was filtered with a 70 µm cell strainer (bd falcon) and centrifuged at 18,000 rpm for 5 min. sperm pellets were resuspended in 500 µl test yolk buffer (refrigeration medium, irving scientific) in a 1.5-ml microcentrifuge tube at room temperature and gradually cooled to 4 °c. the samples were kept at 4 °c until use, or cryopreserved in liquid nitrogen. sperm of transgenic males was obtained by electroejaculation.southern blotting. genomic dna of newborn and spontaneously aborted kittens was analyzed by southern blot hybridization and pcr. total dna was isolated from blood, tail tips and heart using the dneasy blood and tissue kit (qiagen). five micrograms dna was digested with afliii, bamh1 or ndei as indicated. dna fragments were separated by electrophoresis on 0.8% agarose gel and transferred by capillary action to a nytran supercharge membrane (schleicher & schuell bioscience). dna was crosslinked to the membrane using a uv crosslinker (uvc500; hoefer). blots were then hybridized overnight at 42 °c in ultrahyb (ambion) containing an 32 p-labeled egfp probe. after washing at 60 °c with 0.5% sds, 2× ssc followed by 0.5% sds, 0.1× ssc, the blots were exposed to the kodak biomax ms x-ray film (sigma-aldrich) with intensifying screen at -80 °c and developed. bands in figure 5b and the right blot of figure 2b are more widely spaced than bands in the left blot of figure 2b because ndei and bamhi cleave, on average, every 4,096 bp apart, while afliii cuts on average every 1024 nt bp.quantitative rtpcr and semiquantitative pcr. transgenic and control genomic dna samples (pbmc, tail tip and organs) were analyzed by real-time quantitative pcr using the roche faststart dna master sybr green kit i. samples were quantified against a serially-diluted plasmid standard for total gfp using the roche lightcycler and roche lcda software. initial denaturation was at 95 °c for 10 min and a melting step after amplification (40-95 °c, temperature transition rate = 0.05 °c s −1 ). gfp was amplified using 300 nm each sense primer 5′-agaac ggcatcaaggtgaac-3′ and antisense primer 5′-tgctcagg tagtggttgtcg-3′. pcr amplification and analysis was performed as follows; 95 °c for 10 s, 62 °c for 10 s, 72 °c for 10 s, × 35 cycles, temperature transition rate = 5 °c s. as a loading control feline gapdh was quantified using 300 nm each sense primer 5′-accacagtccatgccatcac-3′ and antisense primer 5′-tccaccacccggttgctgta-3′. pcr amplification and analysis was performed using a roche lightcycler as follows: 95 °c for 10 s, 54 °c for 10 s, 72 °c for 18 s, × 35 cycles, temperature transition rate = 5 °c s. semiquantitative analysis for rhesus trimcyp was performed using phusion hot start high-fidelity key: cord-319842-4mnaicki authors: jackson, william t; giddings, thomas h; taylor, matthew p; mulinyawe, sara; rabinovitch, marlene; kopito, ron r; kirkegaard, karla title: subversion of cellular autophagosomal machinery by rna viruses date: 2005-04-26 journal: plos biol doi: 10.1371/journal.pbio.0030156 sha: doc_id: 319842 cord_uid: 4mnaicki infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral rna replication and whose origin is unknown. here, we show that several hallmarks of cellular autophagosomes can be identified in poliovirus-induced vesicles, including colocalization of lamp1 and lc3, the human homolog of saccharomyces cerevisiae atg8p, and staining with the fluorophore monodansylcadaverine followed by fixation. colocalization of lc3 and lamp1 was observed early in the poliovirus replicative cycle, in cells infected with rhinoviruses 2 and 14, and in cells that express poliovirus proteins 2bc and 3a, known to be sufficient to induce double-membraned vesicles. stimulation of autophagy increased poliovirus yield, and inhibition of the autophagosomal pathway by 3-methyladenine or by rna interference against mrnas that encode two different proteins known to be required for autophagy decreased poliovirus yield. we propose that, for poliovirus and rhinovirus, components of the cellular machinery of autophagosome formation are subverted to promote viral replication. although autophagy can serve in the innate immune response to microorganisms, our findings are inconsistent with a role for the induced autophagosome-like structures in clearance of poliovirus. instead, we argue that these double-membraned structures provide membranous supports for viral rna replication complexes, possibly enabling the nonlytic release of cytoplasmic contents, including progeny virions, from infected cells. upon infection by poliovirus and many other positivestrand rna viruses, dramatic changes are rapidly induced in the cellular environment. poliovirus infection causes the massive rearrangement of intracellular membranes, with double-membraned vesicles 200-400 nm in diameter accumulating in the cytoplasm [1, 2] . immunoelectron microscopy has revealed that the cytoplasmic surfaces of these membranous vesicles are the sites of viral rna replication [3, 4, 5] and, indeed, all known positive-strand rna viruses of eukaryotic cells replicate their rna on cytoplasmic membranes. it is thought that one function for membrane localization of viral rna replication proteins is to promote their oligomerization [6, 7] . it has been noted previously that several of the features displayed by the vesicles induced during poliovirus infection [1, 2] are known to be shared with cellular membranous structures termed autophagosomes. during cellular autophagy, cells break down cytoplasmic proteins and organelles within autophagosomes, double-membraned structures that become degradative upon maturation. originally identified as a process induced by cellular starvation, autophagy is now appreciated as a cellular response to a variety of stimuli, including hormone treatment, and is a feature of normal development in several organisms (reviewed in [8] ). in mammalian cells, two chemical inducers of autophagy are tamoxifen [9, 10] and rapamycin [11, 12, 13] . cells that express the estrogen receptor respond to tamoxifen treatment by accumulating large amounts of autophagosomes [9, 10] . rapamycin inhibits the function of the mammalian target of rapamycin, a known repressor of the autophagic pathway (reviewed in [14] ). in yeast, formation and maturation of autophagosomes requires the functions of many genes (reviewed in [15] ). human homologs of several of the yeast autophagy genes have been recently identified, including lc3, the human homolog of atg8p, and it is likely that much of the autophagic pathway is conserved [16, 17, 18] . in mammalian cells, the formation and maturation of autophagosomes involves the stepwise acquisition of proteins from disparate cellular compartments (reviewed in [19, 20, 21] ). nascent autophagosomes form either de novo [22, 23] or from the endoplasmic reticulum (er) [24, 25] and comprise cellular cytoplasm surrounded by two lipid bilayers that fuse from a c-shaped intermediate. although relatively protein-poor [22, 26] , they have been shown to contain a modified, lipidated form of lc3 [16] . as maturation proceeds, the late autophagosome acquires lamp1 before lysosomal fusion [16, 25, 27, 28] , which defines the ''mature autolysosome.'' mature autolysosomes are no longer surrounded by double membranes and, having degraded the inner membrane and much of the cytosolic contents, become electrondense [27, 29] . various intermediates among these stages have also been visualized, including incompletely fused double membranes and incompletely degraded inner membranes [23, 24, 25] . although poliovirus-induced vesicles display several hallmarks of autophagosomes, other origins for the poliovirusinduced vesicles have been suggested [30, 31, 32] . poliovirus rna replication is known to be inhibited by brefeldin a, which has led to the suggestion that the virally induced vesicles might derive from the copi pathway, a known target of brefeldin a [32, 33, 34] . however, this would be inconsistent with the apparent origin of the poliovirus-induced vesicles from the er: many images consistent with the budding of the poliovirus-induced vesicles directly from the er have been reported [3, 31] . furthermore, poliovirus proteins 2bc and 3a induce the formation of vesicles that are biochemically and ultrastructurally similar to those formed in poliovirusinfected cells [35] . the 3a protein and 2c, a proteolytic product of 2bc, localize to the er when expressed in isolation [35, 36] . therefore, it is likely that poliovirus proteins 3a and 2bc, or larger precursors, localize to the er early in infection and subsequently promote vesiculation from er membranes. poliovirus-induced vesicles have been shown to contain human copii proteins sec13p and sec31p early in their formation, leading to the hypothesis that they are modified copii vesicles, subverted from the anterograde transport pathway [31] . although this is an attractive suggestion, it is incongruent with other findings concerning the poliovirusinduced vesicles: their frequently observed double-membraned morphology, their cytosolic contents, and the abundance of components from throughout the secretory pathway, including lamp1, a marker of late endosomes and lysosomes [1, 2, 35] . furthermore, in saccharomyces cerevisiae, components of the copii pathway, encoded by the sec12, 16, 23, and 24 genes, are known to be required for autophagy [37, 38, 39] , making it possible that the sec13p and sec31p proteins could have other functions in addition to their known roles in anterograde traffic. the size of the poliovirusinduced vesicles, at 200-400 nm in diameter, does not immediately suggest whether they are related to the copii or autophagosomal pathways: typical diameters of copii vesicles and autophagosomes are 50-100 nm [40, 41] and 500-1,000 nm, respectively [24, 25] . finally, the idea that poliovirus infection induces autophagosome formation to facilitate viral growth is surprising in view of existing data that autophagy is an effective antimicrobial host response in many cases (reviewed in [42, 43] ). during herpes virus infection, there is good correlation between the induction of autophagy and the presence of effective antiviral responses. the process of autophagy is thought to be destructive to herpes virus because mutant viruses that fail to inhibit autophagy and other antiviral responses showed large decreases in yield [44] . during streptococcus infection, genetic disruption of the autophagy pathway in the infected host cells resulted in increased bacterial yield, consistent with a role for autophagy in bacterial clearance [45] . specific markers for autophagosome formation have become available, facilitating the identification of mem-branes derived from the autophagic pathway (reviewed in [43, 46] ). here, we explore the role of several constituents of autophagosome machinery in poliovirus-and rhinovirusinfected cells by monitoring: the presence of autophagosomal protein lc3 in virally induced vesicles, the acquisition of colocalization of lc3 and lamp1 in virally infected cells, the viral induction of punctate structures that stain with monodansylcadaverine (mdc), and the effects of perturbing the autophagosomal pathway pharmacologically and via rna interference on intracellular and extracellular virus yield. our data support the hypothesis that poliovirus, and likely the closely related rhinovirus, induce the formation of autophagosome-like structures to serve as the membrane scaffolds for rna replication. we further suggest that double-membraned vesicles, by forming a luminal cytoplasmic compartment, may facilitate prelytic viral exit from infected cells. to ask whether the membranes on which poliovirus rna replication complexes assemble contain constituents of autophagosomes, we monitored the localization of both lc3, a specific marker of autophagosomes, and 3a, a critical component of the poliovirus rna replication complex, in infected cells. lc3 was expressed via dna transfection as an amino-terminal fusion with green fluorescent protein (gfp). as can be seen in figure 1 , the punctate gfp-lc3 signal colocalized with that of poliovirus rna replication protein 3a, as visualized by immunofluorescence. no colocalization with poliovirus 3a protein was seen when gfp was not fused to lc3. therefore, the membranous structures to which 3a and other components of the poliovirus rna replication complex localize [3] can recruit lc3. in nonautophagic cells, lc3, originally identified as a microtubule-associated protein [47] , and lamp1, a marker of late endosomes and lysosomes, do not colocalize. however, during autophagy, lc3 has been shown to colocalize with lysosomal protein lamp1, defining the maturation of the nascent autophagosome to the autophagosome [28, 48, 49] . this process can be seen in figure 2a , in which lc3 and lamp1 were shown to colocalize almost completely in mcf-7 cells upon treatment with tamoxifen, a known inducer of autophagy. similarly, rapamycin treatment of hela cells led to the colocalization of gfp-lc3 and lamp-1, while they remained distinct in untreated cells (figure 2a ). by 3 h postinfection with poliovirus, the lamp1 and gfp-lc3 signals in hela cells also began to colocalize ( figure 2b ); this time corresponds to the beginning of viral rna synthesis in infected hela cells [50] . by 4.5 h postinfection, the lamp1 and gfp-lc3 signals had merged almost completely ( figure 2b ). the colocalization of gfp-lc3 and lamp1 characteristic of autophagosome formation was also seen in cells infected with either of two different serotypes of human rhinovirus ( figure 2c ). expression of poliovirus proteins 2bc and 3a in isolation can induce the formation of double-membraned vesicles that display biochemical and ultrastructural similarity to those formed in poliovirus-infected cells [35] . however, expression of poliovirus protein 2bc alone has been shown to induce the formation of single-membraned vesicles in both mammalian cells and s. cerevisiae [4, 35, 51, 52] . expression of poliovirus 3a protein in isolation, on the other hand, reduces the rate of er-to-golgi traffic and distends er membranes [36, 53] . to test whether 2bc, 3a, or both were sufficient to induce colocalization of gfp-lc3 and lamp1 as observed in poliovirus-infected cells, proteins 2bc and 3a were expressed singly or in combination in 293t cells in the presence of gfp-lc3. as shown in figure 3 , colocalization of gfp-lc3 and lamp1 was not observed in control cells or in cells that expressed either 2bc or 3a in isolation. however, coexpression of 2bc and 3a caused gfp-lc3 and lamp1 to colocalize ( figure 3a) , as was seen in cells infected with poliovirus or rhinoviruses treated with tamoxifen or treated with rapamycin (see figure 2 ). to examine further whether the membranes induced during poliovirus infection display additional characteristics of autophagosomes, we employed fluorescent staining with mdc. under specific fixation conditions, mdc is retained in autophagosomal membranes [54, 55] . as shown in figure 4 , treatment of mcf7 human breast tumor cells with tamoxifen caused the accumulation of punctate structures that retained staining with mdc after fixation. similar punctate staining was observed in cells infected for 5 h with rhinovirus 14 ( figure 4a ) or for 3 h or more with poliovirus (see figure 4b ). to test the hypothesis that the mdc-stained structures observed in figure 4 were the same as the gfp-lc3containing structures observed in figure 2 , we monitored the localization of gfp-lc3 with mdc. as a live stain, mdc is a poor marker for autophagosomes, because both autophagic membranes and lysosomes are visualized [46] . however, if cells are subjected to a defined fixation protocol after staining with mdc, the dye can be removed from singlemembraned, but not double-membraned, vesicles [54, 55] . as shown in figure 5 , good colocalization of mdc and lc3 was observed in poliovirus-infected cells, and no evidence of the punctate mdc staining expected of lysosomal membranes could be seen in uninfected cells. to determine whether the autophagosome-like membranes induced during poliovirus infection perform an antiviral function or facilitate viral replication, we tested the effect on poliovirus yield of pretreating h1-hela cells with either tamoxifen or rapamycin, known inducers of autophagy. yield of intracellular virus increased approximately 4-fold when cells were pretreated with tamoxifen, and 3-fold upon pretreatment with rapamycin ( figure 6a and 6b). conversely, when cells were treated with 3-methyladenine, a pharmacological inhibitor of autophagy [56] , yield of intracellular virus was decreased ( figure 6c ). therefore, during poliovirus infection, the activity of the autophagy pathway correlates with productive viral replication, not with viral destruction. to extend the studies with pharmacological agents, we tested the effect of reducing the amount of intracellular autophagy proteins lc3 and atg12p on poliovirus yield, using double-stranded rna oligonucleotides designed to target their mrnas for destruction by rna interference. pools of double-stranded oligonucleotides were synthesized to target atg12 mrna, and to target both lc3a and lc3b mrnas (see materials and methods). as shown in figure 7 , the intracellular abundance of atg12p and lc3 proteins could be reduced to 70% and 90%, respectively, of their abundance in cells treated with control double-stranded small interfering rnas (sirnas) known to target firefly luciferase mrna. these reductions in atg12p and lc3 protein concentration resulted in 3-fold and 4-fold reductions, respectively, in yields of intracellular poliovirus. even stronger effects of the rna interference (rnai)-mediated reduction in autophagy proteins on viral yield were observed, however, when the yields of extracellular virus were examined. the effects of rnai against atg12 and lc3 on extracellular virus were 9-fold and 20-fold, respectively ( figure 7) . two potential mechanisms for this preferential reduction in extracellular, as opposed to intracellular virus, seemed possible. the first possibility was that the rnaimediated reductions in the intracellular concentrations of atg12p and lc3 protein reduced cell lysis and therefore lytic release of virus; links between autophagy and apoptosis have been reported (reviewed in [57, 58] ). the possibility of decreased cellular susceptibility to lysis upon rnai treatment is difficult to exclude. however, early cell lysis in the presence of reduced atg12p or lc3 concentrations seemed unlikely because the amounts of virus released were very small, and the observed reductions in extracellular virus release were seen at early time points, before lysis was expected. a second possibility for the greater reduction in extracellular than intracellular virus is that double-membraned structures with the cytoplasm provide a topologically reasonable mechanism for nonlytic release of cytoplasmic contents. ultrastructural analysis studies of cells infected with poliovirus [1, 2] and with rhinovirus 14 ( figure 8a ) have revealed the presence of virions and other cytoplasmic material within the lumen of double-membraned vesicles. presumably, these are double-membraned vesicles that formed relatively late in infection and were therefore able to trap viruses already present in the nearby cytoplasm. the known proximity of rna replication, protein expression, and virion packaging to the membrane-associated replication complexes [59, 60] should facilitate such events. figure 8b displays an image consistent with the release of the packets of cytosol expected if the outer bilayer of a multilamellar membrane structure were to fuse with the plasma membrane. figure 8c shows that, in poliovirus-infected cells, such blebs can contain lc3, and figure 8d shows that they can also contain vp1, a viral capsid protein. in this work, we have shown that several hallmarks of cellular autophagosomes, including the localization of gfp-lc3 into discrete punctate structures and staining with mdc followed by fixation, can be observed in human cells whether they are treated with tamoxifen or infected with poliovirus or rhinovirus. furthermore, we have employed a new criterion for autophagy, the colocalization of lc3 and lamp1, to demonstrate that this intracellular rearrangement was observed in cells treated with tamoxifen, infected with picornaviruses (poliovirus, rhinovirus 2, or rhinovirus 14) or transfected with plasmids that express poliovirus proteins 2bc and 3a (see figures 2 and 8) . taken together with the previously observed double-membraned morphology, cytoplasmic contents, and complex origin of the membranous structures induced during poliovirus infection [1, 2, 35] , we argue that the viruses utilize components of the autophagosome formation pathway to form the characteristic doublemembraned vesicles seen during infection. do the autophagosome-like membranous structures induced by poliovirus act as scaffolds for rna replication, or are they part of the host antiviral response? the numerous positive correlations between the functional presence of autophagosomal pathways and increased viral yield lead us to conclude that the autophagosome-like structures observed during poliovirus infection are not antiviral. instead, we argue that the double-membraned vesicles induced during poliovirus infection facilitate poliovirus replication, and we hypothesize that poliovirus, rhinovirus 2, and rhinovirus 14 subvert the constituents of the cellular autophagy pathway to form membranous scaffolds on which rna replication complexes can assemble. other positive-strand rna viruses that have been shown to localize their rna replication complexes to double-membraned vesicles in the cytoplasm of infected cells are equine artirivirus [61] , murine hepatitis virus [62] , and sars virus [63] . the membranes associated with murine hepatitis virus infection have been shown recently to contain lc3 protein [64] . strikingly, infection of murine es cell lines deficient for apg5 with murine hepatitis virus was shown to result in a large decrease in the extracellular yield of this enveloped virus; the effect on intracellular viral particles or rna was not reported. nevertheless, the large reduction in yield of extracellular virus seen in the absence of atg5p protein argues that this component of the cellular autophagy pathway is crucial for some step in the formation or egress of infectious virions of this murine coronavirus [64] . for both poliovirus and equine artirivirus, molecular inducers of double-membraned vesicle formation have been identified. specifically, coexpression of poliovirus proteins 2bc and 3a is required to accumulate double-membraned vesicles [35] and to elicit the colocalization of gfp-lc3 and lamp1 that correlates with the formation of autophago-somes (see figure 3a ). for equine artirivirus, the coexpression of viral proteins nsp2 and nsp3 is sufficient to induce the formation of double-membraned vesicles [65] . we anticipate that these viral proteins, likely to be capable of mimicking, intercepting, or corrupting the pathway of cellular autophagy, will prove to be useful tools to decipher its mechanism. recent work has highlighted an important role for autophagy in the innate immune response of vertebrates to intracellular pathogens. for example, induction of autophagy has been shown to promote clearance of mycobacterium tuberculosis from infected macrophage [66] . furthermore, during infection with shigella flexneri, the wild-type function of the bacterial icsb gene was shown to be required to prevent autophagic degradation, a process that the authors argue is specifically induced by a bacterial protein, the product of the virg gene [67] . like s. flexneri, successful microorganisms often display strategies to evade potent host defenses. furthermore, some microorganisms actively subvert otherwise effective host defense responses for their own benefit: for example, the growth of mink focus-forming virus requires apoptotic caspase activity for the maturation of a nonstructural protein [68] , and murine cytomegalovirus encodes a chemokine homolog to attract cells of the immune system to the site of infection, which then promote viral dissemination (reviewed in [69] ). similarly, precedents are beginning to be established in which the autophagic pathway or its constituents may be subverted by intracellular pathogens to benefit their own replication (reviewed in [42, 43] ). as shown in figure 9 , the pathway of autophagosome formation ends with the acquisition of the lysosomal proteases and lipases that render the autophagosome a degradative organelle [27, 28] . mature autolysosomes are no longer bounded by double membranes because the inner membrane and luminal contents are degraded, becoming electron-dense and compact [24] . in legionella infection, several genes, termed dot or icm genes, are required to retard the progression of autophagosome maturation, presumably to benefit bacterial growth within organelles that subvert components of the autophagosome [70, 71, 72] . we argue that poliovirus, rhinovirus 2, rhinovirus 14, equine encephalitis virus [65] , and murine hepatitis virus [64] have similarly evolved a mechanism to accumulate autophagosome-like membranes in the cytoplasm for the duration of the infection. to maintain most of these structures in their double-membraned form, viral infection may both induce their formation and prevent their maturation into degradative organelles. why would a virus choose a double-membraned, autopha-gosome-like vesicle on which to replicate its rna? not all positive-strand rna viruses utilize such structures. for example, flock house virus rna replication complexes assemble on outer mitochondrial membranes [73] . when the rna replication complexes of a subgenomic flock house virus rna were experimentally redirected to the cytoplasmic surface of the er of s. cerevisiae, the yield of replicated rna actually increased [73] . therefore, viral rna replication displayed no mitochondrion-specific requirement for specific lipids, proteins, or processes. presumably, however, there are reasons why particular positive-strand rna viruses target particular intracellular membranes on which to assemble their replication complexes, some of which might not be assayed under single-cycle growth conditions in tissue culture. a larger effect on extracellular than intracellular virus yield was observed when the abundance of autophagy proteins atg12p and lc3 was reduced by rnai (see figure 7 ). possible explanations for the observed preferential decrease in extracellular virions are that the reduction in autophagosome machinery decreased cell lysis early in infection, or that reduced abundance of autophagosomal machinery decreased nonlytic viral escape. although our data do not yet distinguish between these hypotheses, we will discuss the latter possibility because nonlytic delivery of cytosol to the extracellular milieu could be a unique characteristic of multilamellar vesicles, and we were able to obtain ultrastructural images consistent with this interpretation (see figure 8 ). nominally lytic viruses are often assumed to spread exclusively via cell lysis. however, the possibility of nonlytic viral release, even for nonenveloped viruses such as poliovirus, has been suggested from numerous reports of persistently infected cell lines that continuously secreted infectious particles [74, 75, 76, 77] . even more convincingly, when polarized caco-2 cultures, growing as intact monolayers, as shown by resistance to the passage of dyes and electric current between the apical and basolateral surfaces, were infected with poliovirus, newly synthesized virus was shown to emerge from only the apical surface [78] . that the egress of this virus did not correspond to a detectable breach in the monolayer argued that a nonlytic, and polarized exit route of unknown origin had been utilized. we suggest a potential mechanism for this nonlytic release of cytosolic viral particles via the formation of doublemembraned vesicles throughout the course of infection. early in infection, the double-membraned structures would entrap cytosol, but this cytosol would be free of virions. however, at later stages of infection, the cytosol trapped by newly generated double-membraned structures would often contain viral particles. poliovirions and related enteroviruses are relatively resistant to the low ph and active proteolysis that would prevail within the lumen of these vesicles should they figure 3 and prepared for electron microscopy by high-pressure freezing. examples of readily discernable double lipid bilayers are designated with large arrowheads; vesicles that contain intralumenal viral particles are denoted with small arrows. (b and c) cells were transfected with a plasmid that expresses gfp-lc3 and subsequently infected with poliovirus for 5 h as in figure 2 . double-membraned autophagosomes form either from er membrane or de novo, encapsulating cytosol; the action of many gene products, including atg5p and atg12p, are required. lc3 protein (the atg8p homolog) is associated with ''sequestration crescents'' as well as fully formed double-membraned autophagosomes. lamp1 acquisition is a hallmark of the maturation of these structures, which eventually fuse with lysosomes to produce mature autophagosomes with single membranes and electron-dense contents. we hypothesize that infection by poliovirus or rhinovirus induces accumulation of autophagosomes to promote viral rna replication by accelerating the formation of autophagosome-like structures from er membranes, blocking the maturation of these structures into degradative organelles, or both (upper dotted line). the doublemembraned topology makes the extracellular release of virions trapped in the cytosolic lumen topologically plausible, providing a mechanism for viral release in the absence of cell lysis. this could occur either from a double-membraned structure or from one in which only one of the membranes remained (dotted arrows). doi: 10.1371/journal.pbio.0030156.g009 gfp-lc3 was visualized using a secondary antibody coupled to 10-nm gold particles; examples of such particles are denoted with arrows. an arrowhead identifies apparently extracellular packets of cytosol. (d) cells transfected with an gfp-lc3-expressing plasmid and infected with poliovirus as in (c) were immunostained using an antibody directed against vp1, a viral capsid protein and visualized using a secondary antibody conjugated to 10-nm gold particles; examples of such particles are identified with arrows. doi: 10.1371/journal.pbio.0030156.g008 mature [79, 80] . as depicted in figure 9 , the fusion of the outer membrane of an intact double-membraned structure would result in the release of membrane-bound packets of cytoplasm into the extracellular milieu, whereas fusion of the membrane of a mature autophagosome would result in the direct release of cytosolic contents, and incompletely resolved double-membrane structures would result in the formation of more complex topologies. the presence of both lc3 and poliovirus capsid protein vp1 in extracellular structures adjacent to poliovirus-infected cells (see figure 8c and 8d) is consistent with the release of at least partially intact packets of cytoplasm during poliovirus infection. we speculate that the formerly intracellular membranes surrounding these packets of cytoplasm would be short-lived outside the cell, with free virus being the eventual result. recently, hiv has been shown to exit human macrophages via the fusion of multivesicular bodies with the plasma membrane, rather than by directly budding from the cell surface as in hiv-infected t cells [81, 82, 83] . hiv gag protein directs the targeting of hiv particles to multivesicular bodies via direct binding to cellular protein tsg101 [84, 85] , a component of the escrt complex required for the sorting of proteins into endosomes [86, 87, 88] . similarly, the intracellular formation of double-membraned vesicles in poliovirus-infected cells provides a topologically reasonable mechanism for the extracellular delivery of cytosolic contents in the absence of cell lysis. we speculate that a mechanism to spread virus within tissues of infected hosts without cell lysis could provide an advantage to those positive-strand viruses that subvert constituents of the cellular pathway of autophagosome formation. viruses, plasmids, and cells. poliovirus mahoney type 1 was isolated following transfection with an infectious cdna [89] and propagated as previously described [34] . ''tet-off'' mcf7 cells were obtained from clontech (palo alto, california, united states) and propagated in dmem ã¾ 10% fbs; mcf7 cells are highly inducible for autophagosome formation in response to tamoxifen [9] . poliovirus stocks were titered on both hela h1 and mcf7 cells. the multiplicities of infections indicated refer to titers on the appropriate cell lines. h1-hela cells were used due to their permissiveness for rhinovirus infection. rhinovirus stocks were obtained from the american type culture collection (atcc, manassas, virginia, united states). virus stocks were prepared in h1-hela cells (atcc) grown in emem supplemented with 0.2 m hepes and 0.1 m mgcl 2 , and viral titers were measured by tcid 50 as described previously [90] . to construct the gfp-lc3 fusion protein-expressing plasmid, lc3b sequences were amplified by pcr from a human lung library (resgen, carlsbad, california, united states) using targeted primers containing ecori sites (actgaattcccatgccgtcggagaag and tttgaattcttacactgacaatttca). the lc3a coding region was then inserted into the ecori site of pegfp-c3 (clontech, palo alto, california, united states) to create an egfp-lc3 fusion protein under the control of the cmv immediate-early promoter. expression of poliovirus proteins 2bc and 3a was performed as described previously [35] . the 293t cells, which express sv40 t antigen, were used to facilitate expression from the sv40 promoter in these plasmids. immunofluorescence. cells were fixed using freshly made 4% formaldehyde in pbs for 10 min at room temperature. poliovirus protein 3a and cellular protein lamp1 were visualized by indirect immunofluorescence. for visualization of poliovirus 3a protein in the presence of gfp-lc3, cells were washed twice in pbs and incubated in a pbs solution that also contained 0.5% saponin, 10 mm sodium azide, 0.125% bsa, 3a monoclonal tissue culture supernatant at a dilution of 1:30, and rhodamine-linked antimouse secondary antibody (sc-2084, santa cruz biotechnologies, santa cruz, califor-nia, united states) at a dilution of 1:200. cells were incubated at 4 8c for 45 min, washed twice with pbs, and placed under vectashield mounting medium (vector laboratories, burlingame, california, united states). visualization of lamp1 was performed using a monoclonal lamp1 antibody (transduction laboratories, lexington, kentucky, united states) at a dilution of 1:200. mdc staining and costaining. mdc (sigma, st. louis, united states) was stored at ã�20 8c under desiccant. a fresh stock solution of 5 mm mdc was made in 1:1 dmso/etoh immediately prior to adding to cultures. at 1 h before fixation, fresh medium that contained either 10 lm mdc in dmso/etoh or an equivalent volume of dmso/etoh was added to the cells. the cells were then fixed using a freshly made 4% formaldehyde solution in pbs for 10 min at room temperature and imaging was performed immediately. to ensure that membrane vesiculation induced by methods other than the induction of autophagy did not show similar mdc staining patterns, golgi vesiculation was induced with 5 lm ilimaquinone [91] or 5 lm nocodazole [92] ; the disappearance of intact golgi was confirmed using bodipy-c 5 ceramide (molecular probes, eugene, oregon, united states). under these conditions, punctate mdc staining was not observed (data not shown). microscopy and deconvolution analysis. microscopic analysis was carried out on an olympus ix70 at 100x magnification. images were captured and deconvolved using softworx 2.50 on an sgi octane workstation. mdc staining was detected at 360 nm excitation/457 nm emission. gfp-lc3 expression was detected at 490 nm excitation/ 528nm emission. rhodamine was detected at 555 nm excitation/617 nm emission. individual images from each stack were saved as tiff files and processed in adobe photoshop 7.0. rna interference to reduce intracellular concentrations of lc3 and atg12. sirna smartpools, consisting of four rna duplexes targeting the gene of interest, and a control sirna targeting firefly luciferase, were purchased from dharmacon (lafayette, colorado, united states). for lc3, both lc3a and lc3b rnas [93] were targeted, bringing the total number of transfected duplexes to eight. pools consisted of an equal amount of each duplex. the sirna sequences are given as sense/antisense pairs. the sirna sequences for atg12 were: gggaaggacuuacggauguuu/59p-acaucc-guaaguccuucccuu; gaacaccaaguuucacuguuu/59p-acagugaaacuugguguucuu; gcaguagagcgaacac-gaauu/59p-uucguguucgcucuacugcuu; and uguugcag-cuuccuacuucuu/59p-gaaguaggaagcugcaacauu. the sirna sequences for lc3a were: ggacggcuuccucuauauguu/ 59p-cauauagaggaagccguccuu; cggugaucaucgagcg-cuauu/59p-uagcgcucgaugaucaccguu; acaugagcga-guuggucaauu/59p-uugaccaacucgcucauguuu; and cgcccaucgcggacaucuauu/59p-uagauguccgcgaugggc-guu. the sirna sequences for lc3b were: caaaguuccuuguac-cugauu/59p-ucagguacaaggaacuuuguu; gauaauagaac-gauacaaguu/59p-cuuguaucguucuauuaucuu; guagaa-gauguccgacuuauu/59p-uaagucggacaucuucuacuu; and aggagacguucgggaugaauu/ 59p-uucaucccgaacgucuc-cuuu. cells were grown to densities of 1-5 3 10 5 per 6-cm dish in 2.5 ml emem without antibiotics, and transfected using lipofectamine 2000 (invitrogen, carlsbad, california, united states) according to manufacturer's instructions. for each 6-cm dish, 100 total pmol of pooled sirna was diluted in 250 ll of serum-free optimem medium (invitrogen) and, separately, 5 ll of lipofectamine 2000 was diluted in 250 ll of optimem. after an incubation of 5 min at room temperature, the diluted rna and lipofectamine 2000 were combined and incubated for 20 min at room temperature. the 500-ll mixture was then added to each dish and gently rocked to spread the lipid-rna complexes. growth curves and immunoblots were performed 48 h after transfection. total cell extracts were made using rsb-np40 extraction buffer (10 mm tris [ph 7.5], 10 mm nacl, 1.5 mm mgcl 2 , 1% np-40) supplemented with protease inhibitors (roche, mannheim, germany). extract was separated on a 15% laemmli gel and transferred to a pvdf membrane for western blotting. anti-lc3 immunoblotting was performed using rabbit antibody raised commercially (anaspec, san jose, california, united states) against a peptide comprising the first 16 amino acids of murine lc3 (mpsektfkqrrsfeqr). anti-atg12p and anti-gapdh immunoblotting was performed using antibodies from zymed (south san francisco, california, united states) and research diagnostics (flanders, new jersey, united states), respectively. antibodies were diluted 1:3,000 in a pbs solution that also contained 0.1% tween-20 and 2% bsa, and detected with alkaline-phosphatase conjugated goat antirabbit antibody, at a dilution of 1:10,000 using the ecf reagent from amersham biosciences (piscataway, new jersey, united states). high-pressure freezing, freeze-substitution, and electron microscopy (em). for cryofixation and em analysis, h1-hela cells were grown in emem supplemented with 0.2 m hepes and 0.1 m mgcl 2 in flasks. cells were infected with virus at an moi of 50 pfu/cell for poliovirus or 50 tcid 50 /cell for rhinovirus, then washed three times with pbs, trypsinized, and collected by centrifugation. the cell pellet was resuspended in 0.15 m mannitol in pbs and collected by centrifugation. aliquots of the resulting pellet were frozen in a balzers hpm 10 high-pressure freezing apparatus as described previously [2] and stored in liquid nitrogen. to observe cellular ultrastructure, samples were freeze-substituted in 0.1% tannic acid in acetone at ã�80 8c, rinsed in acetone, then warmed at ã�20 8c in the presence of 2% osmium tetroxide in acetone for 16 h, followed by incubation at 4 8c for 4 h. after rinsing in acetone at 4 8c, samples were embedded in epon-araldite resin. thin sections were stained with 2% uranyl acetate and lead citrate and imaged at 80 kv in a jeol 100c or philips cm10 em. for immunostaining, high-pressure frozen samples were freeze-substituted in 0.1% glutaraldehyde-0.05% uranyl acetate in acetone, embedded, stained, and visualized as described previously [35] . the swissprot (http://us.expasy.org/sprot/) accession numbers for the gene products discussed in this article are 2bc and 3a (p03299), atg12p (o94817), atg8p (p38182), lamp1 (p11279), lc3 (q9gzq8), mtor (p42345), sec12p (p11615), sec13p (p5573), sec16p (p48415), sec23p (p15303), sec24p (p40482), and sec31p (o94979). electron microscopic study of the formation of poliovirus cellular origin and ultrastructure of membranes induced during poliovirus infection association of polioviral proteins of the p2 genomic region with the viral replication complex and virusinduced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography membrane rearrangement and vesicle induction by recombinant poliovirus 2c and 2bc in human cells formation of the poliovirus replication complex requires coupled viral translation, vesicle production and viral rna synthesis visualization and 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poliovirus infection of human neuroblastoma cells persistent infection of human erythroblastoid cells by poliovirus persistent echovirus infection of mouse cells expressing the viral receptor vla-2 expression of mutated poliovirus receptors in human neuroblastoma cells persistently infected with poliovirus vectorial release of poliovirus from polarized human intestinal epithelial cells a physico-chemical subgrouping of the mammalian picornaviruses variability in the integrity of human enteroviruses exposed to various simulated in vivo environments infectious hiv-1 assembles in late endosomes in primary macrophage hiv-1 egress is gated through late endosomal membranes cell-type-dependent targeting of human immunodeficiency virus type 1 assembly to the plasma membrane and the multivesicular body tsg101 control of human immunodeficiency virus type 1 gag trafficking and release hiv gag mimics the tst101-recruiting activity of the human hrs protein vps27 recruits escrt machinery to endosomes during mvb sorting hrs regulates multivesicular body formation via escrt recruitment to endosomes the human endosomal sorting complex required for transprot (escrt-i) and its role in hiv-1 budding cloned poliovirus complementary dna is infectious in mammalian cells infection of a human respiratory epithelial cell line with rhinovirus. induction of cytokine release and modulation of susceptibility to infection by cytokine exposure complete vesiculation of golgi membranes and inhibition of protein transport by a novel sea sponge metabolite, ilimaquinone golgi dispersal during microtubule disruption: regeneration of golgi stacks at peripheral endoplasmic reticulum exit sites post-translational modifications of three members of the human map1lc3 family and detection of a novel type of modification for map1lc3b we are indebted to patrice codogno and skip virgin for experimental suggestions and advice and to peter sarnow and katherine shim for helpful comments on the manuscript. we are grateful to susan palmieri and jon mulholland with the stanford cell science imaging facility for assistance with deconvolution microscopy. this work was supported by the hutchison consortium for translation research and the national institutes of health. wtj was supported by a national research service award from the national heart, lung, and blood institute and a stanford dean's fellowship.competing interests. the authors have declared that no competing interests exist.author contributions. wtj, thg, rrk, and kk conceived and designed the experiments. wtj, thg, and mpt performed the experiments. wtj and kk analyzed the data. wtj, mpt, sm, mr, and rrk contributed reagents/materials/analysis tools. kk and wtj wrote the paper. key: cord-346554-a98pjtxs authors: uddin, md bashir; lee, byeong-hoon; nikapitiya, chamilani; kim, jae-hoon; kim, tae-hwan; lee, hyun-cheol; kim, choul goo; lee, jong-soo; kim, chul-joong title: inhibitory effects of bee venom and its components against viruses in vitro and in vivo date: 2016-11-26 journal: j microbiol doi: 10.1007/s12275-016-6376-1 sha: doc_id: 346554 cord_uid: a98pjtxs bee venom (bv) from honey bee (apis melifera l.) contains at least 18 pharmacologically active components including melittin (mlt), phospholipase a(2) (pla(2)), and apamin etc. bv is safe for human treatments dose dependently and proven to possess different healing properties including antibacterial and antiparasitidal properties. nevertheless, antiviral properties of bv have not well investigated. hence, we identified the potential antiviral properties of bv and its component against a broad panel of viruses. co-incubation of non-cytotoxic amounts of bv and mlt, the main component of bv, significantly inhibited the replication of enveloped viruses such as influenza a virus (pr8), vesicular stomatitis virus (vsv), respiratory syncytial virus (rsv), and herpes simplex virus (hsv). additionally, bv and mlt also inhibited the replication of non-enveloped viruses such as enterovirus-71 (ev-71) and coxsackie virus (h3). such antiviral properties were mainly explained by virucidal mechanism. moreover, mlt protected mice which were challenged with lethal doses of pathogenic influenza a h1n1 viruses. therefore, these results provides the evidence that bv and mlt could be a potential source as a promising antiviral agent, especially to develop as a broad spectrum antiviral agent. massive growth in human population, immense rise in urbanization, drastic changes in global environment, and improved connectivity worldwide in terms of better transportation facilities have led to the emergence and re-emergence of viral diseases in human population (li et al., 2011) . outbreaks of many rna viruses are still the leading cause of morbidity and mortality in worldwide population. many antiviral agents against diverse viruses have been reported. however, effective antiviral agents which specifically target to some rna viruses such as sars-cov, influenza a (h5n1, h1n1) and measles are in shortage due to genetic variations of the virus and the lack of approved or universally recommended therapies (wen et al., 2003; lorin et al., 2005; triggiani et al., 2006) . hence, there is a global requirement for continued development of new antiviral agents, especially from natural sources to provide several alternatives for the control, prevention, and management of the spread of diseases caused by rna and dna viruses. number of compounds derived from natural sources are reported to have numerous biological activities, thus considered potential sources of various modern pharmaceuticals including antiviral agents (jacobs and coyne, 2013; kim et al., 2016; weeratunga et al., 2016) . bee venom (bv) of the honey bee apis melifera has been utilize as a traditional medicine for the treatment of rheumatism, arthritis, skin diseases, cancerous tumors, and back pains (billingham et al., 1973; son et al., 2007) due to its antibacterial, antiviral and anti-inflammatory effects (habermann, 1972; son et al., 2007; hwang et al., 2015) . such bio activities are explained by the variety of components contain in the bv such as peptides melittin (mlt), adolapin, apamin, and mast cell degranulating peptide; several enzymes including phospholipase a2 (pla2); biologically active amines (histamine and epinephrine); and non-peptide components (carbohydrates, lipids, and free amino acids) (lariviere and melzack, 1996; park et al., 2004; son et al., 2007) . however, antiviral properties of bv have not well investigated and also its mechanisms are not fully understood. in the present study, we investigated the antiviral activity of bv and its components against enveloped and non-enveloped viruses including rna and dna viruses in vitro and attempted to understand the mechanism of this property is mainly owing to the virucidal effect of both bv and mlt. additionally, we evaluated the antiviral activity of mlt against lethal doses of pathogenic influenza a h1n1 viruses in mice in vivo. bv and mlt were supplied by chung-jin biotech co. ltd, korea and compounds were dissolved in phosphate buffered saline (pbs). stock solutions were aliquoted and stored at -20°c. human embryonic kidney; hek293t (atcc ® crl-11268 tm ), human epithelial cervix adenocarcinoma; hela (atcc ® ccl-2tm), ceropithecus aethiops epithelial kidney; vero (atcc ® ccl-81), human epithelial hela contaminant carcinoma; hep-2 (atcc ® ccl-23 tm ) and canis familiaris kidney epithelial; mdck (atcc ® ccl-34 tm , nbl-2) cells were grown and maintained in dulbecco's modified eagle's medium (dmem) (invitrogen) supplemented with 10% fetal bovine serum (fbs) (gibco) and 1% antibiotic-antimycotic solution (gibco) . all cell cultures were incubated at 37°c in a humidified atmosphere supplied with 5% co2. green fluorescent protein (gfp)-fused vesicular stomatitis virus (vsv-gfp) and herpes simplex virus (hsv-gfp) were propagated on confluent vero cells, and enterovirus-71 (ev-71) and gfp-fused coxsackie virus (h3-gfp) were propagated on confluent hela cells. gfp-fused respiratory syncytial virus (rsv-gfp) was propagated on hep-2 cells. gfp-fused influenza a (a/puertorico/8/34) (h1n1) (pr8-gfp) and challenge viruses of influenza a subtype (a/pr/8/34) (h1n1) were propagated in the allantoic fluid of 10-day-old chicken embryos. all the vsv-gfp, pr8-gfp, h3-gfp, rsv-gfp, and hsv-gfp viruses were kindly gifted from dr. j. u. jung, department of molecular microbiology and immunology, university of southern california, usa. a/pr/8/34(h1n1) was provided by dr. y.k. choi, chunbuk national university, cheongju, republic of korea. the cytotoxic effect of bv or mlt in hek293t, mdck, hep-2, vero or hela cell monolayers were determined by quantifying the cell viability using trypan blue exclusion test as described previously (strober, 2001) . after cells grown in 72well plates into a 75-80% confluent monolayer, different concentrations (0.5-10 μg/ml) of bv or mlt was added to the each well. the dilution medium without the samples was used as a control. experiment was performed in triplicate, and all the plates were incubated at 37°c in a humidified 5% co2. clarified cells were then stained with 0.4% trypan blue (invitrogen) (ratio: 1:1) at 24 h post-treatment (hpt) and mounted to the hemocytometer to obtain the percentages of viable cells. concentrations of the bv or mlt was plotted against the cell viabilities for each cell type and cytotoxicity of the bv to each cell type is expressed as the 50% cytotoxic concentration (cc 50 ), the concentration which 50% of cell death occurred relative to non-treated control cells. ec50 value of bv or mlt against various enveloped and nonenveloped viruses were determined. to determine the ec 50 of bv or mlt, hek293t, hela, vero, mdck, and hep2 cells were cultured in 12-well (1 × 10 6 cells/well) plates, and incubated at 37°c in a 5% co 2 atmosphere. after 12 h, viral suspensions were co-incubated with different concentrations of bv or mlt (0.5-10 μg/ml) at 4°c for 30 min and inoculated to the each cell types; bv and mlt: rr8-gfp (moi= 2.0; mdck), rsv-gfp (moi=1.0; hep2), hsv-gfp (moi =1.0; vero), h3-gfp (moi=2.0; hela), and ev-71 (moi =1.0; hela). ec 50 of bv and mlt for the vsv-gfp (moi =0.2) was tested in hek293t and vero cells, respectively. at 2 h post-infection (hpi), the culture medium was replaced with 10% fbs dmem media, and at 24 hpi, cells or supernatants were harvested and virus titer was evaluated as described below. the ec 50 values were calculated as the bv or mlt concentration yielding 50% reduction of titer compared to the control (virus infection without bv or mlt). the experiments were performed in triplicate. to determine the possible antiviral mechanism of bv, three different treatment groups were designed for comparison of the antiviral effect of bv: (1) bv pre-treated group: hek293t cells were pre-treated with 3.0 μg/ml of bv at 37°c for 12 h and then infected with vsv-gfp (moi=0.2). similarly 1,000 units/ml recombinant human interferon-β (rhifn-β, sigma) was treated as a positive control. as a negative controls, dmem medium only, and virus infection without bv treatment were used; (2) bv co-incubation group: hek293 cells were inoculated with mixture of 2.0 μg/ml bv and vsv-gfp (moi=0.2), which was co-incubated at 4°c for 30 min; and (3) post-treated group (virus replication inhibition after entry): hek293t cells were infected with vsv-gfp first at 37°c for 4 h, and then treated with 3.0 μg/ml of bv. to compare the antiviral effects of bv to vsv-gfp infection, the levels of viral replication (which reflected by gfp expression) were photographed (under 200x magnification) at 12 or 24 hpi of vsv-gfp, and calculated the virus titer by standard plaque assay with some modifications. briefly, vero cells were seeded in 12-well (8 × 10 5 cells/well) plates. after 12 h when cells became confluent monolayer, the harvested cell supernatants containing each replicated virus were 10-fold serially diluted and 100 μl of each dilution was added to the 1% fbs containing each wells. the media in the wells were replaced with 1% agar overlay at 2 hpi and 1-2 days later, number of plaques showing gfp were counted under the microscope (200x magnification). virus titer was calculated using the number of gfp showing plaques and the dilution factor (reed and muench, 1938; coil and miller, 2004) , and expressed as log 10 plaque forming unit/ml (log 10 pfu/ml). to determine whether bv could induce type i ifn, ifn-β secretion levels were measured in hek293t cell supernatants of 3.0 μg/ml bv or rhifn-β (positive control) or dmem medium (negative control) treated samples, which was harvested at 24 hpt. the ifn-β levels were measured using commercial human ifn-β elisa kits (tfb inc.) according to manufacturer's protocol. to determine the antiviral effect of bv or mlt, co-incubation experiments were carried out against different viruses (mlt: vsv-gfp; bv and mlt: pr8-gfp, rsv-gfp, hsv-gfp) in several cell lines. vero, hela, mdck, and hep2 cells were cultured in 12-well (8 × 10 5 cells/well) plates, and incubated at 37°c for 12 h. each viral suspensions (vsv-gfp: moi=0.2; pr8-gfp: moi=2.0; rsv-gfp: moi=1.0; hsv-gfp: moi=1.0) were co-incubated with 2.0 μg/ml of bv or mlt at 4°c for 30 min. after the incubation, the mixture were inoculated to the specified cell monolayers in 12 well plates (vero: vsv-gfp, hsv-gfp; mdck: pr8-gfp; hep2: rsv-gfp) using dmem supplemented with 1% fbs. similarly 1,000 units/ml rhifn-β and viruses were co-incubated and inoculated to the cells, as a positive control for bv antiviral activity assay. dmem medium only and virus infection without any bv or mlt treatments were used as negative controls. unabsorbed viruses and residuals were aspirated out with the supernatant at 2 hpi, and dmem supplemented with 10% fbs. the residual virus infectivity in each treated samples (which reflected by gfp expression) was photographed (under 200x magnification) at 24 hpi. cell supernatants (vsv-gfp, rsv-gfp, and hsv-gfp) or cells (pr8-gfp) were collected at 24 hpi, and expressed gfp was quantified by measuring gfp absorbance using glomax detection system (promega). viral replication levels were calculated by standard plaque assay as described. this virus titer was considered as the residual infectivity (log10 pfu/ml) to evaluate the virucidal activity of the bv or mlt against different viruses tested. bv or mlt antiviral activity was tested in hela cells with h3-gfp and ev-71 viruses using standard antiviral activity assays. h3-gfp (moi=1.0) or ev-71 (moi=1.0) viral suspensions were co-incubated at 4°c for 30 min with 2.0 μg/ml of bv or mlt. after the incubation, the mixtures were inoculated to the 12 h cultured monolayers of hela cells (6well plates; originally seeded 1 × 10 6 cells/well). rest of the antiviral assay procedures were similar to the above section. additionally, bv or mlt antiviral activity for ev-71 was determined by evaluating 50% tissue culture infective dose (tcid 50 ) and analysis of mrna expression levels of the ev-71 vp1 gene. the residual infectivity was titrated for the each samples collected at 24 hpi by tcid50, (the amount of virus required to produce cytopathic effect in 50% of inoculated cells) as described previously and expressed as log 10 tcid50/ml . moreover, ev-71 vp1 gene mrna expression levels were determined at 24 hpi. total mrna from the cells, which inoculated after co-incubation of ev-71 (moi=1.0) with bv (2.0 μg/ml) or mlt (2.0 μg/ml) was extracted using rneasy mini kit (qiagen) and cdna was synthesized using reverse transcriptase (toyobo) according to manufacturer's protocol. to determine the ev-71 mrna expression levels, ev-71 vp1 gene was amplified using gene specific primers (ev-71 vp1 forward primer; vp1fw-5 -ttgacaaaaactgaggggtt-3 and the reverse primer; vp1rev-5 -ttgacaaaaactgaggggtt-3 (wen et al., 2003) . gapdh, forward primer 5 -tgacca cagtccatgccatc-3 and the reverse primer 5 -gac ggacacattgggggtag-3 were used as a house keeping gene (seal et al., 1995) . after the real-time polymerase chain reaction (rt-pcr), equal amount of amplified products were run on 1.5% ethidium bromide agarose gels and visualized using geldoc imaging system (bio-rad). the relative vp1 gene fragment band intensity (rbi) was compared with gapdh using band quantification software (bio-rad). to find out the effective time point which mlt exhibits virucidal activity, 2.0 μg/ml of mlt and vsv-gfp (moi=0.2) were co-incubated for 5, 10, 20, and 30 min at 4°c, and then mixture was inoculated to hek293t cell monolayers in 6 well plates. dmem medium only and virus infection without any mlt treatments were used as negative controls. unattached viruses and mlt were aspirated out with the supernatant at 2 hpi, and dmem supplemented with 10% fbs was added to the wells. the virucidal effect of mlt was analyzed by observing the gfp expression under 200× magnification at 24 hpi. gfp absorbance and virus titer was quantified as described in earlier sections. to determine the effect of mlt on virus infection and the possible effect on the initial stages of viral replication cycle in vitro, following treatment groups were designed: (1) virus attachment assay: co-treatment; 2.0 μg/ml of mlt and pr8-gfp (moi=2.0) were simultaneously added to the mdck cells for 1h at 4°c. residual virus or mlt were aspirated out with the supernatant at 2 hpi, and replaced with 10% fbs dmem media; (2) entry assay: post-treatment; mdck cells were infected with pr8 (moi=2.0) first, and after 1 h of adsorption, cells were washed 3 times with pbs to remove unattached viruses and treated the cells with dmem (10% fbs) containing 2.0 μg/ml of mlt. dmem medium only and virus infection without any mlt treatments were used as negative controls. effect of mlt on virus attachment and virus entry to the cells was determined by measuring the gfp expression at 24 hpi, using glomax multi-detection system (promega). virus titer was determined as described in method section and expressed as log10 pfu/ml. sedimentation velocity ultracentrifugation (gastaminza et al., 2006) was carried out to delineate the virucidal mechanism of enveloped viruses. in here, sucrose gradient separation procedure was conducted with some modifications to identify the fractions containing the infectious viral particles and to estimate their size (molecular mass) by immonoblotting. in brief, a discontinuous sucrose density gradient was prepared by layering successive decreasing sucrose density solutions (from 20-60%) upon one another in the polyallomer tubes. once the discrete layers of sucrose was visible, pr8-gfp mixed with pbs or pr8-gfp mixed with mlt, which was co-incubated at 4°c for 30 min were applied on to the top. the tubes were ultra-centrifuged in swinging bucket sw-28 rotor (beckman) at 26,000 rpm at 4°c for 4 h. after ultracentrifugation, without disturb the sucrose layers, total 30 ml of fractions (assuming that some of the fractions containing virus particles) were collected (30 × 1 ml) from all the gradients. in brief, total 6 ml from each gradient (6 × 1 ml) was collected (3 × 1 ml from upper layer and 3 × 1 ml from lower layer from each sucrose gradient). out of these fractions, 2 nd and 5 th fractions from each gradient (2 nd and 5 th fractions of 20%, 30%, 40%, 50%, and 60% denoted sequentially from 1 to 10 in the figs.) were taken for the immunoblotting using polyclonal antibody derived from h1n1 virus infected mouse serum to identify the which fractions containing the virus particles. as an immunobloting positive control (pc), native pr8-gfp which heated at 100°c for 10 min prior to loading was used. moreover, the same fractions which used for immunobloting were examined for the infectivity of the virus particles by observing and measuring the gfp expression at 24 hpi, after infection to mdck cells. all the animal studies conducted under appropriate conditions with the approval of the institutional animal care and use committee of bioleaders corporation, daejeon, south korea, protocol number: bls-abls-13-008 and all the treatment and challenge experiments were conducted in an approved bsl-2 + laboratory facility. in this study, mouse model was chosen to determine whether mlt exhibits antiviral protection in vivo. mice survival rate and the body weight were evaluated after challenge with co-incubated mixture of pbs or mlt and influenza a virus subtype h1n1 (pbs/h1n1 or mlt/h1n1). for the whole experiment, 6-week-old 20 c57bl/6 female mice (17 ± 1 g) were used and for the survival rate analysis, 12 mice were divided into 2 groups; pbs/h1n1 treated (control) (n=6) and mlt/h1n1 treated (n=6). for the lung virus titration, another 8 mice were grouped into 2 groups; pbs/h1n1 (n=4) and mlt/h1n1 treated (n=4). first, 15 μl of mlt (100 ng) was co-incubated with 15 μl of 5´ minimum lethal dose (mld 50 ) of h1n1 for 30 min at 4°c, and then 30 μl from the mixture was intranasally administered per mice (5.9 ng/g body weight). body weight and survival rate were noted up to 8 days post-infection (dpi). mice showing more than 25% body weight loss were considered as an experimental end point and were humanely killed. lung tissues were collected aseptically from euthanized mice at 5 dpi and homogenized with equal volume (1 ml/g) of pbs containing 1% antibiotic/antimycotic solution. samples were centrifuged (10,000 × g at 4°c for 10 min), supernatants were immediately 10-fold serially diluted and inoculated into cultured mdck cells grown to confluence on 96 well tissue culture plates. the lung viral titer was calculated by the reed and muench method and expressed as log10 tcid 50 /lung . statistical analysis was performed using graphpad prism software version 6 for windows (graphpad software). all in vitro assays were carried out in triplicate and the data were expressed means ± standard deviations (sd). differences between groups were analyzed by analysis of variance (anova) or student's t-test. comparison of survival was done by logrank test using graphpad prism 6 version. p < 0.05 were regarded as significant. the different concentrations of bv was tested for all the cell types used in this study to determine whether the bv is cytotoxic to the cells. cc 50 of bv and mlt in vitro is presented in the table 1 . as shown, bv cc50 for the hek293t, hela and mdck cells were 8.61 ± 0.02, 8.98 ± 0.07, and 8.35 ± 0.08 μg/ml, respectively, representing 50% of the cells were viable at this concentration to the cells. lowest cc50 value of bv was obtained for the hep2 (6.25 ± 0.12 μg/ml) among tested cell lines followed by vero cells (7.13 ± 0.1 μg/ml). the cytotoxic concentration of mlt was also tested via tryphan blue staining and cc 50 of mlt for vero, mdck, hep2 and hela cells were 6.23 ± 0.02, 7.66 ± 0.94, 5.02 ± 0.17, and 4.36 ± 0.54 μg/ml, respectively. according to the cc50 values, mlt dose which showing more than 90% cell viability was selected for the experiments; 2.0 μg/ml or low mlt dose for vero, mdck, and hep2 cells, and 1.0 μg/ml or low mlt dose for hela cells. ec 50 of bv and mlt in vitro is presented in the table 1 . ec 50 value of bv against vsv-gfp, pr8-gfp, rsv-gfp, hsv-gfp, h3-gfp, and ev-71 were 0.5 ± 0.06, 1.81 ± 0.08, and 1.17 ± 0.09, 1.52 ± 0.11, and 0.5 ± 0.04, respectively and virus replication was inhibited by 50% at this concentrations. moreover, ec 50 of mlt was approximately 1.18 ± 0.09, 1.15 ± 0.09, 0.35 ± 0.08, 0.94 ± 0.07, 0.99 ± 0.09, and 0.76 ± 0.03 μg/ml for vsv-gfp, pr8-gfp, rsv-gfp, hsv-gfp, h3-gfp, and ev-71, respectively. the data demonstrated that ec50 of bv against vsv-gfp and h3-gfp were lower than the ec 50 values of mlt. moreover, bv exhibited higher selectivity index (si) than the mlt against vsv-gfp and h3-gfp (table 1) . considering effectiveness and cytotoxicity, 1.0 or 2.0 μg/ml of bv or mlt was used for the subsequent in vitro antiviral assays and utilized doses were mentioned in the method section where appropriate. from above, we could suggest that the antiviral activity of bv or mlt against the viruses examined, were not resulted from the cytotoxicity of the peptide on the tested cells. to determine the antiviral activity and to understand the possible antiviral mechanism of bv, three different treatments were conducted to vsv-gfp virus in hek293t cells as described in material and methods. bv (3.0 μg/ml) pre-treating at 37°c for 12 h before infecting the vsv-gfp to the cells (virus/peptide pre-treatment) showed significant reduction of virus replication at 24 hpi (354-fold), which the pattern was comparable to the rhifnβ pre-treating, compared to the only vsv-gfp infected cells (fig. 1a) . moreover, ifn-β levels were measured in the bv (3.0 μg/ml) treated cell supernatant of hek293t (fig. 1b) . bv significantly induced higher levels of secreted ifn-β compared to the control (medium only), which was similar to the pattern observed in rhifn-β treated hek293t cell supernatant. thus, data indicated that bv can mediate the antiviral innate immune responses in epithelial cells by triggering the expression of type i ifn, which may stimulate the antiviral state in the cells. as shown in fig. 1c , bv (2.0 μg/ml) co-incubating with vsv-gfp at 4°c for 30 min before inoculating to the cells (virus/peptide co-incubation) also showed prominent reduction in virus titer at 24 hpi (5,370-fold) compared to the only vsv-gfp infected cells. although in a low degree, hek293t cells infected with vsv-gfp and treated with 3.0 μg/ml of bv (post-treatment) at 4 hpi also showed inhibition of virus replication at 12 (16136-fold) or 24 hpi (63-fold) (fig. 1d ). both bv and rhifnβ post-treated cells showed similar virus titer, espe-cially at 12 hpi, and it was significantly lower than the only vsv-gfp infected cells. based on the three treatment modes carried out, we speculate that anti-viral activity of bv attributed to stimulating type i ifn signaling, virucidal activity, and ability for the inhibition of virus replication after virus enter to the cells. after investigation the anti-vsv efficacy of the bv, its effectiveness against other enveloped viruses were investigated. pr8-gfp, rsv-gfp, and hsv-gfp co-incubating with bv (2.0 μg/ml) at 4°c for 30 min before inoculating to the cells (virus/peptide co-incubation) showed reduction in the virus infectivity (fig. 2) . bv treated pr8-gfp ( fig. 2a) , rsv-gfp (fig. 2b) , and hsv-gfp (fig. 2c) showed distinct reduction in gfp expression (3-fold, 2-fold, and 7-fold, respectively) and virus titer (175-fold, 7-fold, and 9-fold, respectively) at 24 hpi compared to the untreated virus infected groups. this suggest that bv could disrupt the infectivity of broad spectrum of enveloped viruses, which further convincing its virucidal effect. . gfp expression levels were measured by gloma multi dectection luminometer (promega). the virus titers were determined by standard plaque assay. virus titers were expressed as mean ± sd. scale bars, 100 μm. error bars indicate the range of values obtained from triplicate in three independent experiments (*p < 0.05 and **p < 0.01 indicates a significant difference between groups compared by student's t-test). after investigated the different antiviral effect of the bv, several compounds in bv were tested to find out which component of the bv responsible for its virucidal effect. among those, only mlt showed the similar effects as bv. for further confirmation, the virucidal effect of mlt against enveloped rna (vsv-gfp, pr8-gfp, and rsv-gfp) and dna (hsv-gfp) viruses were tested. in all the tested cells, there was a direct effect of the mlt to the virus which shows reduced virus infectivity (fig. 3) . different viral suspensions were coincubating with mlt (2.0 μg/ml) at 4°c for 30 min before inoculating to the cells showed marked reduction in gfp expression at 24 hpi, whereas the untreated virus infected groups showed significantly high levels of gfp expression for vsv-gfp (fig. 3a) , pr8-gfp (fig. 3b) , rsv-gfp (fig. 3c ), and hsv-gfp (fig. 3d) . in relation to this, mlt treated groups showed reduced viral supernatant titers for vsv-gfp (1598-fold), pr8-gfp (5-fold), rsv-gfp (82-fold), hsv-gfp (16-fold) compared to the only virus infected group. collectively, it suggests that mlt has the anti-viral activity by the virucidal effect against broad spectrum of enveloped viruses. after identifying that bv has virucidal effect on broad spectrum of enveloped viruses, its efficacies against non-enveloped rna viruses such as h3-gfp and ev-71 were also evaluated. h3-gfp and ev-71 viral suspensions were coincubated for 30 min at 4°c with 2.0 μg/ml bv or rhifn-β, and the mixture (bv/h3-gfp and rhifn-β/h3-gfp or bv/ ev-71 and rhifn-β/ev-71) was inoculated to the monoev-71/vp1 gene mrna expression was evaluated using vp1 gene specific pcr primers and all the samples normalized using gapdh. rbi (gene/ gapdh) was determined using geldoc imaging system band quantification software (c and f). virus titers were expressed as mean ± sd. scale bars, 100 μm. error bars indicate the range of values obtained from triplicate in three independent experiments (*p < 0.05 and **p < 0.01 indicates a significant difference between groups compared by student's t-test). layers of hela cells. at 24 hpi, marked reduction of gfp expression was observed in bv/h3-gfp treated cells compared to untreated cells (fig. 4a) . in relation to this, significantly lower (977-fold) virus titer was detected in bv/h3-gfp treated cells than the only h3-gfp infected cells (fig. 4a) and was comparable to virus titers of the rhifn-β/h3-gfp treated cells. moreover, ev-71 induced cpe was markedly inhibited and the cell viability was higher in the bv/ev-71 treated cells than the only ev-71 infected group (data not shown). the only ev-71 infected cells showed significantly higher (131-fold) tcid 50 than bv/ev-71 treated cells (fig. 4b ). ev-71 vp1 mrna expression levels were decreased in bv/ev-71 treated cells compared to the only ev-71 infected cells by 2-fold (fig. 4c) , exhibiting lower rbi of the vp1 gene. interestingly, mlt co-incubated with h3-gfp also showed lower gfp expression (1.5-fold) and virus titers (5-fold) in comparison to the only h3-gfp infected cells (fig. 4d) . ev-71 exhibited significant reduction in cpe and high cell viability in the mlt/ev-71 treated cells than the only ev-71 infected group (data not shown). additionally, only ev-71 infected cells showed significantly higher (236-fold) tcid50 than mlt/ev-71 treated cells (fig. 4e) . moreover, ev-71 vp1 mrna expression levels were decreased in mlt/ev-71 treated cells compared to the only ev-71 infected cells by 4-fold (fig. 4f) , exhibiting lower rbi of the vp1 gene. collectively, data demonstrated that bv or mlt exerts its virucidal antiviral activity not only against enveloped viruses, but also to non-enveloped viruses. . gfp expression levels were measured by gloma multi dectection luminometer (promega). the virus titers were determined by standard plaque assay. virus titers were expressed as mean ± sd. scale bars, 100 μm. error bars indicate the range of values obtained from triplicate in three independent experiments (*p < 0.05 and **p < 0.01 indicates a significant difference between groups compared by student's t-test). to determine the effective time needed for mlt to act on virus, 2.0 μg/ml mlt was incubated with 0.2 moi vsv-gfp at 4°c at different times. as shown in fig. 5a , infectivity of vsv-gfp was dramatically suppressed with the increasing time (5-30 min) of incubation. virus infectivity initiated to reduce from 5 min incubation and during 30 min incubation, it showed high virucidal effect similar to our previous data. this suggests that mlt can inactivate or inhibit virus replication even it was contact with the virus for a short period of time and inactivate the infectivity. to further understand the effect of mlt on viral replication cycle, virus-cell attachment and virus-entry assay were performed. as shown in fig. 5b , gfp absorbance and virus titers of mlt treated groups were not significantly different compared to the control (only virus infected) in both attachment and entry assays. these results indicate that mlt could not interfere the initial steps of the virus life cycle and unable to inhibit virus infection and replication, once the virus is infected to the cells. it evident from the data that mlt involves in the steps occurring prior to virus-cell attachment, mainly may direct interaction of the virus and mlt, and in turn inhibiting the virus infectivity. to clarify the virucidal activity of the mlt against enveloped viruses, sedimentation velocity ultracentrifugation was conducted using continuous sucrose gradient for pr8-gfp viruses co-incubated with pbs or mlt and subjected to immunoblotting as described in materials and methods. as shown in the fig. 6a , surface viral proteins such as hemagglutinnin (ha), nucleoprotein (np), and neuraminidase (na) of pr8-gfp co-incubated with pbs were detected in the 40% sucrose . 6 . virucidal mechanism study of mlt by sedimentation velocity ultracentrifugation. pr8-gfp virus was co-incubated with the pbs or mlt for 30 min at 4°c. samples were loaded on the top of the 20-60% continuous sucrose density gradient and placed into ultracentrifugation with sw-28 rotor at 26,000 rpm for 4 h in 4°c. fractions were collected and 2 nd and 5 th fractions out of six layers from each sucrose gradient were subjected to immunoblotting. 2 nd and 5 th fractions of 20%, 30%, 40%, 50%, and 60% numbered sequentially from 1 to 10. (a) pr8-gfp viral proteins (ha, na, and np) containing in each fraction was detected using polyclonal antibody derived from h1n1 virus infected mouse serum. native pr8-gfp was loaded as an immunobloting positive control (pc). (b and c) the same fractions which used for immunobloting were examined for the infectivity of the virus particles by observing and measuring the gfp expression at 24 hpi, after infection to mdck cells. gfp images were obtained under fluorescence microscopy (200x magnification). gfp expression levels were measured by gloma multi dectection luminometer (promega). gfp expression levels were expressed as mean ± sd. scale bars, 100 μm. error bars indicate the range of values obtained from triplicate in three independent experiments (*p < 0.05 and **p < 0.01 indicates a significant difference between groups compared by student's t-test). cushion (center fractions number 5 and 6), corresponding to the protein band sizes of the pr8-gfp positive control (pc). in contrast, those proteins of the pr8-gfp co-incubated with mlt were observed mainly at the upper sucrose gradient fractions (20% sucrose cushion) (number 1 and 2) followed by slightly in the 30% (fraction number 3 and 4), suggesting that some structural destabilization has occurred due to the mlt, however, no effect to the virus structural proteins. moreover, pr8-gfp viruses treated with pbs fractions (40% sucrose cushion) showed high virus infectivity in vitro, however, viruses co-incubated with mlt did not show the infectivity at over 40% sucrose cushions ( fig. 6b and c) . cells that were treated with the fractions from 20 to 30% sucrose cushion were very weakly infected, probably due to the remained intact virus particles that has not been interacted with the mlt. from the mass differences between native pr8-gfp and the mlt co-incubated with pr8-gfp fractions, it demonstrates that mlt has direct effect on structure destabilization of the virus particle, thus interrupts the virus infectivity. to evaluate the efficacy of mlt in vivo, mlt (100ng) were incubated with 5mld50 of h1n1, for 30 min at 4°c and the mixture (pbs/h1n1 or mlt/h1n1) was inoculated intranasally and monitored daily for morbidity. no toxicity was observed in mlt/h1n1 treated mice, as indicated by weight loss (fig. 7a ) and the survivability (fig. 7b) . mlt/h1n1 treated mice showed slightly decreased body weight until 2 dpi and regained its weight until 8 dpi, whereas, pbs/h1n1 treated mice exhibited severe weight loss and most of all (5mld50/mouse) mixture co-incubating for 30 min at 4°c were intranasally administrated to six-week-old c57bl/6 female mice (17±1 g/mouse). body weight (a) and survival rate (b) were monitored up to 8 dpi (n=6/each group). lung tissues from the infected mice were collected at 5 dpi and viral loads in the lung were measured by standard plaque assay (n=4/each group) (c). (**p < 0.01 indicates a significant difference between groups compared by student's t-test). the mice succumbed to infection and started to die by 7 dpi (showing more than 25% body weight loss) and all were died at 8 dpi. moreover, the control group mice exhibited several respiratory disease clinical signs such as decreased activity, hunched posture, huddling, and ruffled fur. mlt/ h1n1 treated mice showed increased protection and body weight with no clinical signs indicating mlt can protect the mice from lethal virus infections. moreover, influenza virus transmitted mainly through the respiratory system of mice and replicated most efficiently in lungs (bouvier and lowen, 2010) , lung titer was evaluated in pbs/h1n1 or mlt/h1n1 treated mice. mlt/h1n1 treated mice showed lower lung viral titer (log10 tcid50 1.53 ± 0.25) than the pbs/h1n1 treated mice (log 10 tcid 50 4.22 ± 0.27) at 5 dpi (fig. 7c) . these results indicate that mlt has ability to inhibit viral replication against lethal infections of influenza a viruses. animal venom and their derivatives are potential natural agents for innovative drug development against several diseases, however, many of their components and functions are fully unexplored. bv is a cocktail of natural peptides with diversity of biological activities, hence bv and its components are gaining interests to consider valuable resource for use in drug design and development. in this study, potential antiviral activities of bv against vsv-gfp virus infection were first evaluated using pre-treatment, co-incubation or post-treatment experiments ( fig. 1 ) interestingly, bv inhibited the replication of vsv-gfp in all the experiments. among the three different modes of antiviral activities of bv studied, we mainly focused on the antiviral effect of bv by the co-incubation experiments. bv mainly constitutes mlt, pla2, biologically active amines (apamin, adolapin), diverse peptides and non-peptide components (habermann, 1972; lariviere and melzack, 1996; park et al., 2004; son et al., 2007) , thus we investigated antiviral activity of bv main constituents using the co-incubation experiment. as a result, it was found that mlt the only compound that showed the similar effects with bv (data not shown). mlt constitutes almost 40-50% of dry bv and is an α-helical cationic peptide with hemolytic activity in human erythrocytes (tosteson et al., 1985) and other eukaryotic cells. it has a broad antimicrobial spectrum due to its ability to disrupt lipid cell membranes (habermann, 1972; tosteson et al., 1985) . mlt rapidly binds to human red blood cells already in sub micro-molar concentrations, induces release of hemoglobin, increase membrane permeability, and finally lysed the cells (degrado et al., 1982) . mlt disrupts cell membranes and kills gram-negative and gram-positive bacteria in micro-molar concentration range (maher and mcclean, 2006) . besides its powerful antimicrobial and lytic activity, mlt exhibits a variety of other biological effects. mlt hinder transport pumps, such as (h + + k + ) atpase (cuppoletti et al., 1989) and (na + + k + ) atpase (cuppoletti and abbott, 1990) and accelerate the permeability of cell membranes to ions. it induces inhibition and aggregation of membrane proteins (clague and cherry, 1988; mahaney and thomas, 1991) , and also mlt stimulates activity of phospholipase a2 (kuchinka and seelig, 1989) . furthermore, mlt could interfere with the activity of cellular enzyme, na+, k+ atpase, which responsible in membrane fusion process and suppresses cell fusion mediated by hsv-1 syncytial mutants (baghian and kousoulas, 1993) . matanic and castilla (2004) speculate that broad spectrum of antiviral actions might be due to ability of mlt to affect ion gradients across cell membrane. previous studies also showed that mlt display anti-viral activity against animal and plant viruses such as murine retroviruses (esser et al., 1979) , hsv-1 and hiv-1 (wachinger et al., 1992 (wachinger et al., , 1998 baghian et al., 1997) and tobacco mosaic virus (tmv) (marcos et al., 1995) , nevertheless, the mechanism of mlt activity has been not fully discovered in most cases. in this study, antiviral effect of mlt was examined against broad spectrum of viruses. mlt exhibited antiviral activity against enveloped viruses such as influenza a virus (pr8), vesicular stomatitis virus (vsv), respiratory syncytial virus (rsv), and herpes simplex virus (hsv). additionally, mlt also inhibited the replication of non-enveloped viruses such as enterovirus-71 (ev-71) and coxsackie virus (h3). based on our data, virus infectivity in the cells has decreased as the incubation time of the virus and mlt increased, and in the attachment or entry assay, mlt was unable to inhibit virus infection and replication (fig. 5 ). velocity sedimentation ultracentrifugation followed by immunoblotting convinced that mass of the pr8-gfp co-incubated with mlt was reduced compared to native pr8-gfp (fig. 6) . consequently, it could suggest that antiviral activity of mlt was mainly occurred due to involvement of direct interaction with the virus surfaces. this direct effect may involves to destabilize the virus structure and inactivates the viral particles (virucidal activity) which interrupts the virus infectivity. these results suggest that mlt may binds to the virus surface of the enveloped virus by surface charge interaction in virus and decrease the infectivity of the viruses (li et al., 2011) . previously, esser et al. (1979) showed that the antiviral activity of mlt has been attributed to direct lysis of viral membranes, which demonstrated to murine retroviruses. although electron microscopy or crystal structure analysis of the mlt interacting with the viral membranes has not been conducted in this study, previous information of the mlt crystal structure analysis and bio physical properties (a-helical, amphipathic, hydrophobic, and cationic) (dempsey, 1990) , it could assume that mlt may also able to interact with the phospholipid bilayer of viral envelope and cause alterations of lipid organization in the membrane as reported for hemolysis (degrado et al., 1982; terwilliger et al., 1982) or forms of ion-permeable channels in the way of the voltage gated 'pore' (tosteson et al., 1985) or micellize disc formation in the membranes which could diffuse cell contents through the holes produced (dufourc et al., 1986a (dufourc et al., , 1986b . however, the exact mechanism involves not yet identified and needs to be elucidated in future studies. moreover, one of the interesting data obtained from this study was that mlt can inactivate not only enveloped viruses, but also non-enveloped viruses such as enterovirus-71 (ev-71) and coxsackie virus (h3). however, mlt effects to which constituent of the virus and extended virucidal activity mechanism against non-enveloped viruses needs to be investigated in detail. additionally, apart from the virucidal activity of the bv, it was found that pre-treatment or post-treatment of bv can inhibit the virus replication. especially, pre-treatment experiment demonstrated that bv could induce the type i ifn (ifn-β) secretion in hek293t cells (fig. 1b) . this data suggest that bv can mediate the antiviral responses in the epithelial cells by triggering the antiviral state, playing a crucial role in inhibition of viral replication. moreover, post-treat-ment experiment of bv also showed the inhibition of vsv-gfp replication. however, mlt did not show the induction of type i ifn at 2 and 3 μg/ml (data not shown). from these results, it could assume that biological active components present in the bv other than the mlt can inhibit the specific viral replication steps of vsv. consequently, it could expect that bv or specific compounds of bv might play a role for viral replication step and various innate immune pathways that can be involved in bv anti-viral activity, especially, stimulation of type 1 ifn signaling, however, mlt has no effect on the stimulation of type 1 ifn. the detail relationship between the mechanisms of the antiviral effects of bv and active compounds present, which has not been identified yet in the bv should be investigated further. cytotoxicity of the peptide is one of the main difficulty rise during the clinical usage of therapeutic peptide, hence, cytotoxicity was evaluated for the bv and mlt and it exhibited range of cc50 (4.36-8.98 μg/ml) for tested cells (table 1) . it could suggest from the data that the antiviral activity of bv or mlt against the viruses examined, were not resulted from the cytotoxicity of the peptide on the tested cells. further, in vivo studies also showed no toxicity signs in the mouse during challenge experiments (fig. 7) and mice protected from lethal infection of influenza a virus (h1n1). this suggest that mlt can be used safely as an antiviral agent against broad spectrum of viruses with in maximum efficacy dependent on the cell type, however, its safety in concern and mechanism of action needs to be elucidated more in detail in future. in summary, this study demonstrated that bv and mlt derived from bv exhibited potent antiviral activity against various enveloped and non-enveloped viruses in vitro and influenza virus in the in vivo mouse model. moreover, its antiviral mechanism has been confirmed to involve direct interaction with the viral surface. apart from bv virucidal activity, bv can stimulate type i ifn, which subsequently could stimulate the antiviral state in the host cell and also inhibit the viral replication. taken together, these results suggest that bv or mlt has potential to become prophylactic or therapeutic agent for infectious viral diseases. md bashir uddin, byeong-hoon lee designed and executed all virus infection experiments; chamilani nikapitiya, jae-hoon kim, tae-hwan kim and hyun-cheol lee executed all cell biological experiments; choul goo kim and jong-soo lee analyzed the data. jong-soo lee and chul-joong kim designed the overall study and wrote the paper. all animal experiments were approved by the institutional animal care and use committee of bioleaders corporation (reference no. bls-absl-13-008), and were performed in accordance with the guide for the care and use of laboratory animals from the us national institutes of health. none of the authors have any financial or personal relationships with other people or organizations that could inappropriately influence or bias this study. polyphenols and disease risk in epidemiologic studies an amphipathic alpha-helical synthetic peptide analogue of melittin inhibits herpes simplex virus-1 (hsv-1)-induced cell fusion and virus spread role of the na + , k + pump in herpes simplex type 1-induced cell fusion: melittin causes specific reversion of syncytial mutants with the syn1 mutation to syn + (wild-type) phenotype letter: an anti-inflammatory peptide from bee venom animal models for influenza virus pathogenesis and transmission glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus comparison of p25 presequence peptide and melittin. red blood cell haemolysis and band 3 aggregation phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus interaction of melittin with the (na + + k + )atpase: evidence for a melittin-induced conformational change kinetics and mechanism of hemolysis induced by melittin and by a synthetic melittin analogue the actions of melittin on membranes morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles molecular details of melittin-induced lysis of phospholipid membranes as revealed by deuterium and phosphorus nmr disassembly of viral membranes by complement independent of channel formation differential biophysical properties of infectious intracellular and secreted hepatitis c virus particles bee and wasp venoms therapeutic effects of bee venom on immunological and neurological diseases can thousands of years of ancient medical knowledge lead us to new and powerful drug combinations in the fight against cancer and dementia? inhibitory effects of an aqueous extract from cortex phellodendri on the growth and replication of broad-spectrum of viruses in vitro and in vivo interaction of melittin with phosphatidylcholine membranes. binding isotherm and lipid headgroup conformation the bee venom test: a new tonic-pain test virucidal activity of a scorpion venom peptide variant mucroporin-m1 against measles, sars-cov and influenza h5n1 viruses a paediatric vaccination vector based on live attenuated measles vaccine effects of melittin on molecular dynamics and ca-atpase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro inhibition of a plant virus infection by analogs of melittin antiviral activity of antimicrobial cationic peptides against junin virus and herpes simplex virus antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of nf-kappab through interaction with the p50 subunit a simple method of estimating fifty percent endpoints increased sensitivity of sars-coronavirus to a combination of human type i and type ii interferons characterization of newcastle disease virus isolates by reverse transcription pcr coupled to direct nucleotide sequencing and development of sequence database for pathotype prediction and molecular epidemiological analysis therapeutic application of anti-arthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds trypan blue exclusion test of cell viability the structure of melittin in the form i crystals and its implication for melittin's lytic and surface activities melittin lysis of red cells activation of human inflammatory cells by secreted phospholipases a2 antimicrobial peptides melittin and cecropin inhibit replication of human immunodeficiency virus 1 by suppressing viral gene expression influence of amphipathic peptides on the hiv-1 production in persistently infected t lymphoma cells interferon-mediated antiviral activities of angelica tenuissima nakai and its active components comparative study of enterovirus 71 infection of human cell lines the key: cord-103868-iwpiti2h authors: harrison, angela r.; moseley, gregory w. title: the ebola virus interferon antagonist vp24 undergoes active nucleocytoplasmic trafficking date: 2020-08-11 journal: biorxiv doi: 10.1101/2020.08.10.245563 sha: doc_id: 103868 cord_uid: iwpiti2h viral interferon (ifn) antagonist proteins mediate evasion of ifn-mediated innate immunity and are often multifunctional, having distinct roles in viral replication processes. functions of the ebola virus (ebov) ifn antagonist vp24 include nucleocapsid assembly during cytoplasmic replication and inhibition of ifn-activated signalling by stat1. for the latter, vp24 prevents stat1 nuclear import via competitive binding to nuclear import receptors (karyopherins). many viral proteins, including proteins from viruses with cytoplasmic replication cycles, interact with the trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis. despite established karyopherin interaction, the nuclear trafficking profile of vp24 has not been investigated. we find that vp24 becomes strongly nuclear following overexpression of karyopherin or inhibition of nuclear export pathways. molecular mapping indicates that cytoplasmic localisation of vp24 depends on a crm1-dependent nuclear export sequence at the vp24 c-terminus. nuclear export is not required for stat1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism while export mediates return of nuclear vp24 to the cytoplasm for replication functions. thus, nuclear export of vp24 might provide novel targets for antiviral approaches. importance ebola virus (ebov) is the causative agent of ongoing outbreaks of severe haemorrhagic fever with case-fatality rates between 40 and 60%. proteins of many viruses with cytoplasmic replication cycles similar to ebov interact with the nuclear trafficking machinery, resulting in active nucleocytoplasmic shuttling important to immune evasion and other intranuclear functions. however, exploitation of host trafficking machinery for nucleocytoplasmic transport by ebov has not been directly examined. we find that the ebov protein vp24 is actively trafficked between the nucleus and cytoplasm, and identify the specific pathways and sequences involved. the data indicate that nucleocytoplasmic trafficking is important for the multifunctional nature of vp24, which has critical roles in immune evasion and viral replication, identifying a new mechanism in infection by this highly lethal pathogen, and potential target for antivirals. transport, with key roles in pathogenesis. despite established karyopherin interaction, the 23 nuclear trafficking profile of vp24 has not been investigated. we find that vp24 becomes 24 strongly nuclear following overexpression of karyopherin or inhibition of nuclear export 25 pathways. molecular mapping indicates that cytoplasmic localisation of vp24 depends on a 26 crm1-dependent nuclear export sequence at the vp24 c-terminus. nuclear export is not 27 required for stat1 antagonism, consistent with competitive karyopherin binding being the 28 principal antagonistic mechanism while export mediates return of nuclear vp24 to the 29 cytoplasm for replication functions. thus, nuclear export of vp24 might provide novel targets 30 for antiviral approaches. hek293t cells ( figure s1 ). thus, gfp-vp24 can localise into the nucleus in complexes with 149 k1, indicating that cytoplasmic localisation, which is required for roles in nucleocapsid 150 assembly/condensation (4, 5, 7), derives from active nuclear export. can diffuse through the npc and lacks nlss or ness, was diffusely localised between the dependent ness ( figure 2a , figure s2a ). importantly, the fn/c for gfp-vp241-251 in lmb-169 treated cells was higher than that for gfp alone ( figure 2c ), indicative of accumulation. thus, 170 vp24 localisation appears to be dynamic, involving nuclear entry and rapid nuclear export via 171 crm1 interaction. 172 to determine which of the predicted ness is/are responsible for nuclear export, we generated 175 constructs to express truncated vp24 proteins comprising n-terminal (vp241-88), central 176 (vp2489-172) and c-terminal (vp24173-251) portions fused to gfp; each of these contained one 177 or more of the potential ness ( figure 2a ). the truncated proteins were designed to be of 178 similar length and to avoid disruption of key structural elements (e.g. alpha helices and beta 179 sheets), based on the vp24 crystal structure (20). all proteins were predominantly cytoplasmic 180 at steady state ( figure 2b ). localisation of the n-terminal fragment was largely unaffected by 181 lmb treatment, and lmb produced only a small (≤ 1.4 fold) increase for the fn/c of the central 182 fragment ( figure 2b ,c). in contrast, a consistent and substantial increase (> 2 fold) in the fn/c 183 for the c-terminal fragment was observed following lmb treatment. vp24173-251 also displayed 184 a consistently reduced fn/c at steady state compared with the other truncated proteins. thus, it 185 appeared that prominent discrete crm1-dependent nes activity is located in the c-terminal 186 region of vp24. 187 9 notably, only full-length vp24 displayed accumulation into the nucleus following lmb 189 treatment, with all truncated proteins remaining significantly less nuclear than gfp alone. this 190 suggests that the full protein sequence is required for efficient nuclear accumulation, such that 191 truncations remove key sequences or otherwise impact conformation to affect important 192 interactions. the crystal structure of vp24 bound to k5 indicates that three regions contact 193 the k (cl1 and cl2/3, separated by 40-60 residues, figure 2a induced only a small increase in fn/c for gfp-ul44nls-vp2489-172 ( figure 3a ,b), suggestive 209 of cytoplasmic retention or nuclear export mediated largely via an alternative mechanism to 210 crm1-dependent export. however, lmb induced substantial nuclear localisation of gfp-211 ul44nls-vp24173-251 (> 4.6 fold increase in fn/c; figure 3b ) that clearly exceeded nuclear 212 localisation of gfp-vp24173-251 ( figure 2c ), consistent with a classical crm1-dependent nes 213 counteracting the activity of the heterologous ul44 nls. the fn/c for gfp-vp2489-251 was 214 also markedly increased by lmb treatment but did not attain an fn/c similar to that of full-215 required for efficient nuclear localisation. nevertheless, these data clearly indicate that vp24 217 contains classical crm1-dependent nes activity and that the principal nes is within vp24173-218 251. the c-terminal crm1-dependent nes is the principal sequence mediating nuclear export of 221 vp24 222 to confirm that the c-terminal nes is the major sequence driving crm1-dependent export of 223 vp24, we used site-directed mutagenesis to disable the nes motif. analysis of the vp24 c-224 terminal region identified residues 241-251 (comprising the c-terminal 11 residues) as 225 containing a sequence strongly conforming to a nes ( to further examine effects of altered vp24 nuclear trafficking on stat1 responses, we 263 assessed nuclear import of stat1 using clsm analysis of cos7 cells expressing gfp-vp24 264 and immunostained for stat1 following treatment without or with ifn- and/or lmb. in 265 agreement with results of the luciferase reporter assays, we observed that despite substantial 266 re-localisation of gfp-vp24 to the nucleus in lmb-treated cells, ifn--dependent stat1 267 nuclear localisation remained clearly inhibited ( figure s3 ). together, these data indicate that 268 nuclear export of vp24 is not required for inhibition of stat1 responses, consistent with k 269 binding representing the major antagonistic mechanism. thus, it appears that active in this study we have shown that ebov vp24 undergoes active trafficking between the nucleus 275 and cytoplasm involving crm1-dependent nuclear export via a nes at the vp24 c-terminus. 276 the acquisition of active nuclear trafficking sequences is consistent with a requirement for 277 highly regulated/dynamic localisation; furthermore, since vp24 is reported to oligomerise 278 (potentially as tetramers) (38), it is likely that active nuclear trafficking is required for transport 279 of vp24 multimers. the identified nes was not resolved in vp24 crystal structures (20, 47, 280 48) but localisation at the c-terminal end would be consistent with exposure and accessibility 281 to crm1 (20), and the predominantly cytoplasmic localisation of gfp-vp24 in resting cells 282 suggests that the nes is the dominant trafficking signal at steady state. intriguingly, previous 283 studies indicated that a mutated vp24 protein defective for k-binding was more cytoplasmic 284 than wt protein (49). this would be consistent with karyopherin binding mediating import; 285 one might thus speculate that vp24 would require export mechanisms to enable cytoplasmic 286 localisation/functions. our findings are the first to confirm this is the case. notably, the ebov 287 matrix protein vp40 has also been reported to localise to the nucleus in infected and transfected 288 cells (16, 50); however, a direct role for active trafficking pathways to regulate localisation, 289 distinct from mechanisms such as diffusion or interaction with other host factors, has not been 290 defined. thus, our data provides, to our knowledge, the first direct demonstration of a filovirus 291 protein exploiting specific host trafficking machinery for nucleocytoplasmic transport, 292 identifying a new mechanism in infection by these highly lethal pathogens. 293 294 although the nucleus is not directly involved in the replication processes of most rna viruses, 295 proteins of a number of these viruses are reported to encode nuclear trafficking sequences, 296 indicative of a requirement for dynamic regulation or specific accumulation in particular 297 compartments. for example, the rabv ifn antagonist p protein encodes several nlss and 298 ness (32, 43, 51-53), with regulatory mechanisms including co-localisation or overlap of the 299 sequences, enabling co-regulation by mechanisms including phosphorylation (51-53). 300 although our data identify the c-terminal nes as a principal determinant of nucleocytoplasmic 301 localisation of full-length vp24, the differential localisation and lmb sensitivity of vp241-88 302 and vp2489-172, and the finding that vp2489-251 does not recapitulate nuclear accumulation of 303 full-length vp24, suggest the presence of alternative regulatory sequences/mechanisms, 304 potentially exposed by truncation. for example, vp24 is reported to associate with membranes 305 (38), which might result in tethering within the cytoplasm under certain conditions. 306 interestingly, a recent study reported that sumoylation of residue k14 of vp24 enhances k 307 binding and ifn antagonistic function (54). in contrast, ubiquitination, including at residue 308 k206 within cl3 (figure 2a ), appears to negatively regulate ifn antagonist activity (54). 309 intriguingly, k14 is distal to cl1-3 but is within a predicted nes motif (figure 2a) . whether mechanisms will be of interest in defining the processes controlling immune evasion and 312 replication by ebov. 313 while some viral ifn antagonists use ness to facilitate immune evasion, including through 315 mislocalisation of associated stats (33, 34), vp24 uses a mechanism of competitive binding 316 to ks. our finding that vp24 nuclear export is not required for stat antagonism is consistent 317 with this, and indicates that export relates to cytoplasmic roles including in nucleocapsid 318 assembly and transport (4-14). the requirement for efficient translocation out of the nucleus is 319 consistent with interaction of vp24 with k (see above), that underpins distinct functions in 320 immune evasion. this is further supported by our finding that the c-terminal nes motif is 321 conserved among vp24 of several filovirus species that have been shown to bind to ks or 322 have conserved cl sequences (20, 26, 45), but not in mabv vp24 ( figure 4e , figure s2c thus, targeting vp24 regulatory mechanisms, including its nuclear export, may provide novel 353 targets for anti-ebov drug design. 354 355 the construct to express the minimal nls from human cytomegalovirus ul44 protein 358 (residues 425-433) fused to gfp was generated by subcloning from pepi-gfp-ul44425-433 (42, 359 43) into the pegfp-c1 vector c-terminal to gfp (clontech). constructs to express full-length 360 or truncated ebov-vp24 protein fused to gfp or gfp-ul44nls were generated by pcr to express flag-tagged k1 was a kind gift from c. basler (georgia state university). other 366 constructs have been described elsewhere (40). discovery of an ebolavirus-like filovirus 446 in europe filoviruses: ecology, molecular biology, and 448 evolution descriptive analysis of ebola virus proteins infection of naive target cells with virus-like 453 particles: implications for the function of ebola virus vp24 knockdown of ebola virus vp24 impairs viral nucleocapsid assembly and 456 prevents virus replication the assembly of ebola virus nucleocapsid 458 requires virion-associated proteins 35 and 24 and posttranslational modification of 54 contribution of ebola virus 608 glycoprotein, nucleoprotein, and vp24 to budding of vp40 virus-like particles both matrix proteins of ebola 611 virus contribute to the regulation of viral genome replication and transcription ebola virus 614 (ebov) vp24 inhibits transcription and replication of the ebov genome vp24 is a molecular determinant of ebola virus virulence 618 in guinea pigs a single phosphorodiamidate morpholino 621 oligomer targeting vp24 protects rhesus monkeys against lethal ebola virus infection fn/c) for gfp (mean ± sem, n  52 637 cells for each condition; results are from a single assay representative of three independent 638 assays). statistical analysis (student's t-test) was performed using graphpad prism software a) schematic of full-642 length vp24 and truncated vp24 proteins generated. location of potential ness are shown in 643 yellow. location of clusters (cl1-3) of residues that interact with ks in the vp24:kα5 644 complex crystal structure (20) are shown in red. numbering indicates residue positions in full-645 length vp24; sequences of potential ness are shown above. (b) cos7 cells representative images are shown. (c) images such 648 as those shown in b were analysed to calculate the fn/c for gfp (c; mean ± sem; n  31 cells 649 for each condition statistical analysis used student's t-test. **** from a single assay representative of two independent assays). statistical analysis used 657 student's t-test the authors declare that they have no conflicts of interest with the contents of this article. 441 key: cord-003705-ekhj8ae8 authors: azkanaz, maria; rodríguez lópez, aida; de boer, bauke; huiting, wouter; angrand, pierre-olivier; vellenga, edo; kampinga, harm h; bergink, steven; martens, joost ha; schuringa, jan jacob; van den boom, vincent title: protein quality control in the nucleolus safeguards recovery of epigenetic regulators after heat shock date: 2019-06-14 journal: nan doi: 10.7554/elife.45205 sha: doc_id: 3705 cord_uid: ekhj8ae8 maintenance of epigenetic modifiers is of utmost importance to preserve the epigenome and consequently appropriate cellular functioning. here, we analyzed polycomb group protein (pcg) complex integrity in response to heat shock (hs). upon hs, various polycomb repressive complex (prc)1 and prc2 subunits, including cbx proteins, but also other chromatin regulators, are found to accumulate in the nucleolus. in parallel, binding of prc1/2 to target genes is strongly reduced, coinciding with a dramatic loss of h2ak119ub and h3k27me3 marks. nucleolar-accumulated cbx proteins are immobile, but remarkably both cbx protein accumulation and loss of prc1/2 epigenetic marks are reversible. this post-heat shock recovery of pan-nuclear cbx protein localization and reinstallation of epigenetic marks is hsp70 dependent. our findings demonstrate that the nucleolus is an essential protein quality control center, which is indispensable for recovery of epigenetic regulators and maintenance of the epigenome after heat shock. the epigenetic landscape of a cell is fundamentally important for various dna metabolic processes including gene transcription, dna replication and dna repair (tessarz and kouzarides, 2014) . proper maintenance of the epigenome is essential for cell viability, and increasing evidence suggests that changes in the chromatin landscape are causally related to aging-associated functional decline of a cell and ultimately cell death (booth and brunet, 2016) . maintenance of the epigenetic landscape can only be guaranteed by correct positioning and activity of epigenetic modifiers across the genome, and may be threatened by proteotoxic stress. importantly, misregulation of epigenetic modifiers (i.e. mutations, misexpression) is frequently observed in various cancer types, underlining that regulation of the epigenetic landscape is essential for appropriate cellular functioning (dawson and kouzarides, 2012) . the polycomb group (pcg) protein family of epigenetic modifiers warrants proper regulation of stem cell self-renewal and cell lineage specification. pcg proteins reside in the canonical polycomb repressive complexes 1 (prc1) and 2 (prc2) (simon and kingston, 2013) . the prc2 complex contains ezh1/2-dependent methyltransferase activity toward histone h3 at lysine 27 (h3k27me3) (cao et al., 2002; ezhkova et al., 2011; kirmizis et al., 2004; kuzmichev et al., 2002; shen et al., 2008) . prc1 can ubiquitinate histone h2a at lysine 119 (h2ak119), by means of its e3 ligase subunit ring1a/b (de napoles et al., 2004; wang et al., 2004) . prc1 and prc2 frequently colocalize at target genes and initially a hierarchical model was proposed for prc1/2 function, where the cbx subunit of prc1 recognizes the prc2 mark h3k27me3, placing prc1 function downstream of prc2. however, recent studies have shown that prc1 and prc2 recruitment to chromatin, and associated histone modifying activities, can also be independent of each other (for a review see blackledge et al., 2015) . alternatively, non-canonical prc1-deposited h2ak119ub was shown to independently sequester prc2 complexes. work from many labs, including ours, has underlined the importance of (non-) canonical prc1 complexes for regulation of cellular identity of normal hematopoietic stem cells and leukemic stem cells (iwama et al., 2004; lessard and sauvageau, 2003; park et al., 2003; rizo et al., 2008; rizo et al., 2010; rizo et al., 2009; van den boom et al., 2016; van den boom et al., 2013) . it is therefore evident that preservation of polycomb-mediated epigenetic regulation is essential to maintain cell identity and prevent cellular transformation and that, in case of cellular stress induced proteotoxicity, the functionality of the epigenetic machinery is guaranteed. in this study, we investigated the stability of the epigenetic machinery in response to heat shock (hs). hs is known to lead to a general shutdown of transcription. cellular stressors like hs and proteasome inhibition induce a quick depletion of the free ubiquitin pool and this coincides with a quick reduction of ubiquitinated histone h2a (h2ak119ub) in the cell dantuma et al., 2006; mimnaugh et al., 1997) . these data suggest that the epigenome may be affected by hs. a study in drosophila cells indeed showed that hs leads to dramatic alterations of the 3d chromatin architecture as a consequence of weakening insulators between topologically elife digest all cells in our bodies contain the same sequence of dna, hence the same genes, in a compartment called the nucleus. yet different sets of genes are switched on in different types of cells. cells achieve this by a process called epigenetic regulation. proteins known as epigenetic regulators modify dna and its associated proteins in ways that can turn genes on or off. different types of cells contain different epigenetic regulators, and so express different genes. the polycomb group proteins (or pcg for short) turn their target genes off and are important to maintain the identity of a cell. when the target genes of pcg proteins are inadvertently switched on, this may lead to changes in the fate of cells, potentially resulting in diseases such as cancer. so, it is important that cells keep the pcg proteins active where necessary, even in the face of stress. cellular stresses come in several forms but often interfere with the normal activities of proteins. if cells experience high temperatures, they can experience a stress known as heat shock. this can cause proteins, including pcg proteins, to unfold. azkanaz et al. have now investigated what happens to pcg proteins in cells experiencing heat shock, and how these cells try to limit the damage this causes. azkanaz et al. conducted their experiments on healthy and cancerous human blood cells. after exposing the cells to half an hour of high temperature the pcg proteins disappeared from the genes they were switching off. this means that cells exposed to heat shock lose their epigenetic control machinery, which may lead to permanent changes to epigenetic modifications found across the genome when not quickly reinstalled. pcg proteins, and another group of proteins called the heat shock proteins, were found to move to a compartment within the nucleus called the nucleolus. while the cells had returned to body temperature and were recovering from the heat shock, the heat shock proteins helped the pcg proteins fold back into their proper shapes. the pcg proteins then left the nucleolus and returned to their target genes, where they reinstalled the epigenetic marks. these experiments show that heat shock causes a temporary loss of epigenetic regulators from their target genes and that the nucleolus acts as a protein quality control center. future experiments might explore how pcg proteins get to the nucleolus after heat shock and how impaired protein quality control (i.e. upon aging) may lead to alterations of the epigenetic landscape in a cell. deeper knowledge of this process could help us to understand how cells can recover from stress. associating domains (tads) and newly formed architectural protein binding sites (li et al., 2015) . in addition, polycomb complexes were redistributed to active promoters/enhancers and formed inter-tad interactions, likely resulting in transcriptional silencing. for a subset of genes, however, in particular the genes encoding the heat-shock proteins (hsps), hs does not cause a decrease but rather an increase in gene transcription. this response is referred to as the heat shock response and mediated largely by the so-called heat shock transcription factor-1 (hsf-1) (akerfelt et al., 2010) . hsps function as molecular chaperones, not only guiding cotranslational folding under normal conditions but also serving to refold heat-unfolded proteins. if proteins cannot be correctly refolded, they can be poly-ubiquitinated and degraded by the proteasome. importantly, the intracellular pool of 'free' ubiquitin that is used for poly-ubiquitination of proteins is limited . as such, hsps prevent protein dysfunction and aggregation, a hallmark of various age-related neurodegenerative diseases like alzheimer's disease and parkinson's disease (hartl et al., 2011; kampinga and bergink, 2016; morimoto, 2008) . in this study, we specifically investigated the effects of hs on the epigenetic machinery and how this is restored upon return to physiological temperatures. we observed that prc1 and prc2 subunits and various other chromatin modifiers accumulate in the nucleolus upon hs. various labs have reported on reversible accumulation of reporter-proteins in the nucleus upon heat shock (miller et al., 2015; nollen et al., 2001; park et al., 2013) , but whether this also holds true for endogenous proteins, and what could be the physiological relevance of this process, has remained unclear. we find that the nucleolar accumulation of these epigenetic regulators coincides with a displacement of prc1 and prc2 from their target genes and a dramatic loss of h2ak119ub and h3k27me3. most importantly, the nucleolar accumulation is reversible in an hsp70-dependent manner allowing epigenetic recovery. our data demonstrate that the nucleolus is an essential protein quality control (pqc) center that serves to restore the epigenomic landscape after conditions of proteotoxic stress in an hsp-dependent manner. to investigate the effects of thermal stress on the epigenetic machinery, we analyzed the localization of prc1 subunits in response to heat shock (hs). we transduced cord blood cd34 + stem/progenitor cells using a gfp-cbx2 lentiviral vector ( figure 1a) . importantly, gfp pull out experiments in k562 gfp-cbx2 cells confirmed that gfp-cbx2 was properly incorporated in the prc1 complex (figure 1-figure supplement 1a) and chip-seq experiments in k562 gfp-cbx2 and k562 wild-type cells showed that gfp-cbx2 target genes largely overlapped with endogenous cbx2 target genes (based on encode/broad institute data) and h2ak119ub enriched genes (van den boom et al., 2016) . these data underline that the gfp-cbx2 fusion protein is incorporated into a fully functional prc1 complex. next, we studied the localization of gfp-cbx2 in untreated and heat shocked cord blood cd34 + cells. whereas gfp-cbx2 was homogenously distributed throughout the nucleus in untreated cells, cells that received a hs (30 min, 44˚c) displayed strong accumulations of gfp-cbx2 in subnuclear domains, both in cells that were fixed after hs ( figure 1b ) and living cells ( figure 1c) . similarly, k562 leukemic cells also showed hs-induced relocalization of gfp-cbx2 to subnuclear domains ( figure 1-figure supplement 2a) . transmission images suggested that hs induces relocalization of gfp-cbx2 to nucleoli, which was confirmed by immunofluorescence analyses using antibodies against the nucleolar proteins npm1 and fibrillarin ( figure 1d and figure 1 -figure supplement 2b-d). gfp-cbx2 localized directly around fibrillarin, which is confined to the dense fibrillar component (dfc) of the nucleolus, and partially colocalized with the granular component (gc) protein npm1. taken together, these data suggest that gfp-cbx2 is most enriched in the granular component of the nucleolus (boisvert et al., 2007) . the kinetics of hs-induced nucleolar accumulation of gfp-cbx2 were both dependent on the duration and temperature of the hs ( figure 1e and figure 1 -figure supplement 2e-f). cells exposed to a temperature of 42˚c also displayed nucleolar localization of gfp-cbx2 albeit with slower kinetics. to investigate whether hs-induced nucleolar relocalization is common to all prc1-associated cbx paralogs, k562 cell lines were generated expressing gfp-cbx4, gfp-cbx6, gfp-cbx7 or gfp-cbx8. indeed, all these cbx paralogs displayed hs-induced nucleolar accumulation ( figure 1f and figure 1 continued on next page g). importantly, using immunofluorescence, we also observed nucleolar accumulation of endogenous cbx4 upon hs, both in k562 and hl60 cells ( figure 1h and figure 1 -figure supplement 2g). based on these data, we hypothesized that a common domain in these proteins is sufficient for hs-induced nucleolar accumulation. since the homology between these various cbx proteins is confined to the chromodomain (n-terminus) and pc box (c-terminus), we generated gfp-cbx2 and gfp-cbx8 fusions only containing the chromobox (gfp-cbx2 [2-63]) or the chromobox and at hook (gfp-cbx2 [2-96]) ( figure 1-figure supplement 3a) . strikingly, all generated truncated gfp-cbx fusion proteins displayed nucleolar localization after hs, suggesting the presence of the chromodomain is sufficient to induce nucleolar localization after hs (figure 1-figure supplement 3b ). the kinetics of hs-induced relocalization of truncated cbx proteins were slightly slower, suggesting that also non-homologous peptide stretches in cbx proteins contribute to nucleolar relocalization. to verify the hs-induced accumulation of pcg proteins in the nucleolus, we isolated nucleoli from heat shocked and untreated gfp-cbx8 expressing k562 cells ( figure 2a ). microscopic analysis of unfixed isolated nucleoli followed by image analysis showed a robust increase in gfp-cbx8 signal in nucleoli isolated for cells directly after hs ( figure 2b and c). this observation was confirmed by fixing nucleoli and subsequent counterstaining with dapi ( figure 2d ). next, we performed western blot analysis on isolated cytoplasmic, nucleoplasmic and nucleoli fractions from gfp-cbx8 cells, which confirmed the presence of the nucleolar marker fibrillarin in the nucleoli fraction and clearly showed an increase of gfp-cbx8 in the nucleolar fraction after hs ( figure 2e ). to analyze changes in the localization of endogenous prc1 subunits, we isolated nucleoli from wild-type k562 cells and similarly performed western analysis of the cytoplasmic, nucleoplasmic and nucleoli fractions ( figure 2f ). also here fibrillarin was prominently found in the nucleolar fraction and beta-actin was confined to the cytoplasmic fraction. clearly, both endogenous cbx4 and cbx8 were enriched in the nucleolar fraction after hs. in addition, also endogenous ring1b levels were slightly elevated in the nucleolar fraction after hs. to validate these results in an independent cell line, we analyzed cellular fractions isolated from hl60 cells ( figure 2g ). also here, we observed a robust shift of endogenous cbx4, cbx8 and ring1b to the nucleolus after hs. what could be the physiological relevance of such a shift of proteins regulating dna-dependent processes to the nucleolus? it has been known that one of the most dramatic morphological changes in heat treated nuclei is the swelling of nucleoli (welch and suhan, 1986) . whereas initially considered as heat-induced damage, several lines of independent observations have suggested that this might rather reflect a regulated, hsp-dependent process in which the nucleolus serves as a temporal storage site for unfolded proteins during proteotoxic stress (nollen et al., 2001; ohtsuka et al., 1986; welch and feramisco, 1984) . in line with this hypothesis, we also found that both hsp70 and dnajb1 are accumulating in the nucleolus after hs ( figure 2g ), which is in agreement with earlier figure 1 continued cbx2 at continuous exposure at 42˚c or 44˚c. error bars indicate the mean ± sd calculated from independent microscopical images (n = 4; total cell number 70-220). similar results were obtained in independent experiments. (f) gfp-cbx4, gfp-cbx6, gfp-cbx7 and gfp-cbx8 localization in k562 cells in untreated or heat shocked (30 min, 44˚c) cells. scale bar represents 25 mm. (g) quantification of percentage of cells with nucleolar accumulations in designated k562 cell lines after hs (30 min or 2 hr, 44˚c). error bars indicate the mean ± sd calculated from independent microscopical images (n = 3; total cell number 70-100). statistical analysis was performed using student's t-test, **p<0.01 and ***p<0.001. (h) confocal images of untreated and heat shocked k562 cells that were fixed and stained with anti-cbx4 and anti-npm1. scale bar represents 10 mm. doi: https://doi.org/10.7554/elife.45205.003 the following figure supplements are available for figure 1: observations that dnajb1 and hsp70s can translocate to the nucleolus after hs (ohtsuka et al., 1986; welch and feramisco, 1984) . next, we performed label-free quantification on the nucleolar proteome in untreated and heat shocked k562 cells by lc/ms-ms analyses. in total, we identified 1279 proteins, and the nucleolar proteins npm1 and fibrillarin were among the most abundant proteins ( . interestingly, proteins enriched in the nucleolus after hs associated with go terms related to chromatin modification, gene expression, dna repair, histone ubiquitination and protein refolding ( figure 3b ). consistent with our if and biochemical fractionation data, various prc1 subunits, including cbx2, ring1a, ring1b and phc2 were significantly enriched in the nucleolus upon hs ( figure 3c and figure 3 -figure supplement 2b). intriguingly, we also found that prc2 subunits ezh2, suz12 and eed were enriched ( figure 3c and figure 3 -figure supplement 2b) and this hs-induced nucleolar accumulation of ezh2 and suz12 was confirmed using western blot analysis on nucleolar fractions isolated from k562 or hl60 cells ( figure 3d ). hs-induced nucleolar accumulation of ezh2 was independently confirmed using immunofluorescence analyses in k562 cells, hl60 cells, and primary non-transformed cd34 + mobilized peripheral blood stem cells (mpbscs) (figure 3-figure supplement 3a-c). intra-nucleolar levels of h3k27me3 and h2ak119ub were not increased in heat shocked cells versus untreated cells, suggesting that pcg proteins are not involved in polycomb-mediated silencing of nucleolar chromatin ( figure 3e ). in addition to these pcg proteins, many other chromatin and transcription regulating proteins were found to be enriched in the nucleolus after hs, including members of the chromodomain helicase dna-binding (chd) family and the fact chromatin remodeling complex that both can remodel chromatin (marfella and imbalzano, 2007; winkler and luger, 2011) , and the paf complex, which regulates release of rnapii into processive elongation (van oss et al., 2017) ( figure 3f and figure 3 -figure supplement 2c). in addition, accumulation of brd proteins and jmjd6 was observed in the nucleolus after hs ( figure 3g and figure 3 -figure supplement 2d). brd4 and jmjd6 are co-bound to enhancers and regulate promoter-proximal pause-release of rnapii (liu et al., 2013) . taken together, these data show that hs induces strong shifts of various chromatin and transcription regulators toward the nucleolus. in line with our western analysis of cellular fractions, our proteomic analyses showed several chaperone proteins to be enriched in the nucleolus after hs. these included members of the hsp70 chaperone family (hspa1a/b), dnajb1, dnajc7 and the small heat shock protein hspb1 ( figure 3h and figure 3 -figure supplement 2e). strikingly, many other chaperones, including members of the hsp90 family were not or weakly enriched in the nucleolus after hs showing the response is specific and suggesting that this subset of hsps may somehow have functional implications in this response. in addition to the hsps, we also observed a strong accumulation of 26s proteasome subunits in the nucleolus after hs ( figure 3i and consistent with observations using model proteins, which are targeted to the nucleolus for poststress degradation (park et al., 2013) . independent lc-ms/ms analysis with k562 gfp-cbx8 cells confirmed these findings, implying that hs-induced nucleolar accumulation of various chromatin regulators, protein chaperones and proteasomal subunits is a conserved biological phenomenon (figure 3-figure supplement 4a-e, supplementary file 3). our data suggests that upon hs many chromatin remodelers and transcriptional regulators accumulate in the nucleolus which may become a hot spot for protein quality control. in drosophila, nucleolar accumulation of pc has been suggested to contribute to the generalized silencing of most of the genome observed in heat shocked cells (li et al., 2015) . combined with our observations that many chromatin remodeling and transcription regulatory proteins accumulate in the nucleolus after hs, these data prompted us to speculate that such hs-induced redistributions may severely impact on the chromatin bound fraction of various epigenetic regulators. to analyze changes in prc1 complex chromatin binding upon hs we performed chip-qpcrs in k562 cells expressing pcg gfp-fusion proteins and validated prc1 binding to pcg target genes. indeed, upon hs (1 hr, 44˚c) we observed a strong reduction in chromatin binding of gfp-cbx2, bmi1-gfp, mel18-gfp and, to a lesser extent, gfp-ring1b ( figure 4a ). to investigate how hs impacts on chromatin binding of endogenous prc1 subunits, we performed chips using an antibody directed against endogenous cbx8, and also here we observed a quick reduction in cbx8 binding to polycomb target genes after hs ( figure 4b ). moreover, in line with our lc-ms/ms data, chip analysis using an antibody directed against the prc2 subunit ezh2 also clearly showed a loss of endogenous ezh2 binding from target genes after hs, confirming that both prc1 and prc2 show strongly reduced chromatin binding after hs ( figure 4c) . to investigate how this may functionally impact on prc1/2-deposited epigenetic marks, we analyzed h2ak119ub and h3k27me3 levels at pcg target genes. previous studies have shown that hs induces a rapid but reversible loss of ubiquitinated histones mimnaugh et al., 1997) . indeed, we also observed a strong decrease in h2ak119ub levels after hs ( figure 4d ). in addition, h3k27me3 levels were also significantly reduced in heat-shocked cells vs. control cells ( figure 4e ). these data show that hs not only causes displacement of prc1/2 complexes but also leads to a reduction of their respective epimarks. to assay hs-induced loss of prc1 chromatin binding in a genome-wide manner, we performed chip-seq of both endogenous cbx8 (k562 cells) and gfp-cbx2 (k562 gfp-cbx2 cells) in untreated cells or after hs. heat maps and band plots of cbx8 clearly, also here a loss of cbx8 and gfp-cbx2 chromatin association is observed upon hs. tss-localized peaks are associated to genes enriched for development-related go terms, confirming that these are polycomb target genes ( figure 4h) . typical examples of chromatin regions that show a reduction of cbx8 or gfp-cbx2 chromatin binding are shown in figure 4i . next, we analyzed hs-induced changes of other epigenetic modifications. here we find that, in addition to loss of h2ak119ub and h3k27me3, after hs (1 hr, 44˚c) also h3k4me3 levels are reduced, and, likely as a consequence of h3k4me3 loss, h3k4me1 levels are increased ( figure 4j ). the increase in h3k4me1 levels argues that loss of epimarks is not a mere consequence of decreased nucleosome occupancy but truly a consequence of changes in the epigenetic marking of the chromatin. finally, we investigated whether the hs-induced loss of prc1/2-associated epimarks also resulted in loss of silencing of polycomb target genes. indeed, we found that the expression of various polycomb target genes was increased 3 hr after hs, and recovered to pre-hs levels afterwards ( figure 4k ). taken together, these data show that, in parallel to the reallocation of chromatin remodelers and transcriptional regulators to the nucleolus, hs induces a quick reduction in prc1 and prc2 chromatin binding and changes in the epigenetic profile of pcg target genes with consequences for the transcriptional state of these genes. altered gfp-cbx2 protein dynamics in the nucleolus upon hs next, we aimed to determine the physical-dynamic properties of pcg in the nucleoli of heat shocked cells. to achieve this, we stably expressed gfp-cbx2 in hela cells and determined gfp-cbx2 dynamics between the nucleolus and the nucleoplasm by photobleaching gfp-cbx2 in the nucleoli and analyzing the fluorescent recovery after photobleaching (frap) in time ( figure 5a) . strikingly, whereas the kinetics of nucleolar gfp-cbx2 in untreated cells were very quick, nucleolar gfp-cbx2 was highly immobile in heat shocked cells ( figure 5b and c) with limited to no dynamic exchange between the nucleolus and the nucleoplasm. the nucleolus is a membrane-less nuclear body with liquid-like properties; its formation depends on liquid-liquid phase transition (brangwynne et al., 2011; marko, 2012) . analysis of the intranucleolar dynamics of gfp-cbx2 in heat shocked cells using frap/flip (fluorescence loss in photobleaching) analysis ( figure 5a) , revealed that there was very little fluorescence recovery in the bleached region and fluorescence loss in the adjacent nucleolar region ( figure 5d and e), indicating antibody directed against endogenous cbx8 in untreated and heat shocked (1 hr, 44˚c) k562 cells. error bars represent mean ±range (n = 2 independent replicates, *p<0.05). (c) chip-qpcr analyses of polycomb target genes using an antibody directed against endogenous ezh2 in untreated and heat shocked (1 hr, 44˚c) k562 cells. error bars represent mean ± sd (n = 4 independent replicates, *p<0.05, **p<0.01 and ***p<0.001). (d) chip-qpcr analyses of h2ak119ub levels at polycomb target genes in untreated and heat shocked (1 hr, 44˚c) k562 cells. error bars represent mean ± sd (n = 3 independent replicates, *p<0.05 and ***p<0.001). (e) chip-qpcr analyses of h3k27me3 levels at polycomb target genes in untreated and heat shocked (1 hr, 44˚c) k562 cells. error bars represent mean ± sd (n = 3 independent replicates, *p<0.05). (f) chip-seq heatmap of endogenous cbx8 peaks (k562) and gfp-cbx2 peaks (k562 gfp-cbx2) and surrounding regions (à5 to + 5 kb) as identified in untreated cells and the respective signal in heat shocked cells (1 hr, 44˚c) frap and flip regions are indicated in the pre-bleach images. scale bar represents 10 mm. (e) average flip and frap signals in the nucleolus, starting before photobleaching. error bars indicate mean ± sd. (f) gfp-cbx2 localization in k562 gfp-cbx2 cells that were heat shocked (1 hr, 44˚c) and subsequently cultured at 37˚c for 1 hr in the presence or absence of 10% 1,6-hexanediol. scale bar represents 25 mm. (g) gfp-cbx8 localization in k562 gfp-cbx8 cells that were heat shocked (1 hr, 44˚c) and subsequently cultured at 37˚c for 1 hr in the presence or absence of 10% 1,6-hexanediol. scale bar represents 25 mm. doi: https://doi.org/10.7554/elife.45205.015 that gfp-cbx2 might be present in these nucleoli in a more solid-like state. proteins in similar solid states have been shown to be aggregated (patel et al., 2015) . to discriminate between a liquid-like or solid state of cbx protein accumulations, we exposed heat shocked k562 gfp-cbx2 and k562 gfp-cbx8 cells to 1,6-hexanediol, an aliphatic alcohol that disturbs weak hydrophobic interactions (kroschwald et al., 2015; patel et al., 2007) . clearly, both gfp-cbx2 and gfp-cbx8 nucleolar accumulations were 1,6-hexanediol insensitive ( figure 5f and g), in line with a more solid, aggregation-like state. heat-shock proteins modulate cbx protein recovery we next asked whether the hs-induced allocation of pcg proteins to nucleoli serves to allow for a quick recovery of the epigenetic modifiers to restore chromatin binding of epigenetic regulators and associated changes in the epigenetic landscape after heat shock. in addition, we argued that the coappearance of hsps in these nucleoli may be required to recover these regulators from their solidlike, aggregated state. certain mammalian hsps have been demonstrated to be able to disentangle protein aggregates (mogk et al., 2018; nillegoda et al., 2018) , including the hsps (dnajb1 and hsp70) that we identified as part of the nucleolar proteome after heat shock ( figure 3h ). to test the reversibility of nucleolar gfp-cbx2 accumulations after hs, we treated k562 gfp-cbx2 cells with a hs (30 min, 44˚c) and monitored gfp-cbx2 localization at 37˚c afterwards. clearly, within 3 hr after hs the gfp-cbx2 nucleolar accumulations dispersed and gfp-cbx2 regained its original pan-nuclear distribution ( figure 6a) , showing that nucleolar accumulations of pcg proteins are readily reversible. similarly, and in line with our cellular fraction data and lc-ms/ms data, we also observed a reversible hs-induced nucleolar accumulation of dnajb1 ( figure 6-figure supplement 1a ). in addition, also hsp70 showed post-hs nuclear translocation, and localized to the nucleolus in a reversible fashion ( figure 6-figure supplement 1b) . accumulation of hsp70 in the nucleolus was not as prominent as observed for dnajb1, which is in line with our lc-ms/ms data, and may be a consequence of other nuclear activities of hsp70. next, we investigated whether drug-induced inhibition of the hsp70 machinery using the hsp70 inhibitor ver-155008 would impact on the relocalization of pcg proteins to the nucleolus or would delay recovery of gfp-cbx2 from the nucleolus after hs. clearly, post-hs nucleolar accumulation was not impaired upon hsp70 inhibition, suggesting that the hsp70 machinery is not involved in chaperoning pcg proteins to the nucleolus after hs ( figure 6b ). however, hsp70 inhibition led to a clear delay in recovery of gfp-cbx2 from the nucleoli suggesting that hsp70 activity is required to resolve the nucleolar accumulation of gfp-cbx2 ( figure 6b and c) . similarly, a partial knockdown of hspa1a, an abundant heat-inducible hsp70 family member in human cells and identical to hspa1b at the protein level, resulted in a significant delay of gfp-cbx2 recovery from the nucleoli in hek293t gfp-cbx2 cells ( figure 6 -figure supplement 1c-e). next, we investigated whether induction of endogenous hsps accelerates gfp-cbx2 recovery after hs. k562 gfp-cbx2 cells were treated with two consecutive hss with a 3-hr interval ( figure 6d ). hs is known to induce hsp expression (including several hsp70s), resulting in a period of increased thermotolerance. this cellular property allowed us to compare the kinetics of gfp-cbx2 recovery in the presence of basal or hs-induced hsp levels. indeed, whereas recovery of gfp-cbx2 after the first hs required almost 3 hr, recovery after the second hs was almost completed within 30 min ( figure 6e and f) . importantly, the amount of gfp-cbx2 that initially accumulated during the first or second hs did not change dramatically ( figure 6e and f). this implies that increased hsp levels specifically affect the recovery of gfp-cbx2 proteins from the solid phase within the nucleolus. next, we tested whether the kinetics of recovery of gfp-cbx2 from the nucleolus and epigenetic recovery after hs were similar. indeed, we found that h2ak119ub was completely recovered at 4 hr after hs, whereas h3k27me3 recovery was slightly delayed ( figure 6g ). at later time points after hs we observed a complete recovery of gfp-cbx2 and various epigenetic modifications ( figure 6-figure supplement 2a) . interestingly, h2ak119ub recovery was strongly dependent on hsp protein expression. thermotolerant cells that received a second hs displayed a much faster recovery of h2ak119ub compared to cells after the first hs ( figure 6h ). in line with our chip-qpcr data, h3k27me3 was also reduced albeit with slower kinetics. both hsp70 and dnajb1 were strongly induced upon the first hs, whereas ezh2 and cbx8 protein levels were rather stable or slightly reduced. next, we investigated whether post-hs h2ak119ub recovery is dependent on polycomb proteins that are recovered from the nucleolus and not merely on newly translated polycomb proteins. therefore, we pre-treated k562 cells for 1 hr with cycloheximide or dmso and studied h2ak119ub recovery after hs. importantly, h2ak119ub recovery did not depend on de novo protein synthesis as h2ak119ub levels also recovered in cycloheximide-treated cells ( figure 6 -figure supplement 2b). taken together, these data show that molecular chaperones are crucial for recovery of gfp-cbx2 from the nucleolus and that this activity is essential for epigenetic recovery after hs ( figure 6i ). spatial separation of proteins in the cytosol and nucleus upon heat stress has been repeatedly suggested to prevent interference of un-or misfolded proteins with essential cellular processes (escusa-toret et al., 2013; kaganovich et al., 2008; miller et al., 2015) . at the same time, this 'storage' may allow for rapid recovery of proteins to re-initiate the crucial processes they are normally engaged in. for the nucleolus, these experiments have been done with reporter proteins, without any direct connection to physiological cellular processes. despite this drawback, these studies revealed that hs-induced redistribution to the nucleolus is important for both refolding (nollen et al., 2001) or degradation (park et al., 2013) of these reporters. our data are the first to show that numerous endogenous chromatin regulators temporarily accumulate in the nucleolus upon a hs, and, in an hsp70-dependent manner, fully and functionally recover to the chromatin upon return to physiological temperatures. at this stage, we do not know what drives the association of the various pcg proteins to the nucleolus upon hs. given the protein denaturation effects of hs, (partial) protein unfolding is likely a key driver of protein relocalization to the nucleolus. whether it is unfolding of nucleolar proteins that cause retention of pcg proteins or actually partial unfolding of pcg proteins (or both) remains to be elucidated. data from audas and colleagues showed that a ncrna, arising from intergenic stretches in between ribosomal dna repeats, is capable of sequestering and immobilizing various proteins in the nucleolus upon acidosis or hs (audas et al., 2012) . alternatively or in parallel, heat-unfolded nuclear reporter proteins (nollen et al., 2001) or cytosolic proteins (park et al., 2013) have been reported to accumulate in the nucleolus via chaperoned transport. whilst we cannot exclude this possibility here, the appearance of pcg proteins in the nucleolus were independent of the hsp70 machinery. however, like in other studies (nollen et al., 2001; welch and feramisco, 1984) , we did find significant enrichment of hsp70 family members, and co-chaperones such as dnajb1 and hspb1, in the nucleolus after hs. we also show that both hspa1a/hsp70 knockdown and inhibiting hsp70 activity result in a significant delay of gfp-cbx2 recovery from the nucleolus, whereas elevated hsp70 expression accelerates the reallocation of pcg proteins to the chromatin. whereas the nucleolus is a membrane-less organelle with liquid-like properties (brangwynne et al., 2011; marko, 2012) , our finding that post-hs gfp-cbx2 accumulations are 1,6-hexanediol insensitive, suggest that it is present in the nucleolus in a more solid phase. this could relate to the requirement of an active hsp70 machinery for its re-solubilization upon recovery. in fact, both hsp70 and dnajb1 that accumulate in the nucleolus are crucial components of chaperone machines with protein disaggregation power capable of functionally solubilizing proteins (mogk et al., 2018; nillegoda et al., 2018) . it is important to note that the functional recovery of epigenetic control was not dependent on de novo synthesis of the pcg proteins. translation inhibition did not interfere with this h2ak119ub recovery, suggesting that a least a large fraction of nucleolar pcg proteins are re-solubilized and functionally refolded. in addition, a fraction of the nucleolar accumulated proteins may be targeted for proteasomal degradation supported by the hs-induced nucleolar enrichment of the 26s proteasome that we found. in line with these data, a link between the proteasome and the nucleolus was previously suggested and proteasome inhibition leads to nucleolar accumulation of the proteasome (arabi et al., 2003; fátyol and grummt, 2008; latonen et al., 2011) . interestingly, also other types of stress such as transcription inhibition, dna damage induction and viral infection, have been shown to cause major changes in the protein composition of the nucleolus (andersen et al., 2005; boisvert et al., 2010; emmott et al., 2010; lam et al., 2010) . however, whereas hs mainly induced accumulation of proteins in the nucleolus, transcription inhibition using actinomycin d resulted in a release of ribosomal proteins and rna processing factors and an increase of snrnp proteins (andersen et al., 2005) . in contrast, treatment of cells with the proteasome inhibitor mg132, which similarly to hs leads to proteotoxic stress, led to an increase in ribosomal proteins in the nucleolus (andersen et al., 2005) . together, these data suggest that the nucleolus could be an important protein quality control center serving under many different stress conditions. proteotoxic stress-induced loss of h2ak119ub has previously been observed by other groups dantuma et al., 2006; mimnaugh et al., 1997) , and it has been proposed that the reason for this loss is the urgent need for 'free' ubiquitin in cells post-hs. our data suggests that hs-induced redistribution of pcg proteins to the nucleolus has direct implications for histone marking and is not limited to h2ak119ub, but also affects h3k27me3, h3k4me3 and h3k4me1 levels at pcg target genes. other studies have also shown hs-induced loss of h3k27ac from hs-repressed enhancers (chen et al., 2017) and changes in the 3d chromatin structure and epigenetic landscape in drosophila cells (li et al., 2015) . in this latter study, the authors observed a moderate localization of the drosophila polycomb (pc) protein to the nucleolus upon hs. although ribosomal dna transcription in the nucleolus was strongly reduced upon hs, pc binding to ribosomal dna repeats was not increased, suggesting that pc is not involved in repressing ribosomal dna transcription (li et al., 2015) . whereas our hs-induced cbx protein accumulation in the nucleolus is more robust, we did not observe an increase in rdna binding by cbx8 (data not shown). we also did not find increased h3k27me3 and h2ak119ub levels in the nucleolus after hs suggesting that accumulating prc1 and prc2 subunits are functionally inactive. together these findings contradict a hypothetical chromatin regulatory activity of prc1/2 in the nucleolus after hs but rather suggest that their respective subunits are undergoing protein quality control. taken together, our data shows that hs directly affects chromatin binding of pcg proteins and results in a decrease of pcg-related epigenetic modifications. importantly, hsp70-dependent protein disaggregation and refolding enables pcg proteins to quickly re-initiate their epigenetic functions at target genes. it is evident that quick re-installation of pcg epimarks is key to maintain proper epigenetic regulation of pcg target genes. the question remains how often cells will erroneously 'repair' the epigenetic profile after stress-induced epigenetic instability. despite the fact that the majority of epimarks may be properly reinstalled, mistakes will result in epigenetic scars, which may contribute to cellular transformation, loss of cell function and ultimately cell death. various studies have shown that alterations in transcriptional and epigenetic regulation are major contributors to aging-associated loss of cellular function (booth and brunet, 2016) . based on our data, it is tempting to speculate that cellular-stress-induced epigenetic changes may contribute to age-associated epigenetic alterations. importantly, it has been shown that the molecular chaperone system of a cell displays age-associated functional decline (klaips et al., 2018; labbadia and morimoto, 2015) . this may well trigger age-or disease-associated reduction in protein quality control of epigenetic regulators, including pcg proteins, leading to alterations in the epigenome. we propose a model where hs leads to loss of chromatin binding and nucleolar accumulation of pcg proteins and various other epigenetic regulators, likely as a consequence of protein unfolding. loss of pcg chromatin binding leads to a loss of prc1/2-related epigenetic modifications which recovery depends on hsp70 activity in the nucleolus. cord blood (cb) and mobilized peripheral blood stem cells (mpbscs) were obtained from healthy full-term pregnancies and allogeneic blood stem cell donors respectively after informed consent in accordance with the declaration of helsinki at the obstetrics departments at the martini hospital and university medical center groningen. this study was approved by the umcg medical ethical committee. cb cd34 + cells were isolated as previously described (schuringa et al., 2004) . cb cd34 + cells, k562, hela, and hek293t cells were transduced as described previously (horton et al., 2013; schuringa et al., 2004; van den boom et al., 2013) . one round of lentiviral transduction was performed and cells were harvested at day 2 after transduction. for retroviral transductions virus was produced transiently in hek293t cells by transfection of the appropriate pc182 gfp-fusion vector and pcl-ampho at day 1. at day 2 the medium on the hek293t cells was changed to rpmi (incl. 10% fcs and 1% p/s) and at day 3 the supernatant was harvested, filtered and used for infection of cells. to generate stable cell lines gfp-positive cells were sorted out 3 days after transduction. the (human) erythromyeloblastoid leukemia cell line k562 and hl60 cells were cultured in rpmi 1640 (containing l-glutamine) supplemented with 10% fcs and 1% penicillin/streptomycin (paa laboratories). cb cd34 + cells were cultured in imdm, supplemented with 20% fcs, 1% penicillin/ streptomycin, 20 ng/ml scf, and 20 ng/ml il-3. hela cells and hek293t cells were cultured in dmem supplemented with 10% fcs and 1% penicillin/streptomycin. cell lines were all tested mycoplasma-free using a pcr-based assay. for ver-155008 treatment cells were pre-treated with ver-155008 at a concentration of 5 mm for 48 hr. cycloheximide treatments were performed at a concentration of 10 mg/ml. the lentiviral prrl sffv gfp-fusion vector for cbx2 was generated as described previously (van den boom et al., 2013) . other gfp-cbx fusion proteins (pc182 gfp-cbx4, gfp-cbx6, gfp-cbx7 and gfp-cbx8) were expressed from retroviral vectors that were previously described (vandamme et al., 2011) . prrl sffv gfp-cbx2 (aa2-63), prrl sffv gfp-cbx2 (aa2-96), prrl sffv gfp-cbx8 (aa2-62), and prrl sffv gfp-cbx8 (aa2-96) were generated by pcr amplification of the indicated fragment of the cbx2/8 protein using prrl sffv gfp-cbx2 and pc182 gfp-cbx8 as templates, followed by subcloning into the pjet1.2/blunt vector (thermofisher). after sequence validation, these fragments were excised using bsrgi and subcloned into prrl sffv gfp-cbx2 where cbx2 was excised using bsrgi. for immunofluorescence microscopy, cells were cytospinned on glass slides and subsequently fixed using 4% paraformaldehyde in pbs. subsequently, cells were permeabilized using pbs containing 0.1% triton x-100. primary antibodies include anti-fibrillarin (ab5821, abcam), anti-npm1 (fc-61991, thermo fisher scientific), anti-cbx4 (e6l7x, cell signalling technology) and anti-ezh2 (d2c9, cell signalling technology) followed by secondary antibody staining using alexa fluor 488 goat-anti-rabbit (thermo fisher scientific, a-11008), alexa fluor 488 goat-anti-mouse (thermo fisher scientific, a-11001), alexa fluor 647 goat-anti-rabbit (thermo fisher scientific, a-21244), or alexa fluor 647 goat-anti-mouse (thermo fisher scientific, a-21235). images were acquired on a leica dm6000b microscope using a 40x dry objective (hcx pl fluotar, numerical aperture: 0.75) or a 63x immersion objective (pl s-apo, numerical aperture: 1.30) using las-af software (leica). confocal images were acquired on a leica tcs sp8 confocal laser-scanning microscope using a hc pl apo cs2 63x/1.4 oil objective, and excitation with 488 nm (20 mw) and 633 nm (30 mw) laser lines. for frap experiments, a confocal laser-scanning microscope (zeiss lsm780 nlo; carl zeiss microcopy) was used. hela gfp-cbx2 cells were seeded in a 35 mm dishes, no. 15 coverslip, 14 mm diameter (mattek). to measure protein mobility after hs, cells were first heat shocked (30 min, 44c ), followed by frap analysis at 37˚c. to perform frap experiments first the subnuclear location of nucleoli was identified using a transmission image. next, a region of interest covering the whole nucleolus (in the case of frap experiments), or half the nucleolus (frap/flip) was defined. for frap/flip experiments the opposite side of the nucleolus was selected to measure the flip signal. the frap region was bleached for five iterations at the highest intensity of the 488 nm line of a 25 mw argon laser focused by a ec plan-neofluar 40x/1.30 oil dic m27 lens (carl zeiss microcopy). recovery of fluorescence was monitored at 1 s intervals at 0.5% of the laser intensity used for bleaching. for generation of frap and flip curves the background signal was subtracted from the measured fluorescent intensities and subsequently normalized to prebleach levels. finally, the mean and standard deviation were plotted. for hspa1a/hsp70 knockdown, hek293t gfp-cbx2 cells were seeded on poly-l-lysine coated coverslips, and the next day mock or hspa1a/hsp70 sirnas were transfected using lipofectamine 2000 (thermo fisher scientific). two days after transfection, cells were heat shocked and fixed at the indicated time points. cellular fractionation and nucleoli isolation was essentially performed as described (andersen et al., 2002) and http://www.lamondlab.com). briefly, 1 â 10 8 k562 or hl60 cells were spun down and washed using pbs. subsequently, cell pellets were resuspended in 2 ml ice-cold buffer a (10 mm hepes ph 7.9, 1.5 mm mgcl 2 , 10 mm kcl, 0.5 mm dtt, and protease inhibitors. cells were incubated on ice for five minutes and subsequently broken open using a 2 ml dounce homogenizer (10 strokes using a tight pestle). dounced cells were spun down at 4˚c and the supernatant (cytoplasmic fraction) was stored. the pellet was resuspended in 1.2 ml buffer s1 (0.25 m sucrose, 10 mm mgcl2, and protease inhibitors) and layered on 1.2 ml buffer s2 (0.35m sucrose, 0.5 mm mgcl2, and protease inhibitors). the nuclei were spun though the sucrose cushion for 5 min at 1475 x g. next, the nuclei were resuspended in 1.2 ml buffer s2 and sonicated 6 â 10 s on ice using a probe sonicator (soniprep 150, mse). the sonicated nuclei were layered on 1.2 ml buffer s3 (0.88m sucrose, 0.5 mm mgcl2, and protease inhibitors) and centrifuged for 10 min at 2889 x g. the supernatant (nucleoplasmic fraction) was stored and the pellet was washed using 0.5 ml buffer s2 and finally resuspended in 80 microliter buffer s2. subsequently, 20 microliter 5x laemmli sample buffer was added and samples were boiled for 5 min. nucleoli samples were loaded on a 4-12% pre-cast nupage gel (invitrogen), and shortly ran into the gel. gel staining was performed using coomassie dye r-250 (thermo scientific) followed by destaining with ultrapure water. coomassie-stained samples were excised in one gel slice that were further cut into small pieces and destained using 70% 50 mm nh 4 hco 3 and 30% acetonitrile. reduction was performed using 10 mm dtt dissolved in 50 mm nh 4 hco 3 for 30 min at 55˚c. next the samples were alkylated using 55 mm iodoacetamide in 50 mm nh 4 hco 3 for 30 min at room temperature and protected from light. subsequently, samples were washed for 10 min with 50 mm nh 4 hco 3 and for 30 min with 100% acetonitrile. remaining fluid was removed and gel pieces were dried for 15 min. at 55˚c. tryptic digest was performed by addition of sequencing-grade modified trypsin (10 ng/ml in 50 mm nh 4 hco 3 ) and overnight incubation at 37˚c. peptides were extracted using 5% formic acid followed by a second elution with 5% formic acid in 75% acetonitrile. samples were dried in a speedvac centrifuge and dissolved in 5% formic acid. online chromatography of peptides was performed with an ultimate 3000 nano-hplc system (thermo fisher scientific) coupled online to a q-exactive-plus mass spectrometer with a nanoflex source (thermo fisher scientific) equipped with a stainless steel emitter. tryptic digests were loaded onto a 5 mm â 300 mm i.d. trapping micro column packed with pepmap100 5 mm particles (dionex) in 0.1% fa at the flow rate of 20 ml/min. after loading and washing for 3 min, peptides were forward-flush eluted onto a 50 cm â 75 mm i.d. nanocolumn, packed with acclaim c18 pepmap100 2 mm particles (dionex). the following mobile phase gradient was delivered at the flow rate of 300 nl/ min: 3-50% of solvent b in 90 min; 50-80% b in 1 min; 80% b during 9 min, and back to 3% b in 1 min and held at 3% b for 19 min. solvent a was 100:0 h2o/acetonitrile (v/v) with 0.1% formic acid and solvent b was 0:100 h2o/acetonitrile (v/v) with 0.1% formic acid. ms data were acquired using a data-dependent top-10 method dynamically choosing the most abundant not-yet-sequenced precursor ions from the survey scans (300-1650 th) with a dynamic exclusion of 20 s. sequencing was performed via higher energy collisional dissociation fragmentation with a target value of 1e5 ions determined with predictive automatic gain control. isolation of precursors was performed with a window of 1.8 da. survey scans were acquired at a resolution of 70,000 at m/z 200. resolution for hcd spectra was set to 17,500 at m/z 200 with a maximum ion injection time of 50 ms. normalized collision energy was set at 28. furthermore, the s-lens rf level was set at 60 and the capillary temperature was set at 250degr. c. precursor ions with single, unassigned, or six and higher charge states were excluded from fragmentation selection. raw mass spectrometry data were analyzed using maxquant version, 1.5.2.8 (cox and mann, 2008) , using default settings and lfq/ibaq enabled, and searched against the human uniprot/ swissprot database (downloaded june 26, 2016, 20197 entries). the data was further processed using perseus software, version 1.5.8.5 (tyanova et al., 2016) . total rna was isolated using the rneasy mini kit (qiagen), and cdna was generated using the iscript cdna synthesis kit (bio-rad). for quantitative rt-pcr, cdna was amplified using ssoadvanced sybr green supermix (bio-rad) on a myiq thermocycler (bio-rad). primer sequences can be found in supplementary file 6. chip analysis was essentially performed as described previously (frank et al., 2001) . chip reactions were performed using the following antibodies: anti-gfp (ab290, abcam), anti-cbx8 (c15410333, diagenode), anti-ezh2 (d2c9, cell signaling), anti-h2ak119ub (d27c4, cell signaling), anti-h3k27me3 (c15410195, diagenode), anti-h3k27ac (c15410196, diagenode), anti-h3k4me1 (c15410194, diagenode), and anti-h3k4me3 (c15410003, diagenode). chip efficiencies were assessed using qpcr. primer sequences can be found in supplementary file 6. sequencing samples were prepared according to the manufacturer's protocol (illumina). end repair was performed using the precipitated dna using klenow and t4 pnk. a 3' protruding a base was generated using taq polymerase and adapters were ligated. the dna was loaded on gel and a band corresponding to~300 bp (chip fragment + adapters) was excised. the dna was isolated, amplified by pcr and used for cluster generation on the illumina nextseq 500 genome analyzer. the 50 bp tags were mapped to the human genome hg19 using bwa . for processing and manipulation of sam/bam files samtools was used . for each base pair in the genome, the number of overlapping sequence reads was determined and averaged over a 10 bp window and visualized in the ucsc genome browser (kent et al., 2002) . peak calling algorithm macs was used to detect the binding sites at a q-value cut off for peak detection of 0.01. chip-seq tracks were visualized using ucsc genome browser (kent et al., 2002) . identification of genes associated to detected peaks was performed using great (mclean et al., 2010) . the accession number the chip-seq data in this paper is geo: gse121182. tags within a given region were counted and adjusted to represent the number of tags within a 1 kb region. subsequently, the percentage of these tags as a measure of the total number of sequenced tags of the sample was calculated. heatmaps and bandplot profiles were generated using fluff (georgiou and van heeringen, 2016) . gene ontology (go) analysis was performed using bingo (maere et al., 2005) . data availability numerical data of proteomics experiments can be found in supplementary files 1-3. additional data on detected peaks in our chip-seq data sets can be found in supplementary files 4 and 5. sequencing data have been deposited in geo under accession codes gse121182. heat shock factors: integrators of cell stress, development and lifespan directed proteomic analysis of the human nucleolus nucleolar proteome dynamics accumulation of c-myc and proteasomes at the nucleoli of cells containing elevated c-myc protein levels immobilization of proteins in the nucleolus by ribosomal intergenic spacer noncoding rna targeting polycomb systems to regulate gene expression: modifications to a complex story the multifunctional nucleolus a quantitative proteomics analysis of subcellular proteome localization and changes induced by dna damage the aging epigenome active liquid-like behavior of nucleoli determines their size and shape in xenopus laevis oocytes role of histone h3 lysine 27 methylation in polycomb-group silencing microinjection of ubiquitin: changes in protein degradation in hela cells subjected to heat-shock microinjection of ubiquitin: intracellular distribution and metabolism in hela cells maintained under normal physiological conditions paf1 regulation of promoter-proximal pause release via enhancer activation maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification a dynamic ubiquitin equilibrium couples proteasomal activity to chromatin remodeling cancer epigenetics: from mechanism to therapy polycomb group proteins ring1a/b link ubiquitylation of histone h2a to heritable gene silencing and x inactivation quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals changes in the cytoplasmic, nuclear, and nucleolar proteomes in vero cells infected with the coronavirus infectious bronchitis virus spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress ezh1 and ezh2 cogovern histone h3k27 trimethylation and are essential for hair follicle homeostasis and wound repair proteasomal atpases are associated with rdna: the ubiquitin proteasome system plays a direct role in rna polymerase i transcription binding of c-myc to chromatin mediates mitogen-induced acetylation of histone h4 and gene activation fluff: exploratory analysis and visualization of high-throughput sequencing data molecular chaperones in protein folding and proteostasis mll-af9-mediated immortalization of human hematopoietic cells along different lineages changes during ontogeny enhanced self-renewal of hematopoietic stem cells mediated by the polycomb gene product bmi-1 misfolded proteins partition between two distinct quality control compartments heat shock proteins as potential targets for protective strategies in neurodegeneration the human genome browser at ucsc silencing of human polycomb target genes is associated with methylation of histone h3 lys 27 pathways of cellular proteostasis in aging and disease promiscuous interactions and protein disaggregases determine the material state of stress-inducible rnp granules histone methyltransferase activity associated with a human multiprotein complex containing the enhancer of zeste protein the biology of proteostasis in aging and disease proteomics analysis of the nucleolus in adenovirus-infected cells proteasome inhibitors induce nucleolar aggregation of proteasome target proteins and polyadenylated rna by altering ubiquitin availability bmi-1 determines the proliferative capacity of normal and leukaemic stem cells the sequence alignment/map format and samtools widespread rearrangement of 3d chromatin organization underlies polycomb-mediated stressinduced silencing fast and accurate short read alignment with burrows-wheeler transform brd4 and jmjd6-associated anti-pause enhancers in regulation of transcriptional pause release bingo: a cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks the chd family of chromatin remodelers the liquid drop nature of nucleoli great improves functional interpretation of cis-regulatory regions compartment-specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition rapid deubiquitination of nucleosomal histones in human tumor cells caused by proteasome inhibitors and stress response inducers: effects on replication, transcription, translation, and the cellular stress response cellular handling of protein aggregates by disaggregation machines proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging protein disaggregation in multicellular organisms dynamic changes in the localization of thermally unfolded nuclear proteins associated with chaperone-dependent protection intracellular distribution of 73,000 and 72,000 dalton heat shock proteins in hela cells bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells polyq proteins interfere with nuclear degradation of cytosolic proteins by sequestering the sis1p chaperone natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex a liquid-to-solid phase transition of the als protein fus accelerated by disease mutation long-term maintenance of human hematopoietic stem/progenitor cells by expression of bmi1 repression of bmi1 in normal and leukemic human cd34(+) cells impairs self-renewal and induces apoptosis bmi1 collaborates with bcr-abl in leukemic transformation of human cd34+ cells constitutive activation of stat5a promotes human hematopoietic stem cell self-renewal and erythroid differentiation ezh1 mediates methylation on histone h3 lysine 27 and complements ezh2 in maintaining stem cell identity and executing pluripotency occupying chromatin: polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put histone core modifications regulating nucleosome structure and dynamics the perseus computational platform for comprehensive analysis of (prote)omics data nonredundant and locus-specific gene repression functions of prc1 paralog family members in human hematopoietic stem/progenitor cells emerging insights into the roles of the paf1 complex in gene regulation interaction proteomics analysis of polycomb proteins defines distinct prc1 complexes in mammalian cells role of histone h2a ubiquitination in polycomb silencing nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian cells cellular and biochemical events in mammalian cells during and after recovery from physiological stress the histone chaperone fact: structural insights and mechanisms for nucleosome reorganization we thank marcel de vries (interfaculty mass spectrometry center, groningen) for help with mass spectrometry analyses. this work is supported by a grant from the dutch cancer foundation (rug 2014-6832). part of this work was performed at the umcg microscopy and imaging center (umic), which is sponsored by nwo-grant 175-010-2009-023. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. the following dataset was generated: key: cord-299509-7xjdryoq authors: scholte, florine e. m.; tas, ali; martina, byron e. e.; cordioli, paolo; narayanan, krishna; makino, shinji; snijder, eric j.; van hemert, martijn j. title: characterization of synthetic chikungunya viruses based on the consensus sequence of recent e1-226v isolates date: 2013-08-01 journal: plos one doi: 10.1371/journal.pone.0071047 sha: doc_id: 299509 cord_uid: 7xjdryoq chikungunya virus (chikv) is a mosquito-borne alphavirus that re-emerged in 2004 and has caused massive outbreaks in recent years. the lack of a licensed vaccine or treatment options emphasize the need to obtain more insight into the viral life cycle and chikv-host interactions. infectious cdna clones are important tools for such studies, and for mechanism of action studies on antiviral compounds. existing chikv cdna clones are based on a single genome from an individual clinical isolate, which is expected to have evolved specific characteristics in response to the host environment, and possibly also during subsequent cell culture passaging. to obtain a virus expected to have the general characteristics of the recent e1-226v chikv isolates, we have constructed a new chikv full-length cdna clone, chikv ls3, based on the consensus sequence of their aligned genomes. here we report the characterization of this synthetic virus and a green fluorescent protein-expressing variant (chikv ls3-gfp). their characteristics were compared to those of natural strain ita07-ra1, which was isolated during the 2007 outbreak in italy. in cell culture the synthetic viruses displayed phenotypes comparable to the natural isolate, and in a mouse model they caused lethal infections that were indistinguishable from infections with a natural strain. compared to ita07-ra1 and clinical isolate nl10/152, the synthetic viruses displayed similar sensitivities to several antiviral compounds. 3-deaza-adenosine was identified as a new inhibitor of chikv replication. cyclosporin a had no effect on chikv replication, suggesting that cyclophilins -opposite to what was found for other +rna virusesdo not play an essential role in chikv replication. the characterization of the consensus sequence-based synthetic viruses and their comparison to natural isolates demonstrated that chikv ls3 and ls3-gfp are suitable and representative tools to study chikv-host interactions, screen for antiviral compounds and unravel their mode of action. chikungunya virus (chikv) re-emerged in 2004 and has caused unprecedented outbreaks in asia and africa since 2005. the estimated number of cases exceeds 2 million and over a thousand infected travelers have returned to europe and the usa since 2006 [1, 2] . chikv generally causes a fever that resolves within several days, a maculopapular rash, and a characteristic arthralgia that can be extremely painful and may persist for months. during the recent outbreaks also more severe clinical manifestations have been reported occasionally, such as neurological complications and even deaths, usually in the elderly, patients with underlying conditions, and newborns [3, 4] . a licensed vaccine or specific antiviral therapy are currently not available. chikv is an alphavirus with an 11.7 kb positive-stranded rna genome that contains two open reading frames (orfs). the 59 orf encodes the nonstructural polyproteins p123 and p1234. the latter results from translational read-through of an opal termination codon that is present at the end of the nonstructural protein (nsp) 3 coding sequence of most chikv isolates. assuming that chikv follows the typical alphavirus life cycle, proteolytic processing of the nonstructural polyproteins by the protease domain in nsp2 will ultimately lead to the release of nsp1, nsp2, nsp3, and nsp4. these nsps and their precursors possess a variety of functions and the enzymatic activities, including protease, helicase, methyltransferase, and rna-dependent rna polymerase (rdrp) activity that drive chikv replication [5] . in addition to replication of its genomic rna, chikv also transcribes a subgenomic (sg) rna encoding a precursor polyprotein that is processed by viral and cellular proteases into the structural proteins c, e3, e2, 6k and e1. chikv nsps willpresumably together with host factors -assemble into replication and transcription complexes (rtcs) that associate with membrane structures derived from the plasma membrane and/or endosomes, as observed for other alphaviruses [5] [6] [7] . the chikv strains that emerged during the 2005-2006 outbreaks had acquired a mutation (a226v) in the e1 envelope glycoprotein, which facilitated transmission of the virus via a new vector, the asian tiger mosquito aedes albopictus, and consequently dramatically increased the epidemic potential of chikv [8, 9] . later studies suggested that recent indian and indian ocean epidemics have emerged separately as the result of at least three independent events, and that convergent evolution of east-central-south african lineage strains in different geographical regions ultimately led to the emergence of strains with the a226v substitution in e1 [10] [11] [12] [13] . more recently, other amino acid positions and epistatic interactions were also shown to play an important role in the emergence of these new chikv variants, which are now even replacing endemic strains that have been circulating in asia for decades [14] . aedes albopictus also thrives in more temperate climates and its geographical distribution has rapidly expanded. over the past decades, parts of southern europe and large areas of the usa have been invaded by this mosquito, providing imported cases of chikv with a competent mosquito vector, thus paving the road for outbreaks in nonendemic-areas such as the usa and europe. indeed, autochthonous infections have been reported from italy in 2007 and france in 2010 [15, 16] . the recent and ongoing chikv outbreaks are characterized by their rapid geographical spread, high numbers of infected people and high morbidity, emphasizing the need to gain more insight into the replicative cycle of this important human pathogen. infectious cdna clones of viruses have become invaluable tools that allow reverse genetics studies to elucidate the contribution of specific amino acids or rna structures to viraemia, virulence, antigenicity, replication kinetics, interactions with host factors, adaptation to new vectors, and many other aspects of the viral life cycle. the use of cdna clones is also instrumental in mechanism of action studies to pinpoint the viral target of antiviral compounds by selecting for and genotyping compound-resistant viruses, followed by reverse engineering of the identified mutations to assess their individual phenotypic contribution to resistance. finally, the generation of cdna clones of reporter viruses, like those expressing green fluorescent protein (gfp), greatly facilitates high throughput screening, e.g. for antiviral compounds or host factors that affect replication. several chikv cdna clones have been constructed in the past, which -except for the west african lineage strain 37997 strain that was isolated from a mosquito -were all based on clinical isolates from infected humans [17] [18] [19] [20] [21] [22] [23] . each natural isolate is expected to have evolved its own specific characteristics in terms of sequence, virulence and virus-host interactions as a result of specific selective pressures within the infected host (tissue) and possibly also during subsequent passaging in cell culture. intrahost evolution and quasispecies diversity is expected to be substantial, especially compared to the relatively low level of interhost variation when the consensus sequences of chikv genomes isolated from different hosts are aligned. the low level of interhost variation is a typical trait of arboviruses, due to evolutionary constraints imposed by the alternating replication in vertebrate and arthropod hosts. a recent study on the distantly related ross river virus indeed reported a high level of intrahost diversity [24] . the existing chikv molecular clones can be considered to represent a single individual genome (or fragments of several individual genomes) out of the whole spectrum of viruses present in the chikv quasispecies population that has been shaped by intrahost evolution and probably a complex set of environmental factors. in contrast, most deposited chikv genome sequences represent the consensus (or master sequence) of a viral quasispecies population. to obtain a virus that -in terms of virulence, sensitivity to antiviral compounds, and chikv-host interactions -is expected to have the general characteristics of the e1-226v chikv strains that were circulating during the 2005-2009 outbreaks, we have constructed a completely synthetic chikv cdna clone based on the consensus sequence of the aligned genomes of these recent isolates. this new infectious clone, chikv ls3 (leiden synthetic 3), and a variant that expresses the egfp reporter gene under control of a duplicated subgenomic promoter (chikv ls3-gfp), were created by custom dna synthesis. the properties and replicative cycle of the new synthetic viruses were characterized in detail, and comparison with a field isolate (ita07-ra1) from the 2007 chikv outbreak in italy demonstrated that they have similar characteristics. the sensitivity of ls3 to a number of antiviral compounds was compared to those of ita07-ra1 and clinical isolate nl10/152. all compounds tested had a similar antiviral activity against ls3 and the natural isolates. these experiments also identified 3-deaza-adenosine as a novel inhibitor of chikv replication. this study describes a detailed characterization of the chikv replication cycle at the molecular level and demonstrates that a new synthetic infectious clonederived virus is a useful and representative tool to gain more insight into the replicative cycle of chikv, its interactions with the host, and the mode of action of antiviral compounds, which should aid in the development of antiviral strategies against this important human pathogen. vero e6, ae. albopictus c6/36 [25] and 293/ace2 cells [26] were maintained in dulbecco's modified eagle's medium (dmem; lonza), supplemented with 8% fetal calf serum (fcs; paa), 2 mm l-glutamine, 100 iu/ml of penicillin and 100 mg/ml of streptomycin. 293/ace2 cells were grown in the presence of 12 mg/ml blasticidin (paa) and c6/36 medium was supplemented with non-essential amino acids (lonza). bhk-21 cells were cultured in glasgow's modified eagles medium (gibco) supplemented with 7.5% fcs, 10 mm hepes ph 7.4, 8% tryptose phosphate broth (gibco), and antibiotics. the mammalian cell lines were grown at 37uc and c6/36 cells at 30uc in 5% co 2 . chikv strain ita07-ra1 (genbank accession number eu244823) was isolated from ae. albopictus during the 2007 outbreak in ravenna, italy, and was passaged twice on bhk-21 cells. chikv nl10/152 (genbank kc862329) was isolated at the erasmus medical center in rotterdam from the serum of an infected traveler that returned from indonesia and was passaged twice on vero cells. working stocks of chikv were routinely produced in vero e6 cells at 37uc, typically yielding titers of ,10 7 pfu/ml. infections were performed in eagle's minimal essential medium (emem; lonza) with 25 mm hepes (lonza) supplemented with 2% fcs, l-glutamine, and antibiotics. after 1 h, the inoculum was replaced with fresh culture medium. all procedures with live chikv were performed in a biosafety level 3 facility at the leiden university medical center. a cdna clone of the synthetic chikv strain ls3-gfp, which contains a duplicated subgenomic promoter and expresses the egfp reporter gene, was designed in silico as described in the results section. three dna fragments together forming a cdna copy of chikv ls3-gfp were chemically synthesized (geneart, germany). using standard cloning techniques, these fragments were assembled and cloned into the asci-noti sites of vector puc19an, a puc19-derived plasmid in which the original polylinker was replaced by one with asci-ncoi-ecorv-xhoi-noti sites. the resulting plasmid (pchikv-ls3-gfp) contains the genomic cdna of chikv ls3-gfp directly downstream of a phi2.5 promoter and followed by a unique spei linearization site for dna linearization prior to in vitro transcription. the 'wild type' synthetic pchikv-ls3 construct was made by deleting the 920 bp egfp-containing saci fragment from pchikv-ls3-gfp (fig. 1) . a third variant with a duplicated subgenomic promoter and a multiple cloning site behind subgenomic promoter 1 (pchikv-ls3-mcs), which allows the introduction of e.g. a reporter gene, was generated by removing the 737 bp asisi-paci fragment from pchikv-ls3-gfp. the constructs were verified by sequencing. in vitro transcription and rna transfection rna was transcribed from plasmids with the phi2.5 promoter [27] using the ampliscribe t7 high yield transcription kit (epicenter), the m 7 gpppa rna cap structure analog (neb) and 0.7 mg of template dna that had been linearized with spei. after a 3-h reaction at 37uc, template dna was digested with dnasei and rna was purified by precipitation with 7.5 m licl (ambion). the concentration of in vitro transcribed rna was determined with a nanodrop spectrophotometer (thermo scientific) and its integrity was checked by agarose gel electrophoresis. bhk-21 cells (2610 6 ) were electroporated with 1 mg of rna using program t-20 of the amaxa nucleofector and kit t (lonza) according to the manufacturer's instructions. electroporated cells were plated in 6well clusters and incubated at 37uc in the same medium used for chikv infection experiments. chikv rna was isolated from virions using the qiaamp viral rna mini kit. four overlapping amplicons were generated by a two-step reverse transcriptase (rt) pcr. in the first step cdna was synthesized using revertaid h minus reverse transcriptase (fermentas) and primers at-39 (gactgca-gatgcccgccatt), at-41 (cgctcggtccagg-caactct), at-43 (cgtggtgtttgccaacaggc), or at-52 (cgccgttttttttttttttttttttttttt). in the second step 4 pcr products were generated using combinations of primers at-38 (atggctgcgtgagacacacg) and at-39, at-40 (tgcacccaagtgtaccacaa) and at-41, at-42 (caggagagtgcatccatggc) and at-43, or at-44 (gaatgcgcgcagatacccgt) and at-52. the resulting rt-pcr products were purified and directly sequenced (50 ng template) using the bigdye terminator cycle sequencing kit v1.1 (applied biosystems) and a 3130 genetic analyzer automatic sequencer (applied biosystems). pcr conditions and primer sequences are available upon request. viral titers were determined by plaque assay on vero e6 cells. six-well clusters containing confluent monolayers of vero e6 cells were incubated with 0.5-ml volumes of 10-fold serial dilutions of chikv-containing samples. after a 1-h incubation at 37uc, the inoculum was replaced with 2 ml of dmem containing 1.2% avicel rc-581 (fmc biopolymer), 2% fcs, 25 mm hepes, and antibiotics. after a 66-h incubation at 36uc, monolayers were fixed with 3.7% formaldehyde in pbs and plaques were visualized by crystal violet staining. for infectious center assays 10-fold serial dilutions of electroporated cells were added to 6-well clusters already containing a monolayer of 1610 6 bhk-21 cells per well. after a 1-h incubation at 37uc, a dmem/avicel overlay was applied and cells were incubated at 37uc for 48 h. plaques were visualized as described above. total rna was isolated from 7610 5 cells by lysis in 0.5 ml of 20 mm tris-hcl (ph 7.4), 100 mm licl, 2 mm edta, 5 mm dtt, 5% (w/v) lithium dodecyl sulfate, and 100 mg/ml proteinase k. after acid phenol (ambion) extraction, rna was precipitated with isopropanol, washed with 75% ethanol, and dissolved in 1 mm sodium citrate (ph 6.4). samples containing rna from 4.7610 4 cells were mixed with 3 volumes of 67% formamide, 23% formaldehyde, 6.7% glycerol, 13 mm mops (ph 7.2), 6.7 mm naac, 2.7 mm edta, 0.07% sds, and 0.03% bromophenol blue. after denaturation for 15 min at 75uc, rna was separated in 1.5% denaturing formaldehyde-agarose gels using the mops buffer system as described [28] . rna molecules were detected by direct hybridization of the dried gel with 32 p-labeled oligonucleotides essentially as described previously [29] . positive-stranded genomic and subgenomic chikv rnas were visualized with probe chikv-hyb4 (59-tgtgggttcggagaatcgtg-gaagagtt-39) that is complementary to the 39 end of the genome. negative-stranded rna was detected with probe chikv-hyb2 (59-aacccatcatggatcctgtgtacgtg-ga-39) that is complementary to the 39 end of the minus strand. 18s ribosomal rna (loading control) was detected with the oligonucleotide probe 59-atgcccccggccgtccctct-39. probes (10 pmol) were labeled with 10 mci [c-32 p]atp (perki-nelmer) in a 1h reaction using 10 u of t4 polynucleotide kinase (invitrogen) in 10 ml of the supplied forward reaction buffer (invitrogen). prehybridization (1 h) and hybridization (overnight) were done at 55uc in 56 sspe (0.9 m nacl, 50 mm nah 2 po 4 , 5 mm edta, ph 7.4), 56 denhardt's solution, 0.05% sds, and 0.1 mg/ml homomix i. hybridized gels were washed twice in 56 sspe with 0.05% sds before they were exposed to storage phosphor screens. after scanning with a typhoon-9410 scanner (ge healthcare), quantification of rna levels was done with quantity one v4.5.1 (biorad) and corrections for loading variations were made based on the quantity of 18s ribosomal rna in the same lane. the results of two or three independent experiments were quantified (one representative experiment is shown in figures). total protein samples were prepared by lysing 7610 5 cells in 0.5 ml of 46 laemmli sample buffer (100 mm tris-hcl, ph 6.8, 40% glycerol, 8% sds, 40 mm dtt, 0,04 mg/ml bromophenol blue). proteins were separated by sds-page in 12% polyacrylamide gels and were transferred to hybond-lfp membranes (ge healthcare) by semi-dry blotting. after blocking with 1% casein (sigma) in pbs with 0.1% tween-20 (pbst), membranes were incubated overnight with rabbit antisera against chikv nsp1 (raised using the peptide eveprqvtpndhan), nsp4 (raised using the peptide assrsnfeklrgpv) or e2 [30] in pbst with 0.5% casein. mouse monoclonal antibodies against b-actin (sigma), or the transferrin receptor (zymed) were used for detection of loading controls. biotin-conjugated swine-a-rabbit (dako) or goat-a-mouse (dako), and cy3-conjugated mousea-biotin (jackson) were used for fluorescent detection of the primary antibodies with a typhoon-9410 scanner (ge healthcare). at various time points post infection approximately 2610 5 chikv-infected or mock-infected 293/ace2 cells in 12-well clusters were incubated with 40 mci of 3 h-uridine in medium and incorporation was allowed to proceed for 60 minutes at 37uc. total rna was isolated and analyzed in a denaturing agarose gel as described above. for fluorographic detection of 3 h-labeled rna, the gel was soaked in methanol for 1 hour (one change) and then incubated with 3% 2,5-diphenyloxazole in methanol for at least 3 hours. after incubation in milliq for 30 minutes, the gel was dried and a fuji rx film was placed on top. films were developed after a 1-4 day exposure at 280uc and scanned with a biorad gs-800 densitometer. to check equal sample loading, the gel was hybridized with a 32 p-labeled 18s ribosomal rna-specific probe as described above. in addition, incorporation of 3 h-uridine into rna was quantified by analyzing 2-ml samples of isolated total rna with a liquid scintillation counter (beckman ls 6500 ic). in control samples, cellular transcription was inhibited by adding actinomycin d (sigma) to a final concentration of 5 mg/ml. at various time points post infection approximately 2610 5 chikv-infected or mock-infected 293/ace2 cells in 12-well clusters were starved in dmem lacking l-methionine and lcysteine (invitrogen) for 30 min., and subsequently incubated with 44 mci easytag express 35 s protein labeling mix (perkinelmer) for 30 min. total protein samples were analyzed by sds-page as described above. gels were stained with coomassie to check equal sample loading and 35 s-labeled proteins were detected by drying the gels and exposing them to a storage phosphor screen, which was scanned 1-2 days later with a typhoon-9410 scanner (ge healthcare). chikv-or mock-infected vero e6 cells grown on coverslips were fixed with 3% paraformaldehyde in pbs. after quenching with 10 mm glycine in pbs, cells were permeabilized with 0.1% triton in pbs for 10 min. and coverslips were incubated with primary antibodies diluted in pbs with 5% fcs for 1 h. doublestranded rna was detected with a 1:200 dilution of mouse monoclonal antibody j2 (english & scientific consulting). chikv e2 was visualized with a 1:8000 dilution of a polyclonal rabbit antiserum [30] . detection of primary antibodies was done with donkey-a-mouse-cy3, goat-a-rabbit-cy3 or goat-a-rabbit-alexa488 (1:500; jackson). nuclei were stained with hoechst 33342. the coverslips were mounted with prolong (invitrogen) and analyzed using an axioskop2 mot plus fluorescence microscope with axiocam hrc camera and axiovision software (zeiss). mouse monoclonal antibodies raised against chikv particles of strain ita07-ra1 (izsler, brescia, italy) were heatinactivated for 30 min. at 56uc. two-fold serial dilutions of the neutralizing monoclonal antibody 1h7 and non-neutralizing control antibody 3h9 [31] were incubated with an equal volume of medium containing 100 pfu of chikv. these mixtures were incubated for 60 min. at 37uc and transferred to 96-well clusters containing 2610 4 vero e6 cells per well. after incubation at 37uc for 2 days, the wells were fixed with 3.7% formaldehyde and cpe was detected by staining with crystal violet. chloroquine, 6-aza-uridine and ribavirin were dissolved in pbs. cyclosporin a and 3-deaza-adenosine were dissolved in dmso. mycophenolic acid was dissolved in ethanol. all compounds were obtained from sigma. for cpe reduction assays, 96-well clusters with ,1610 4 vero e6 cells/well were incubated with 50 pfu of virus per well, corresponding to a multiplicity of infection (moi) of 0.005, and 2-fold serial dilutions of the compound in medium. wells without cells, uninfected cells, infected untreated cells and infected cells treated with solvent alone were included as controls. four days post-infection cell viability was assessed using the celltiter 96h aqueous non-radioactive cell proliferation assay (promega). cpe reduction experiments with ribavirin were done with bhk-21 cells in a similar way, except that viability was assessed 2 days post infection. for egfp reporter gene assays, ,1610 4 vero e6 cells/well in black 96-well plates were infected with chikv ls3-gfp at an moi of 0.05. after a 42-h incubation in medium containing the compound, the cells were fixed with 3% paraformaldehyde in pbs. egfp expression was quantified using a berthold mithras lb 940 plate reader, with excitation and emission wavelengths of 485 and 535 nm, respectively. the fluorescence in wells containing mock-infected cells was used to correct for background signal. ic 50 and cc 50 values were calculated with graphpad prism 5 using the nonlinear regression model. all animal experiments described in this paper were carried out in the bsl3 facilities of the erasmus medical center in accordance with the dutch guidelines for animal experimentation and were approved by the institute's independent animal ethics committee. twelve-day old c57bl/6 mice were injected intraperitoneally with 100 tcid 50 of chikv s27, chikv ls3 or chikv ls3-gfp. after the challenge the mice were monitored daily for signs of illness or death. the infection was considered lethal when the animals reached humane end-points and needed to be euthanized. viral rna was extracted from brain samples using the automated magnapure method (total nucleic acid isolation kit, roche diagnostics, the netherlands) according to the manufacturer's instructions, and quantified using a one-step rt-pcr taqman protocol (ez-kit, applied biosystems) and an abi prism 7500 detection instrument. the primers and probe used for chikv rna quantification were essentially as described [32] except that probe fam-ccaatgtcttcagcctgga-caccttt-tamra was used. dilutions of virus suspensions of known titer were included to make a calibration curve, which was used to express results as tcid 50 equivalents per gram of brain tissue. all animal experiments described in this paper were carried out in the bsl3 facilities of the erasmus medical center in accordance with the dutch guidelines for animal experimentation. a dutch government-approved and independent animal experimentation ethical review committee (stichting dec consult) approved the animal studies (permit nr. emc2838/122-12-29). the genbank accession numbers for the full-length cdna clones pchikv-ls3, pchikv-ls3-gfp and pchikv-ls3-mcs are jx911334, jx911335, and jx911336 respectively. the genbank accession numbers for the genomic rna sequences of chikv ls3, ls3-gfp, lcs3-mcs and nl10/152 are kc149888, kc149887, kc149889, and kc862329, respectively. the complete genomes of the 13 chikv strains carrying the e1-a226v mutation ( table 1 ) that were available in genbank at the time of in silico design (november 2009) were aligned using mafft [33] and the resulting consensus sequence formed the basis for the synthetic full-length cdna clones. a 40 nucleotide polya tail was added to the 39 end of the consensus sequence and an a7435g point mutation was introduced to create a translationally silent saci restriction site required for cloning. the virus encoded by the resulting sequence was termed chikv ls3 (genbank accession kc149888). variants containing a duplicated subgenomic promoter and a multiple cloning site (chikv ls3-mcs; genbank kc149889) or an egfp reporter gene (chikv ls3-gfp; genbank kc149887) were also designed. the egfp reporter gene was placed under control of the native subgenomic promoter and upstream of a second subgenomic promoter that drives expression of the viral structural polyprotein, as this configuration was previously reported to result in a more stable reporter gene expression [18] . the chikv cdnas were placed downstream of a phi2.5 t7 promoter, and a unique spei site for linearization prior to in vitro transcription directly followed the polya tail. the phi2.5 promoter was used because the 59 ends of capped transcripts generated from this promoter with t7 polymerase and the m 7 gpppa cap analog are identical to the 59 end of the genomes of naturally occurring chikv strains. in contrast, capped rnas generated by in vitro transcription from the frequently used sp6 promoter will contain m 7 gpppg at their 59 terminus, i.e. will contain an additional 59terminal g residue. however, existing chikv cdna clones that contain the sp6 promoter also efficiently yield infectious virus and it is assumed that the additional 59-terminal g residue is removed during subsequent rounds of replication. in line with this, in vitro transcribed rna from pchikv-ls2, a variant of plasmid pchikv-ls3 in which the phi2.5 promoter was replaced with the sp6 promoter also yielded infectious virus. plasmid pchikv-ls3-gfp, the infectious clone encoding the egfp-expressing reporter virus, was created by assembling the chemically synthesized dna fragments as described in the materials and methods section. plasmid pchikv-ls3, the infectious clone encoding the synthetic 'wild type' strain chikv ls3, and plasmid pchikv-ls3-mcs were generated from pchikv-ls3-gfp by deleting specific restriction fragments, as described in materials and methods (fig. 1) . in the original alignment, strains drde-07 (genbank u372006) and d570/06 (genbank ef012359) shared the highest sequence similarity with ls3, with 3 amino acid differences (table 2) . however, a blast search performed in march 2013, three years after the design of chikv ls3, and alignment of the retrieved complete chikv genomes revealed that strains ind-06-ap3 (genbank ef027134), ind-gj53 (genbank fj000065), and chik31 (genbank eu564335) share the highest degree of nucleotide sequence identity with chikv ls3 (.99.9%), with only 5-7 nucleotide differences respectively (table s1). interestingly, these indian strains were not included in the original alignment on which the ls3 sequence was based, as they do not contain the e1-a226v mutation (table 1) . however, nsp1234 of ls3 is identical to that of ind-06-ap3. at the amino acid level, chikv ls3 differs at 4 positions from lr2006_opy1 and at 3 positions from ita07-ra1 (table 2 ). to determine whether infectious virus could be generated from the synthetic chikv clones, in vitro transcribed rna was electroporated into bhk-21 cells. strong egfp fluorescence was readily detected 16 h after transfection of chikv ls3-gfp rna. for chikv ls3 and ls3-gfp rna specific infectivities of approximately 10 5 pfu/mg of rna were found in infectious center assays, which is similar to what has been found for other chikv cdna clones [17, 18] . virus titers in cell culture supernatants 16 h after electroporation, were generally in the range of 10 5 -10 6 pfu/ml. this is lower than the peak viral titers that are obtained during infection experiments, but can be explained by the early time point of harvesting and the fact that not all cells were transfected. as expected, electroporation of bhk-21 cells with uncapped chikv rna did not result in the release of infectious virus. to assess the stability of egfp reporter expression, chikv ls3-gfp was serially passaged (moi 0.5) in both 293/ace2 and vero e6 cells. virus harvested during each passage was used to infect vero e6 cells at an moi of 0.2 and immunofluorescence microscopy revealed that at passage 10, over 95% of the e2positive foci still displayed robust egfp expression. sequencing of cdna obtained by rt-pcr amplification of rna extracted from extracellular virions revealed that, after 3 passages on vero e6 cells, the consensus genome sequence of chikv ls3-gfp was identical to the original in silico designed sequence. these results demonstrated that the synthetic viruses are viable, genetically stable, and able to retain stable expression of the reporter gene. since we aim to use chikv in sirna screens and proteomics studies to identify host factors involved in replication, various human cell lines were evaluated for their ability to support chikv replication. chikv ls3-gfp was able to productively infect hela, mrc-5, huh7, 293, and 293/ace2 cells (data not shown). infection of hela and huh7 cells was not very efficient and these cells were therefore not used for any further experiments. 293/ace2 cells were selected for this study, as they supported high levels of chikv ls3-gfp replication, could be efficiently transfected with sirnas, and -unlike regular 293 cellsadhered well to tissue culture plastics. 293/ace2 cells stably express angiotensin-converting enzyme 2 (ace2), the receptor for sars-coronavirus. ace2 expression is not required for chikv infection, but these cells were chosen because of the aforementioned advantages and the fact that they have been previously used in our lab in sirna screens for host factors that affect corona-and arterivirus replication ( [34] ; de wilde et al. submitted; wannee et al., in preparation). using these cells in similar sirna screens with chikv and other alphaviruses would allow direct comparison of data sets with those obtained for corona-and arteriviruses, which could lead to the identification of common (broad spectrum) pro-and antiviral host factors. to determine whether the synthetic viruses behave like natural isolates, their growth kinetics in vero e6, 293/ace2, and c6/36 cells were compared to those of ita07-ra1, which was isolated during the 2007 chikv outbreak in italy ( fig. 2a-c) . the growth curves of chikv ls3 on all three cell lines were found not to differ significantly from those of ita07-ra1, with virus titers reaching a maximum 14-18 h post infection (p.i.). peak virus titers on mosquito cells were approximately 1-log higher than those on mammalian cells. chikv ls3-gfp replicated slightly slower than the other viruses in all three tested cell lines, which is not unusual for recombinant reporter viruses. egfp expression could be detected as early as 6 h p.i. and peaked around 22 h p.i. the plaque morphology of the synthetic viruses was similar to that of ita07-ra1 (fig. 2d) . chikv ls3 induced a cytopathic effect (cpe) indistinguishable from the natural isolate. on vero e6 cells early signs of cpe started to appear around 12 h p.i. and cpe was complete by 24 h p.i. (fig. 2e) . to study chikv-induced transcriptional host shut-off, the incorporation of 3 h-uridine into cellular and viral rna was analyzed by metabolic labeling of infected 293/ace2 cells at various time points post infection (moi of 5). a strong reduction in the incorporation of 3 h-uridine into rna was observed at 10-12 h p.i. in cells infected with chikv ls3 or ita07, as determined by liquid scintillation counting of total rna samples table 2 . comparison of the amino acid sequences of chikv ls3 and various natural isolates. the chikv ls3 amino acid sequences were aligned with those of several highly similar natural isolates, and clinical isolate nl10/152. only amino acid differences are indicated and identity is represented by dots. the numbering is based on the sequence of ls3, which is also equal to that of lr2006_opy1. it is important to note that -like all other chikv strains in this ( fig. 3a) . inhibition of cellular transcription with actinomycin d for 30 min. prior to metabolic labeling at 12 h p.i. revealed the contribution of viral rna synthesis to the total signal. fluorographic detection of 3 h-labeled rna analyzed in denaturing gels also showed a decrease in cellular transcription during the course of the infection, while the synthesis of chikv rna became clearly detectable by 6 h p.i (fig. 3b) . transcriptional shut-off occurred around 10-12 h p.i. and was induced by chikv ls3 and ita07 with similar kinetics, although ls3 seemed to act slightly faster. to examine chikv-induced translational shut-off, the synthesis of 35 s-labeled viral and cellular proteins during the course of chikv ls3 infection was analyzed by metabolic labeling of infected 293/ace2 cells with 35 s-met and 35 s-cys (fig. 3c) . a clear shut-off of host translation was observed 8-9 h p.i. beyond 9 h p.i. the bulk of newly produced protein appears to be of viral origin, likely c, e1, e2 and their precursors (indicated with * in fig. 3c ). chikv ita07 and ls3 induced translational host shut-off in a similar manner (only results obtained with ls3 are shown in fig. 3c ). both chikv ita07-ra1 and the synthetic viruses established non-cytopathic persistent infections in c6/36 mosquito cells. all characterization experiments have been performed in both 293/ ace2 and vero e6 cells, with similar results. for simplicity only the results for 293/ace2 cells are shown, except for immunofluorescence experiments, which were done with vero e6 cells as they had a more suitable morphology. the replication cycle of the synthetic viruses and ita07-ra1 was characterized in more detail to assess whether the synthetic viruses behaved like their natural counterpart. the kinetics of rna synthesis was analyzed by isolating total rna from 293/ ace2 cells infected with chikv ls3, ls3-gfp, or ita07-ra1 at various time points post infection. negative-and positivestranded rnas were detected by hybridization with 32 p-labeled oligonucleotide probes (fig. 4a ). both negative-and positivestrand rnas were readily detected in cells infected with the various strains starting at 6 h p.i. the negative-strand rna was less abundant than the positive strand, it was easily detected relatively early in infection ( fig. 4a top panel, fig. 4b) , and appeared to decrease at later time points as has also been observed for other alphaviruses. this apparent decrease is probably not only due to degradation of minus strands, but at least partly due to a hampered detection caused by the large excess of positive-strand rna present at late time points. this excess of positive-strand rna competes with the radioactively labeled minus-strand specific probe. in support of this, we observed that mixing rna isolated from chikv-infected cells at 6 h p.i. with in vitro transcribed positive-strand rna reduced the amount of negative strand that could be detected (data not shown). furthermore, when samples taken at 6 and 14 h p.i. were treated with singlestrand-specific rnase a/t1 before hybridization, the negativestrand levels at the late time point were approximately 70% of that at 6 h p.i, instead of the approximately 50% in untreated samples (data not shown). using a positive-strand-specific probe, both the 49s genomic and 26s sgrna could be detected, and both rnas accumulated until 12 h p.i (fig. 4a middle panel, fig. 4c ). the ratio of genomic to sgrna varied between 1:3.5 and 1:5.5 during the course of infection, similar to the ratios reported for semliki forest virus and sinv [35] . the kinetics of rna synthesis and rna accumulation levels were similar in chikv ls3-and ita07-ra1-infected cells. in cells infected with chikv ls3-gfp, the additional subgenomic rna encoding the egfp reporter gave rise to an extra band above the 26s rna band, and its expression level was calculated to be approximately half of that of the 26s rna. the individual levels of the two sgrnas expressed by chikv ls3-gfp were lower than those of ita07 or ls3, but their combined abundance was comparable to that of the viruses expressing a single sgrna (fig. 4c ). to monitor viral protein expression, 293/ace2 cells were infected with chikv ls3, ls3-gfp, or ita07-ra1 and total protein was isolated at various time points post infection. these samples were analyzed by western blotting with antisera against the nonstructural proteins nsp1 and nsp4, and the structural protein e2. expression of nsp1, e2, and the e3e2 precursor could be detected as early as 6 h p.i. and the proteins accumulated over time, reaching a plateau around 12 h p.i. (fig. 5) . the rdrp nsp4 could not be detected in infected cells using a chikv nsp4specific antiserum capable of detecting the purified bacterially expressed protein. this was probably due to the low affinity of the antibody, the low expression level and relative instability of nsp4 in infected cells, as was also observed for other alphaviruses [36] . in addition, a quantitative proteomics study on chikv-infected cells also suggested that at 10 h p.i. the amount of nsp4 was at least 200-fold lower than that of nsp1 (treffers, tas, de ru, van veelen, snijder and van hemert, manuscript in preparation). indirect immunofluorescence analysis of vero e6 cells infected with chikv ls3, ls3-gfp, or ita07-ra1 at various time points showed that the localization and expression kinetics of e2 and dsrna were similar for the natural isolate and the synthetic viruses (fig. 6) . double-stranded rna, which is assumed to be generated during replication of chikv in infected cells [37] , could be detected as early as 4 h p.i. and remained clearly visible throughout the infection. the dsrna localized to foci throughout the cytoplasm. the e2 protein could be detected from 6 h p.i. onwards with maximum expression reached by 12 h p.i. the e2 protein mainly localized to the plasma membrane of infected cells. egfp produced by the reporter virus was visible from 6 h p.i. onwards, reaching a maximum level around 12 h p.i. (fig. 6c ). chikv ls3 and ita07-ra1 were compared in a neutralization assay using the neutralizing monoclonal antibody 1h7 that was raised in mice against chikv ita07-ra1 virions, and appears to recognize a linear epitope in e2 [31] . the nonneutralizing mab 3h9 was used as a control. both the natural isolate and chikv ls3 were neutralized with similar characteristics by 1h7, while their infectivity was not affected by 3h9 (fig. 7) . newborn mice are highly susceptible to chikv infection and they develop symptoms as lethargy, dragging of hind limbs, flaccid paralysis, and reduced weight gain [38] . 12-day old mice were injected intraperitoneally with 100 tcid 50 of chikv ls3, ls3-gfp or prototype strain s27 as a control. the animals were euthanized when their humane end points were reached 3 or 4 days post infection and viral rna levels in brain tissue were analyzed (fig. 8) . both synthetic viruses behaved like the natural isolate in vivo, causing lethal infections with similar kinetics (fig. 8a ). in addition, the viral titers in the brains of chikv s27-infected mice were similar to those of mice infected with the synthetic viruses (fig. 8b ). to evaluate their suitability for analyzing the potency and mechanism of action of antiviral compounds, the sensitivity of chikv ls3 and ls3-gfp to a number of such compounds was determined and compared to ita07-ra1. cyclosporin a, which through its effect on cellular cyclophilins inhibits the replication of a variety of viruses, had no specific effect on chikv replication, not even at a (cytotoxic) dose of 32 mm (data not shown). the compounds 3-deaza-adenosine, 6-aza-uridine, chloroquine, and mycophenolic acid were tested in cpe reduction assays with vero e6 cells infected at an moi of 0.005 and analyzed 4 days p.i. they were all found to inhibit chikv replication with ic 50 s in the low micromolar range and with minimal cytotoxicity (fig. 9a-d) . no substantial differences were observed between the ic 50 values calculated for ita07-ra1, ls3 and ls3-gfp. the four compounds also clearly reduced egfp reporter gene expression in vero e6 cells infected with chikv ls3-gfp (fig. 9f) . slightly lower ic 50 values were obtained for 6-aza-uridine and chloroquine, and a significantly higher ic 50 was observed for 3-deazaadenosine in this assay, compared to the cpe-based assay. this might be due to the mode of action of 3-deaza-adenosine and/or due to differences in experimental set-up compared to the cpebased assay (moi 0.05 vs. 0.005; measurement 42 h p.i. vs. 4 d p.i). ribavirin is a known inhibitor of chikv replication, but in our cpe reduction assay with vero e6 cells it was not very effective in inhibiting replication of the various strains, as ic 50 values of over 400 mm were obtained (fig. 9e, gray lines) . this is likely due to the inefficient conversion of ribavirin to its active phosphorylated form in vero e6 cells [39] . therefore, we have also analyzed the effect of ribavirin in a 2-day cpe reduction assays with bhk-21 cells, which are able to metabolize ribavirin [40, 41] and found ic 50 s of 15-21 mm for the various strains. clinical isolate nl10/152 was also included in the assays and appeared to be somewhat more sensitive to the antiviral compounds than ls3 and ita07-ra1. however, the slower replication kinetics of this strain made it impossible to directly compare nl10/152 and ls3 in the same cpe reduction assays, despite the fact that virus yields and cytopathic effect of nl10/152 and ls3 were comparable (data not shown). the massive chikv outbreaks that have been occurring in asia and the indian ocean region since 2005 are associated with the emergence of strains with the a226v substitution in the e1 glycoprotein, which allowed their transmission by a novel mosquito vector, aedes albopictus [8] [9] [10] [11] [12] [13] . these east-central-south african lineage-derived strains even appear to be replacing the the relative abundance of rna was calculated as before, except that data were normalized to the value measured for ls3 sgrna at 12 h p.i (100%). genomic rna levels are indicated with black lines, sgrna levels with gray lines. the total level of both sgrnas expressed by ls3-gfp is indicated with the gray dotted line. doi:10.1371/journal.pone.0071047.g004 asian lineage chikv strains that have been endemic in the region for decades. since the 1980s, the geographic distribution of aedes albopictus has dramatically expanded and now also includes large parts of the usa and several european countries. this creates concern for locally transmitted outbreaks in europe and the usa, which could be initiated by viraemic travelers arriving from countries where chikv is endemic, like india and indonesia. locally transmitted chikv infections have indeed already been reported from italy in 2007 and france in 2010 [15, 16] and recent studies suggest that also the usa is at risk for locally transmitted chikv outbreaks [42, 43] . besides its large medical and societal impact in endemic countries, the increased risk of chikv outbreaks in europe and the usa underlines the importance of studying the replication of this important human pathogen and its interactions with the host to develop safe and effective vaccines and antiviral therapy. infectious cdna clones have proven to be important tools to study many aspects of the viral life cycle, and molecular clones of a variety of natural isolates have been instrumental in several recent chikv studies [17] [18] [19] [20] [21] [22] [23] . the existing chikv molecular clones can be considered to be derived from a single genome (or fragments of single genomes) out of the whole spectrum of viruses present in a chikv quasispecies population. in contrast, most of the complete chikv genome sequences that have been deposited in genbank represent the consensus (or master sequence) of a viral quasispecies population. the diversity (and evolution) of a chikv quasispecies population has probably been shaped by the characteristics of the individual host and the specific tissue source (serum) from which it was isolated. for ross river virus it was observed that the level of intrahost genetic variation in patient serum samples, was considerably larger than that observed at the epidemiological scale, which can be explained by the purifying selection for replication in both arthropod and vertebrate hosts [24] . advances in sequencing techniques now allow a more detailed view on quasispecies diversity and intrahost evolution, and also for chikv a recent study has provided more insight into quasispecies dynamics and the effect of purifying selection by host alternation [44] . a link was observed between increased fitness as a result of alternating passaging and reduced quasispecies complexity, which restricted adaptability to novel selective pressures like antiviral treatment or antibody-mediated neutralization [44] . individual chikv isolates or molecular clone derived viruses could have their specific properties in terms of replication kinetics, vector specificity, dissemination within the host, virulence, virus-host interactions or sensitivity to antiviral compounds. we were interested in studying the general characteristics of the life cycle and virus-host interactions of the e1-226v chikv strains that were circulating during the 2005-2009 outbreaks. therefore, we have constructed a fully synthetic cdna clone, chikv ls3, based on the consensus sequence of the aligned genomes of these recent e1-226v isolates, rather than on a single genome from a clinical isolate. in addition, a variant that expresses the egfp reporter gene under control of a (duplicated) subgenomic promoter was created (chikv ls3-gfp). the current possibilities of gene synthesis allowed the design of these clones in silico, with sequences tailored to our requirements, e.g. already containing a reporter gene under control of a duplicated subgenomic promoter and including (translationally silent) mutations to create restriction sites that facilitate cloning and reverse genetics studies. alignment of all 148 complete chikv genomes that were in genbank by june 2013 yielded a consensus sequence that differed only at 3 nucleotide positions from the sequence of ls3 that was designed 3 years earlier. these were position 7,435 at which we introduced a translationally silent restriction site (g7435a), a synonymous urc substitution at position 3,397, and position 10,670, which is a c in 68% of all deposited genomes (strains with e1-226a), while the remaining (e1-226v) strains have a u at this position. an interesting observation was that 6% of the sequenced chikv strains, including the prototype strains s27 and ross, contain an arginine codon instead of the opal stop codon that is present between the nsp3-and nsp4-coding regions of most chikv isolates. the presence or absence of this opal codon might be influenced by the passage history of the isolate as has been observed for other alphaviruses [45] . this might also explain why the sequence of the original clinical isolate of lr2006-opy1 (genbank dq443544.2) contains the opal termination codon near the end of the nsp3 coding region, while the infectious clone of this strain (genbank eu224268.1) contains an arginine codon at this position. to assess whether the synthetic viruses are representative models, their characteristics were compared to those of the natural strain ita07-ra1. like the natural isolate, the synthetic viruses caused cytopathic infections in vero e6 and 293/ace2 cells (fig. 2e) , whereas non-cytopathic persistent infections were observed in the mosquito cell line c6/36. in vertebrate cells all strains caused a shut-off of cellular translation around 8-9 h p.i. and a strong inhibition of cellular transcription by 10-12 h p.i. the accumulation of negative-and positive-strand viral rna, the kinetics of non-structural and structural viral protein expression, as well as the growth kinetics and plaque morphology of the synthetic viruses were indistinguishable from those of chikv ita07-ra1 ( fig. 2-6 ). in addition, the synthetic viruses caused lethal infections in 12-day old mice, with virus spreading to the brain, as observed for natural isolates (fig. 8) . although this demonstrates that the synthetic viruses replicate in vivo, this mouse model does not allow comparison of strains for more subtle differences in virulence and pathogenesis. the genomic stability of chikv ls3-gfp was assessed and after 3 passages its (consensus) sequence was found to be identical to the original in silico designed sequence. the expression of the egfp reporter gene was stable for at least 10 passages, making the synthetic viruses suitable tools for highthroughput screens for antiviral compounds, (reverse genetics) studies into their mechanism of action, and systematic functional genomics screens for host factors affecting chikv replication. to evaluate whether chikv ls3 and ls3-gfp are suitable to analyze the potency and mechanism of action of antiviral compounds, their sensitivity to a number of such compounds was determined and compared to ita07-ra1. the lysosomotropic agent chloroquine and nucleoside analog 6-aza-uridine inhibited the replication of the synthetic viruses and natural isolates with ic 50 s that were in the same range and comparable to values previously reported by others [46] [47] [48] [49] [50] [51] . the inhibitory effect of chloroquine on the replication of many viruses including alphaviruses has been known for decades. for chikv it is a useful reference compound in cell-based studies, but a small scale clinical trial on the island of la reunion suggested it is not effective in the treatment of chikv infections in patients [46] . the nucleoside analog 6-aza-uridine has previously been reported to inhibit the replication of a variety of viruses, including chikv [47, 51] . the compound could interfere with cellular utp metabolism and may be incorporated into chikv rna, leading to chain termination and/or increased error frequency, ultimately resulting in 'error catastrophe'. mycophenolic acid is a noncompetitive inhibitor of inosine monophosphate dehydrogenase (impdh), causing a depletion of the intracellular guanosine pool. it is a known inhibitor of various viruses, including chikv [47, 52] . ribavirin is a synthetic nucleoside analog with broad spectrum antiviral effect due to potential effects on the cellular impdh enzyme, viral rna synthesis and capping [39] . however, not all cell lines are able to perform the necessary conversion of this compound to its active phosphorylated form, explaining the contradictory reports on the antiviral activity of this compound [40, 41, 53] . in our hands, ribavirin inhibited chikv replication in bhk-21 cells with an ic 50 of around 18 mm, while it was hardly effective in vero e6 cells, with ic 50 values of over 400 m. the ic 50 that we obtained with bhk-21 cells is in the same range as those previously reported for the antiviral effect of ribavirin on chikv replication [47, 51] . cyclosporin a, which through its effect on the cellular cyclophilins, inhibits the replication of a variety of viruses (for recent review see [54] ), had no effect on chikv replication. we identified 3-deaza-adenosine as a novel inhibitor of chikv replication with an ic 50 of approximately 6 mm and a cc 50 .50 mm. this compound has previously been identified as inhibitor of a broad spectrum of viruses, although many other +rna viruses appeared to be rather insensitive or not affected at all (reviewed in [55] ). the antiviral activity of 3-deazaadenosine was attributed to its inhibitory effect on the cellular enzyme s-adenosylhomocysteine hydrolase, leading to an accumulation of s-adenosylhomocysteine, which inhibits s-adenosylmethionine-dependent methylation reactions [55] . in this manner the enzyme plays a key role in s-adenosylmethionine-dependent methylation reactions and inhibition of viral methylation reactions (e.g. of viral rna) apparently can be achieved at compound concentrations that do not notably interfere with cellular methylation reactions. our observation warrants a more detailed analysis of the mode of action of 3-deaza-adenosine and analogs, also to evaluate their potential for use in antiviral therapy to treat chikv infections. overall, no large differences were observed between the ic 50 values calculated for ita07-ra1, ls3 and ls3-gfp, indicating that the synthetic viruses are suitable for use in antiviral screens. for most compounds, a faster and simpler assay with chikv ls3-gfp reporter virus showed a good dosedependent response that correlated well with results obtained in the cpe-based assay. clinical isolate nl10/152 exhibited slightly slower replication kinetics and appeared to be more sensitive to antiviral compounds than ita07 and the synthetic viruses. differences in the sensitivity to antiviral compounds among clinical isolates is not an uncommon phenomenon. nl10/152 differs at 7 amino acid positions from ls3 and it will be interesting to determine the contribution of these mutations, in particular the r88s substitution in nsp4, to the slower replication kinetics (and higher sensitivity to antivirals). taken together the detailed characterization of the chikv replication cycle at the molecular level demonstrated that our new synthetic consensus-based viruses behave like natural isolates and are suitable tools to study various aspects of the chikv life cycle, which should ultimately provide a basis for the development of antiviral therapy. table s1 comparison of chikv ls3 with the genome sequences of various closely related natural isolates. only differences between ls3 and each of the other strains are summarized. dots indicate that the nucleotide at that position is identical to that at the corresponding position in the sequence of ls3. genomes were aligned with mafft and analyzed in jalview. numbering is based on the sequence of lr2006_opy1 (and is equal to ls3 numbering). the nucleotide at position 10670 (indicated in gray) determines whether the strain has the a226v mutation in the e1 protein. strains with a t at this position have the a226v mutation. differences not included in the comparison are the 35 nt, 5 nt and 23 nucleotides that are missing from the 3'utr of the sequences of drde-07, d570/06 and ita07-ra1, respectively. the missing first 19 nt, missing last 13 nt and the insertion of an a after position 11564 in the sequence of ind-06-ap3 were also not included in this comparison. 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mediated resistance of vesicular stomatitis virus and sendai virus to ribavirin cyclophilin involvement in the replication of hepatitis c virus and other viruses carbocyclic adenosine analogues as s-adenosylhomocysteine hydrolase inhibitors and antiviral agents: recent advances we are grateful to dr. gorben pijlman (wageningen university, the netherlands) for his generous gift of chikv e2 antiserum and to adriaan de wilde and emmely treffers for helpful discussions and sharing their unpublished data. key: cord-253709-frauasts authors: lan, dongming; huang, guangrui; shao, hongwei; zhang, lichun; ma, lixin; chen, shangwu; xu, anlong title: an improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases date: 2011-08-15 journal: analytical biochemistry doi: 10.1016/j.ab.2011.04.034 sha: doc_id: 253709 cord_uid: frauasts abstract proteins fused to the elastin-like polypeptide (elp) tag can be selectively separated from crude cell extract without chromatography. to avoid the interference of the elp tag on properties of the target protein, it is necessary to remove the elp tag from target protein by protease digestion. therefore, an additional chromatographic purification step is required to remove the proteases, and this is timeand labor-consuming. here we demonstrate the utility of the elp-tagged proteases for cleavage of elp fusion proteins, allowing one-step removal of the cleaved elp tag and elp-tagged proteases without chromatography. proteins fused to the elastin-like polypeptide (elp) tag can be selectively separated from crude cell extract without chromatography. to avoid the interference of the elp tag on properties of the target protein, it is necessary to remove the elp tag from target protein by protease digestion. therefore, an additional chromatographic purification step is required to remove the proteases, and this is time-and laborconsuming. here we demonstrate the utility of the elp-tagged proteases for cleavage of elp fusion proteins, allowing one-step removal of the cleaved elp tag and elp-tagged proteases without chromatography. ó 2011 elsevier inc. all rights reserved. it has been an urgent desire to find a simple reliable, and economic method for protein purification during the postgenomic era. for this purpose, a lot of protein purification methods have been explored. among them, meyer and chilkoti reported a nonchromatographic method for purification of recombinant proteins using an elastin-like polypeptide (elp) 2 tag [1] . the elp tag is an artificial biopolymer consisting of repeating pentapeptides, val-pro-gly-xaa-gly (vpgxg), where xaa can be any amino acid except pro [2] . the elp can undergo a reversible phase transition from soluble forms into aggregates by increasing the salt concentration or heating. the elp aggregates can be recovered by centrifugation and resolubilized in buffer at a temperature below the transition temperature. importantly, target proteins fused to the elp tag still retain the phase transition behavior [1] . this characteristic transition allows the recombinant elp fusion protein to be isolated from the cell lysate by repeated steps of aggregation, centrifugation, and resolubilization without chromatography. this nonchromatographic method for protein purification was termed as inverse transition cycling (itc) [1, 3] . recombinant elp fusion proteins at different expression levels can be efficiently recovered by the itc method [4, 5] . in addition to simple separation of recombinant proteins, ultra-high-level recombinant proteins can be produced by fusion with the elp tag [6] . due to the attractive advantages of the elp tag, various proteins of interest have been expressed and purified using the itc method [7] [8] [9] [10] [11] . for crystal structural and biochemical characterization, however, it is necessary to remove the elp tag from the target proteins. cleavage of fusion proteins using proteases is feasible, but an additional purification step is needed to remove the proteases, and this is time-consuming. to solve this problem, self-cleaving elp tags have been devised for purification of the tag-free recombinant proteins without using proteases [12, 13] . here we report the application of elp-tagged proteases for cleavage of the elp fusion proteins to improve the elp-tag-based nonchromatographic purification method. the principle using elp-tagged proteases for the separation of tag-free target proteins is shown in fig. 1 . the human rhinovirus 3c protease and small ubiquitin-related modifier (sumo) protease from yeast were used to demonstrate the proof-of-principle for this method. the human rhinovirus 3c protease is a specific protease that recognizes a peptide levlfq/gp and cleaves between the gln and gly residues [14] . sumo protease can recognize the tertiary structure of sumo moiety instead of a peptide sequence and cleave in the diglycine motif at the junction between sumo and the target proteins [15, 16] . both proteases have been expressed and purified from escherichia coli [16, 17] . an elp gene encoding 90 repeating vpgxg was synthesized as described previously [1] and inserted into the plasmid pet23a initially, the elp-3c protease was expressed and purified. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) analysis of the purified products for each step is shown in fig. 2a , and the page gel was stained with coomassie brilliant blue g-250 [18] . elp-3c protease was produced at a high level with an apparent molecular weight of approximately 58 kda. to aggregate elp-3c protease, solid nacl was added into the cleared lysate to a final concentration of 2 m at room temperature to trigger the phase transition of elp. the elp-3c protease aggregation was precipitated by centrifugation at 14,000g for 10 min at room temperature. no obvious trace of elp-3c protease was found in the supernatants ( fig. 2a, lane 3) , indicating that elp-3c protease was fully recovered. the precipitated elp-3c protease was then dissolved in cooled 50 mm tris-hcl (ph 8.0) buffer at 4°c. the insoluble materials were removed by additional centrifugation at 4°c and 14,000g for 10 min, and the purified elp-3c protease was recovered from the supernatant. the final yield of elp-3c protease was approximately 22.5 mg/l after two rounds of itc. to test the activity of elp-3c protease and the feasibility using elp-tagged protease to purify the tag-free recombinant proteins, the elp-gfp fusion protein was selected as a model protein that contains a 3c protease recognition site between the elp tag and gfp. the elp-gfp was purified by itc as described above. it was shown that highly purified elp-gfp was recovered after two rounds of itc (fig. 2b, lane 4) . to release the elp tag from the gfp, the elp-gfp was digested using elp-3c protease at a protease-to-substrate ratio of 1:100 (w/w) in 50 mm tris-hcl (ph 8.0) buffer (150 mm nacl, 1 mm ethylenediaminetetraacetic acid [edta] , and 1 mm dithiothreitol [dtt]) at 5°c for 16 h. sds-page analysis showed that the elp-gfp was efficiently digested by elp-3c protease. after enzymatic cleavage, nacl was added into the digestion mixtures to trigger the phase transition of elp, resulting in aggregation of the cleaved elp tag and elp-3c protease. after centrifugation at 14,000g at room temperature for 10 min, the supernatants and pellet were analyzed by sds-page. only the gfp band was found in the supernatants (fig. 2b, lane 7) . the cleaved elp tag, elp-3c protease, and undigested elp-gfp were separated from the gfp and kept in the pellet (fig. 2b, lane 6) . the purified gfp that we obtained was approximately 25.6 mg/l. the fluorescence of purified gfp was detected by a fluorescence microscopy. the same method was successfully used to purify another tested protein, maltose binding protein (mbp) (fig. 2c) . because it is efficient to purify the tag-free recombinant protein using elp-3c protease, we purified several other proteins with various sizes, including glutathione s-transferase, thioredoxin, and severe acute respiratory syndrome-associated coronavirus (sars-cov) nucleocapsid (data not shown). the use of elp-3c protease for enzymatic cleavage of fusion protein has several advantages. first, elp-3c protease is easy to produce in ordinary laboratories because the procedure for preparation of elp-3c protease is simple, and this can save the cost of buying commercial proteases. second, elp-tagged protease as well as the cleaved elp tag can be removed from the target proteins easily by centrifugation after the triggering phase transition of the elp, so it saves time, labor, and cost. third, higher yield of the recombinant proteins can be achieved compared with that of the utility of the self-cleaving elp tag given that there is no risk of premature cleavage of the fusion protein containing a protease recognition site in vivo. besides 3c protease, the principle can also be applied to other proteases for various purposes. unlike 3c protease, sumo protease from yeast recognizes the tertiary structure of the sumo tag and releases the target protein with no additional amino acids at the n terminus, which is considered as a robust protease [15, 16] . therefore, elp-sumo protease has also been expressed and isolated by itc from e. coli (fig. 2d ). the yield of purified elp-sumo protease was approximately 11.8 mg/l. elp-sumo protease could efficiently digest the elp-sumo-gfp to release the gfp from the elp-sumo tag (fig. 2e, lane 5) . but a band of elp-sumo was observed in addition to the gfp band in the page gel after itc (fig. 2e, lane 7) , suggesting that the elp-sumo was not completely precipitated. to solve this problem, the free elp protein was added to the digestions to facilitate the aggregation of elp-sumo. after triggering the transition of elp, elp-sumo and free elp were coagfig.1 . schematic map of illustrating purification of recombinant proteins using elp-tagged proteases. the elp fusion protein was separated by itc from the lysate and digested by elp-tagged protease to release the elp tag from the target protein. after enzymatic cleavage reaction, another round of itc was carried out to precipitate the cleaved elp tag and elp-tagged protease. thus, the tag-free recombinant protein with high purity was recovered from cell lysate without chromatography. gregated and completely separated from gfp by centrifugation (data not shown). the final yield of gfp was approximately 17.1 mg/l. elp-tagged sumo protease can be applied to isolate target proteins with a native n terminus. in summary, we have developed a greatly improved nonchromatographic method for isolation of recombinant proteins using elp-tagged protease. this method provides an opportunity for efficient and cost-effective purification of a broad range of tag-free proteins for high-throughput analysis of proteins. purification of recombinant proteins by fusion with thermally-responsive polypeptides free energy transduction in polypeptides and proteins based on inverse temperature transitions protein purification by fusion with an environmentally responsive elastin-like polypeptide: effect of polypeptide length on the purification of thioredoxin purification of recombinant proteins from escherichia coli at low expression levels by inverse transition cycling simultaneous phase transition of elp tagged molecules and free elp: an efficient and reversible capture system ultra-high expression of a thermally responsive recombinant fusion protein in e. coli expression and purification of recombinant proteins from escherichia coli: comparison of an elastin-like polypeptide fusion with an oligohistidine fusion thermally triggered purification and immobilization of elastin-oph fusions functional expression of a biologically active fragment of soluble gp130 as an elp-fusion protein in transgenic plants: purification via inverse transition cycling heterologous expression and optimized one-step separation of levansucrase via elastin-like polypeptides tagging system effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity? simple bioseparations using self-cleaving elastin-like polypeptide tags selfcleavable stimulus responsive tags for protein purification without chromatography substrate requirements of human rhinovirus 3c protease for peptide cleavage in vitro ulp1-sumo crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast sumo fusions and sumo-specific protease for efficient expression and purification of proteins efficient expression and purification of a protease from the common cold virus, human rhinovirus type 14 blue silver: a very sensitive colloidal coomassie g-250 staining for proteome analysis protein samples are separated by 12% sds-page and analyzed by coomassie brilliant blue g-250 (20% methanol, 10% phosphoric acid, 10% ammonium sulfate, and 0.12% g-250) staining. (a) expression and purification of elp-3c protease molecular weight marker; lane 1: crude extract of cell expressing elp-3c protease; lane 2: cleared lysate; lane 3: supernatant after precipitation of the elp-3c protease; lane 4: purified elp-3c protease. (b) expression and purification of elp-gfp and gfp. lane m: marker; lane 1: crude extract of cell expressing elp-gfp; lane 2: cleared lysate; lane 3: supernatant after precipitation of elp-gfp; lane 4: purified elp-gfp; lane 5: cleavage product of elp-gfp; lane 6: precipitation of cleaved elp, elp-3c protease, and undigested elp-gfp supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2011.04.034. key: cord-316814-9fv9xrln authors: li, hong-ye; chye, mee-len title: use of gfp to investigate expression of plant-derived vaccines date: 2009 journal: viral applications of green fluorescent protein doi: 10.1007/978-1-59745-559-6_19 sha: doc_id: 316814 cord_uid: 9fv9xrln plants are low-cost bioreactors for the production of various biopharmaceuticals including oral vaccines. plant-derived oral vaccines are potentially useful in combating viral infections involving mucosal immunity. transgenic plants have been generated to successfully produce mucosal vaccines against cholera, hepatitis b, foot-and-mouth disease, and norwalk virus. as a first step toward the generation of oral vaccines against the severe acute respiratory syndrome coronavirus (sars-cov), we have expressed a recombinant s1 protein of the sars-cov in transformed tobacco. since plant transformation and regeneration of stable transformants require considerable time, we initially used a green fluorescent protein (gfp) to tag the antigen in transient expression. gfp was fused to the carboxy-terminus of s1 for expression of s1-gfp to show expression of recombinant s1 by agroinfiltration of tobacco leaves. the gfp tag enables a relatively quick confirmation of antigen expression in plant cells by fluorescent microscopy. such analysis using gfp that precedes stable plant transformation will enable the rapid screening of multiple constructs to attain optimal recombinant protein expression. furthermore, this approach determines the subcellular localization of the recombinant protein in plant cells, providing information on optimal subcellular targeting for production in plant bioreactors. transgenic plants have already been generated by nuclear transformation to express mucosal vaccines against cholera, norwalk virus, hepatitis b, foot-and-mouth disease, and severe acute respiratory syndrome, sars (1, 2) . these viral antigens generated in transgenic plants are effective in inducing mucosal and serum immune responses in animals. in the case of sars, nuclear-transformed tobacco and tomato expressing the sars-cov viral antigens induced immunogenic responses in mice; sars-cov-specific iga increased in mice following oral ingestion of s1-expressing tomato fruits (2) . such edible vaccines deliverable as fruits (e.g., tomato), leaves (e.g., lettuce), tubers (e.g., potato), seeds (e.g., rice or corn), stems, or roots, would obliterate purification procedures required for microbial-derived recombinant vaccines. further added advantages include the easy storage, transport, and administration by direct ingestion of these oral vaccines. represented in the form of plant organs (fruits, leaves, tubers, seeds, stems, or roots), these vaccines can be easily grown and distributed, omitting the need for refrigeration in delivery or the requirement for injection by trained health personnel, thereby reducing the costs of immunization programs in developing countries. green fluorescent protein (gfp) has been routinely used in laboratories worldwide for tagging proteins in both plant and animal cells. it has been proven easy and convenient to use in transient expression and stable transformation of plant cells. further, it is pertinent to note that in its application for tagging of viral antigens in plants, gfp can be processed by the ubiquitinproteasome pathway (3) since ubiquitinated antigens are a prerequisite to processing into antigenic peptides for presentation to the major histocompatibility complex (4) . we describe here several techniques that utilize agrobacterium tumefaciens for the introduction of foreign dna into plant cells. in nature, agrobacterium tumefaciens is a gram-negative soil bacterium that transfers and incorporates its transfer-dna (t-dna) into the plant nuclear genome. this results in an expression of genes on the t-dna that will cause crown gall in the infected plant (5) . it has been demonstrated in agrobacteriummediated transformation that following co-cultivation of tobacco by-2 cells with an agrobacterium harboring a gfp fusion construct, the rate of fusion protein synthesis markedly increased between 24 and 36 h, after which rapid decrease occurred (6) . agroinfiltration is a more recent technique that can be applied to investigate transient expression in plant cells by which an agrobacterium liquid culture is infiltrated into intact plant leaves (7) . the feasibility of rapid analyses of foreign protein expression in transiently expressed leaves following agroinfiltration presents a major advantage in its use. the transiently expressed foreign protein is probably derived from transiently synthesized t-dna containing the construct that had been mobilized from agrobacterium cells to the tobacco nuclei but has yet to be integrated into the tobacco nuclear genome (8) . such analysis using agrobacterium-mediated transient expression with gfp-tagged constructs will enable the rapid screening of multiple constructs so that the construct that gives optimal recombinant protein expression can be quickly identified. upon the identification of such a high-expressing construct, it can then be selected for use in stable plant transformation to generate transgenic plants, a more time-consuming process. this approach by using gfp as an autoflourescent tag can further determine the subcellular localization of recombinant-tagged protein expressed in plant cells, thus providing information for optimal subcellular targeting of such recombinant proteins produced in plant bioreactors. 1. plasmid pcrii-s1, a pcrii (invitrogen) derivative containing the sars-cov s1 cdna (encoding amino acids 1-658 of the s protein) amplified by the polymerase chain reaction 2. agrobacterium tumefaciens strain lba4404 (10) . 3. agrobacterium culture medium: yeast extract peptone (yep) medium (ph 7.0) containing 10 g/l yeast extract, 10 g/l bacto-peptone, and 5 g/l nacl supplemented with 10 mm mgso 4 , 100 μ g/ml of streptomycin and, in the case of a derivative harboring pgd, plus 50 μ g/ml of kanamycin. 1. fluorescence microscopy or confocal laser-scanning microscope equipped with helium/neon lasers. 2. image processing software lsm 510 (zeiss) and photoshop 6.0 (adobe). 1. liquid nitrogen. 3. protein homogenizing buffer: 50 mm tris-hcl , ph 7.5, 2 mm edta, and freshly add 28 mm β -mercaptoethanol and 2 mm phenylmethanesulfonylfluoride (pmsf, sigma). 1. sodium dodecyl sulfate (sds)-polyacrylamide gel electrophoresis (page). 7. sample loading buffer (5×): 250 mm tris-hcl, 10% sds, 0.5% bromophenol blue, 50% glycerol, ph 6.8. 8. mini-protean ii slab gel unit (bio-rad) or comparable apparatus. 9. rainbow tm colored protein molecular weight marker (amersham). 10. hybond-c (amersham) supported nitrocellulose membranes. the gfp tag enables relatively quick confirmation of antigen expression in plant cells by fluorescent microscopy. by simple infiltration of agrobacterium cells carrying appropriate gene constructs into tobacco plants leaves, transient expression assays can be performed within 3 days without using expensive instruments or complicated procedures. two days after agroinfiltration, expression and subcellular localization of the gfp fusion proteins in tobacco leaves can be determined by simple observation under fluorescence or confocal laser scanning microscopy. in addition, rna and protein can be extracted from the agroinfiltrated leaves for reverse transcription-polymerase chain reaction (rt-pcr), northern blot analysis, and western blot analysis. the sars-cov s1 cdna (encoding amino acids 1-658 of the s protein) was amplified by pcr using template derived from total rna of vero cells infected with the sars-cov (11) . plasmid pcrii-s1 containing the sars-cov s1 cdna was used as a mutagenesis template for generating derivatives with optimized codon usage for heterogeneous gene expression in plants. 3. transform supercompetent escherichia coli dh5 α bacteria using 1 μ l of the dpni-treated dna from each reaction. 4. select clones for extraction of plasmid dna to verify the change in dna sequence. 5. the oligonucleotides we used in site-directed mutagenesis are shown in table 1 . 6. a total of 13 nucleotide changes were incorporated for an "optimized" s1. use the optimized s1 for cloning into nuclear transformation vector. 7. generate plasmid pcv12 (fig. 1 ) or similar nuclear transformation vector for expression of a fusion protein consisting of the sars-cov s1 and gfp. this was generated by cloning a 0.8-kb xhoi-spei gfp fragment from plasmid pgfp2 in the sali-xbai sites of pcrii-s1 to create an s1-gfp fusion (12) . subsequently, table 1 oligonucleotides used for site-directed mutagenesis of s1 for codon optimization (reproduced from ref. affected residue sequence of primer nucleotides in italics are mutated; the altered codons are underlined fig. 1 . plasmid pcv12 that was used for expression of s1 tagged to gfp in plant cells. the plasmid pcv12 is a plant nuclear transformation vector used for the expression of a protein fusion consisting of the sars-cov s1 protein fused translationally with gfp. rb and lb represent right and left borders of t (transfer)-dna for random insertion into the plant nuclear genome; p35s, cauliflower mosaic virus 35s promoter; tnos, nos terminator . t nos lb rb gfp a 2.8-kb bamhi-apai fragment containing the s1 cdna (encoding amino acids 1-643) and gfp cdna was cloned into the bglii-apai sites of a plant binary vector, pgd (12) . production of tobacco leaves transiently expressing a protein fusion consisting of the sars-cov s1 protein fused with the gfp was carried out using agrobacterium-mediated transformation with plasmid pcv12. 1. grow agrobacterium lba4404/pcv12 for infiltration overnight with shaking (200 rpm) at 28 °c in yep medium supplemented with 10 mm mgso 4 , 100 μ g/ml of streptomycin and, in the case of pgd, plus 50 μ g/ml of kanamycin. 2. subsequently, initiate a new culture by inoculating fresh medium with the overnight culture at 1:10 dilution. 5. make some tiny wounds using a needle in the region where infiltration will apply. using a syringe lacking a needle, press the syringe tip on the wounds against the lower side of a tobacco leaf, meanwhile applying counterpressure to the upper leaf, so that the bacterial suspension can be efficiently introduced through the stomata to the leaf lamina. it generally takes a few minutes to do one infiltration. there is no need to wash off the bacterial solution outside the leaves following the procedure. freshly expanded young leaves are preferentially used for better expression ( see notes 1 -4 ). 1. two days after agroinfiltration, the expression and subcellular localization of transiently-expressed gfp fusion proteins can be determined. representative tobacco leaf epidermal cells are selected and observed by fluorescence microscopy or confocal microscopy. agrobacterium lba4404 harboring plasmid pcv12 (expressing the s1:gfp-fusion protein) and those agroinfiltrated with lba4404 harboring pgdg (control expressing gfp alone). 3. detect fluorescence and collect digital images using a zeiss lsm 510 inverted confocal laser-scanning microscope equipped with helium/neon lasers or comparable microscope. gfp fluorescence excites at 488 nm, is filtered through a primary dichroic (uv/488/543), a secondary dichroic of 545 nm, and subsequently through bp505-530 nm emission filters to the photomultiplier tube detector. 4. the images can be processed using the lsm 510 software (zeiss) and photoshop 6.0 (adobe) or comparable software. images of transient expression of s1:gfp antigen in the cytosol of agroinfiltrated tobacco leaves are shown in fig. 2 . expression of the construct with gfp alone was not confined to the cytosol. nuclear expression is seen because of the accumulation of passive diffusion of gfp into the nucleus (13) . the s1:gfp fusion was localized to the cytosol, and also to the periphery of the nucleus as well, implicating that s1:gfp was secreted through the endoplasmic reticulum network. depending on total experimental conditions, the expression of the target gene at the infiltrated area can vary. under optimal conditions, almost all cells at the infiltrated area show expression, like in fig. 2 . generally, it should not be difficult to get expression in more than 50% of plant cells at the infiltrated area. to confirm the transient expression of s1:gfp in tobacco leaves following agroinfiltration, western blot analysis was carried out using antibodies against gfp. total proteins were extracted from tobacco leaves infiltrated with plasmid pgdg expressing gfp alone or plasmid pcv12 expressing the s1:gfp fusion. 1. harvest plant tissues and immediately freeze in liquid nitrogen. grind using a mortar and pestle in protein homogenizing buffer. use about 10 ml of ice-cold homogenizing buffer per 1 g of tissue. 2. boil ground tissue at 100 °c for 10 min, centrifuge at 14,000 rpm for 5 min, and transfer the supernatant into a new eppendorf and store at -20 °c. determine the protein content. 3. denature the protein samples in 1× sds sample loading buffer by heating at 95 °c for 5 min before loading onto the gel. separate protein samples (20 μ g) by sds-page using a 10% gel using a mini-protean ii slab gel unit or comparable apparatus. 4. use rainbow tm colored protein molecular weight markers in gels destined for transfer to hybond-c membrane. 5. electrophoretically transfer proteins to hybond-c membrane from sds-page gel using a trans-blot cell at 50 v for 1.5 h in cold transfer buffer. a piece of hybond-c membrane, cut to the size of the gel and wetted with distilled water, was placed on top of the gel without inclusion of any air bubbles. the gel and the membrane were then sandwiched between two pieces of whatman no. 3 paper that were also cut to the size of the gel and were presoaked in transfer buffer ( see note 5 ). 6. for detection using anti-gfp antibodies in western blot analysis, incubate the blot for 1 h at room temperature (rt) in blocking solution with gently shaking. 7. after washing the blots twice for 10 min in ttbs, then incubated for 2 h at room temperature in monoclonal anti-gfp antibodies diluted 1:5,000 in ttbs with gentle agitation. 8. wash the blot twice for 10 min with ttbs, followed by incubation in anti-mouse igg alkaline phosphatase diluted 1:30,000 in ttbs for 1 h at room temperature with gentle shaking. 9. wash in ttbs four times for 10 min, and stain the blot in freshly prepared color development solution at room temperature until the reacting protein bands are visible. 10. stop the reaction by washing the membrane twice for 5 min in water. results of western blot analysis using antibodies against gfp of total plant proteins extracted from tobacco leaves infiltrated with plasmid pcv12 (expressing the s1:gfp fusion) are shown in fig. 2e (lane 2 shows an expected band of calculated size 99.1 kda, demonstrating that the s1:gfp fusion protein was successfully expressed). 1. the technique is suitable for leaves or petals. agrobacterium has to be introduced into the leaves through the stomata or via wounding. in transient gene expression, the levels in expression may be age dependent and may differ in leaf tissues at varying stages of development. generally, the highest level of transient expression is observed in tissues that contain recently expanded leaf cells. 2. agroinfiltration can also be used in conjunction with vacuum infiltration on either detached or attached plant leaves. with the use of detached leaves, the leaves should be best incubated on wet filter paper rather than on solidified ms medium following agroinfiltration since incubation on ms often gave lower expression levels (8) . 3. the ph of the agrobacterium resuspension can be within a range of ph 5.0-5.8. we usually use ph 5.5. the concentration of agrobacterium cells is important for infiltration. the optimal concentration of the agrobacterium suspension is od 600 = 0.4-0.6. the agrobacterium culture is grown to log phase and then resuspended to od 600 = 0.4-0.6 for better expression. if the bacterial cells are out of log phase, the efficiency could be much decreased. 4. acetosyringone is usually added in the agroinfiltration medium to increase the efficiency. it has been demonstrated in chinese cabbage that agroinfiltration can be improved by adding hydrogen peroxide (14) . since the plasmid vector for agroinfiltration does not necessarily carry an antibiotic-resistance selectable marker gene, a smaller vector can be considered for use to enhance t-dna transfer in plant cells by agroinfiltration. 5. make sure there are no air bubbles between nitrocellulose membrane and gel or else certain regions will not be transferred to the membrane. alternatively, the blot can be assembled under transfer buffer to avoid trapping bubbles. plant cell factories and mucosal vaccines severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine short-lived green fluorescent proteins for quantifying ubiquitin "proteasome-dependent proteolysis in living cells generation of major histocompatibility complex class i antigens: functional interplay between proteasomes and tppii agrobacterium -mediated plant transformation: the biology behind the "gene-jockeying transient gene expression in plant cells mediated by agrobacterium tumefaciens : application for the analysis of virulence loci silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigation of sequence requirements for gene silencing an agrobacteriummediated transient gene expression for intact leaves pgd vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves a binary plant vector strategy based on separation of vir and t-region of the a. tumefaciens ti plasmid accumulation of recombinant sars-cov spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines nucleocytoplasmic transport transient expression in chinese cabbage by hydrogen peroxide-aided agroinfiltration use of gfp to investigate expression of plant-derived vaccines key: cord-000933-nn9gj0z1 authors: krzyzaniak, magdalena anna; zumstein, michael thomas; gerez, juan atilio; picotti, paola; helenius, ari title: host cell entry of respiratory syncytial virus involves macropinocytosis followed by proteolytic activation of the f protein date: 2013-04-11 journal: plos pathog doi: 10.1371/journal.ppat.1003309 sha: doc_id: 933 cord_uid: nn9gj0z1 respiratory syncytial virus (rsv) is a highly pathogenic member of the paramyxoviridae that causes severe respiratory tract infections. reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. to study the entry process in human tissue culture cells (hela, a549), we used fluorescence microscopy and developed quantitative, facs-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. a variety of perturbants were employed to characterize the cellular processes involved. we found that immediately after binding to cells rsv activated a signaling cascade involving the egf receptor, cdc42, pak1, and downstream effectors. this led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. if these effects were inhibited using compounds targeting na(+)/h(+) exchangers, myosin ii, pak1, and other factors, no infection was observed. the rsv was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. rather than fusing with the plasma membrane, the viruses thus entered rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. rab5 was required for infection. to find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, f, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. this cleavage by a furin-like protease removed a small peptide from the f1 subunits, and made the virus infectious. human respiratory syncytial virus (rsv) belongs to the paramyxoviridae, a family of enveloped viruses with a negativestranded rna genome. it is a ubiquitous human pathogen that causes severe respiratory tract infections affecting mainly children and the elderly worldwide. despite ongoing efforts, there are no available vaccines or treatments except passive immunoprophylaxis [1] . a better understanding of virus/host cell interactions is critical for the development of new therapeutic strategies. rsv particles produced in tissue culture are heterogeneous in size and shape. some are rounded with a diameter of 100-300 nm, others filamentous with a length up to 10 mm [2] . the nucleocapsid is helical and contains in addition to the rna the nucleoprotein n, the viral polymerase l, its cofactor-phosphoprotein p, and the transcription processivity factor m2-1. the matrix protein m is believed to form a layer on the inside of the viral envelope [3] . the lipid envelope is derived from the plasma membrane (pm) of the infected host cell, and contains three viral glycoproteins; the major attachment protein g, the fusion protein f, and a small hydrophobic protein sh. cell attachment of rsv is mediated by g and f, which bind to cellular glycosaminoglycans [4] . that g and sh are not essential for replication in cell culture [5] , indicates that the f protein can support both attachment and fusion. in vivo, rsv targets airway epithelial cells, and in the human mucociliary epithelium it infects ciliated cells from the apical surface [6] . previous studies on rsv entry employing a lipid-dequenching assay suggested that rsv, as most other paramyxoviruses, fuses its membrane directly with the pm of target cells [7] . that rsv entry is ph-independent is consistent with this view [8] . on the other hand, kolokoltsov and coworkers concluded, that rsv uses clathrin-mediated endocytosis (cme) to infect hela cells because a targeted sirna screen revealed clathrin light chain, eps-15, and ap-2 as important cellular factors in rsv infection [9] . in a recent publication, san-juan-vergara et al. [10] argued that in primary nheb cells rsv entry is a two-step process; rsv docks to cholesterol-rich pm domains facilitating hemifusion between the viral envelope and the pm followed by endocytosis and complete fusion in endosomes. to determine the pathway of rsv entry into hela and a549 cells, we developed quantitative fluorescence-activated cell sorting (facs) assays and complemented them with confocal microscopy to monitor cell binding of rsv, endocytosis, fusion, and infection. we tested the effects of inhibitors and other perturbants. our results indicated that rsv infected the cells by an endocytosismediated mechanism that fulfilled the criteria of macropinocytosis. after uptake into macropinosomes, a second proteolytic cleavage in f served as a 'cue' for penetration by membrane fusion. in our studies, we used a recombinant rsv strain called rgrsv that expresses gfp [11] enabling us to quantify infection by facs. the virus was grown in hep-2 cells, and to minimize exposure to broken cells, harvested from the cell supernatant before cytopathic effects were observed. the quality of virus purified by gradient centrifugation was confirmed by sds-page (fig. 1a) . when the purified particles were examined by indirect immunofluorescence (iif) using antibodies to the f and the n proteins, we found three different particle populations. half of the particles represented intact virions because in addition to f (green) they contained n (red) (fig. 1b) . of these, 30% also stained with phalloidin (blue, pseudocolored white) indicating the presence of actin filaments as previously reported (fig. 1b arrowheads) [12] . the remaining particles constituted capsid-free envelopes (vlps). they stained for f but not for n. since we did not detect free capsids that would stain only for n or p (data not shown), we used the presence of the capsid antigens to distinguish between intact rsvs and vlps. when purified virus preparations were incubated with hela cells at 4uc, immunoblotting after sds-page showed that more than half of the input n and p associated with the cells indicating that rsv binding in the cold was efficient (fig. 1c) . to measure infection, rsv was added to hela cells for 1 h and infection was continued for additional 3-8 h before measuring gfp expression by facs (fig. 1d ). the fraction of cells respiratory syncytial virus (rsv) is a highly pathogenic paramyxovirus. we developed assays for rsv endocytosis, intracellular trafficking, membrane fusion, and infection. the results showed that rsv was rapidly and efficiently internalized, and that acid-independent membrane fusion occurred intracellularly after endocytosis. cell biological studies demonstrated that endocytosis was macropinocytic, and that it was required for infection. the process involved activation of the egf receptor and its downstream effectors including cdc42, pak1, and myosin ii. rsv induced transient actin rearrangements accompanied by plasma membrane blebbing, elevated fluid uptake, and internalization of intact rsv particles into large macropinosomes. expression of a dominant negative rab5 mutant but not rab7 decreased infection indicating that rsv penetration is intracellular, and takes place in rab5 positive macropinosomes before fusion with endolysosomal compartments. the reason why rsv, unlike most paramyxoviruses, depended on endocytic entry was found to be the need for activation of the f protein by a second proteolytic cleavage. it occurred after endocytosis, and involved most likely a furin-like, vacuolar enzyme. . gradient purifier rsv (,10 5 particles/50 ml) was resolved on the sds-page, followed by the blue silver gel staining. (b) . after binding to a glass slide, purified rsv was stained with anti-f-af488 (green), anti-n-af594 (red), and phalloidin-af647 (pseudocolored white). the particles (n = 457) were imaged with a confocal microscope and analyzed for colocalization by imaris. arrowheads show particles with all three stains. (c). equal volumes of the virus input (moi 10), the cell bound virus lysates, and the unbound virus (sup) were resolved by a sds-page. western blots were developed with anti-p or anti-n rsv specific antibody. (left) representative western blots. (right) densitometry quantification of the p-and n-protein bands intensities for the virus input and cell bound virus samples. (d). hela cells were infected with rsv moi (3-10) for 1 h at 37uc. virus inoculum was replaced with medium and the infection was carried for indicated times. the percentage of infected cells expressing gfp was measured by facs. doi:10.1371/journal.ppat.1003309.g001 expressing gfp increased with time and with increasing multiplicity of infection (moi). in cells infected at moi of 10, gfp expression was detected as early as 3 h post-infection (hpi) (28% gfp positive cells) and after 5 hpi 80% of the cells were infected. at a moi of 3, gfp expression was delayed by about 3 h. to follow the fate of the cell-bound particles in the cold after warming to 37uc, iif with anti-f and anti-n antibodies was used. actin filaments were labeled with phalloidin to visualize cell boundaries. confocal z-stack image series in the orthogonal view revealed that after 30 min virus particles containing n and f were present not only on the cell surface but also deep inside the cytoplasm ( fig. 2a) . this indicated that viral particles and vlps were endocytosed. after binding in the cold, cell-associated viruses and vlps can be removed from the cell surface by brief incubation with trypsin in the cold that does not affect cell attachment (fig. 2b , 0 min) [10] . we found that when cells after virus binding in the cold were incubated at 37uc, an increasing fraction of the cell-associated particles became trypsin resistant (fig. 2b , 15-60 min). quantitation using spot detection software imaris showed that after 60 min, 77% vlp and 70% rsv-containing spots were, in fact, resistant to trypsin (fig. 2c ). that the total number of rsv-and vlp-containing spots decreased over time was probably caused by the accumulation of multiple particles in common endocytic vacuoles that represented single spots. of the anti-f stained spots, 47% stained for n indicating that they were intact viruses. to confirm that rsv was endocytosed in an intact form, it was important to determine whether the endocytosed particles also contained the viral lipid envelope. purified rsv was therefore labeled with a lipophilic fluorescent dye, dioc, which partitions into the viral membrane. it is fixable with formaldehyde, and can be quenched by the membrane-impermeable dye, trypan blue (tb) [13] . after labeling, 80% of re-purified particles contained detectable dioc (data not shown). when added to cells and incubated at 37uc, the rsv-dioc particles were visible as discrete fluorescent spots, and of these some were quenched when tb was added (fig. 3a) . facs analysis showed that, 50% of the fluorescence was resistant to tb after 30 min, and full resistance was reached in about 180 min ( fig. 3b top) , indicating that internalization of rsv and vlps was rapid and complete. importantly, when the intracellular accumulation of f and n proteins was measured in parallel ( fig. 3b bottom), internalization of both antigens and dioc (fig. 3b top) followed similar kinetics. to monitor fusion of rsv with cellular membranes, we used a method developed by sakai and coworkers [14] . in this case, rsv was labeled with two fluorescent lipids, r18 (red) and dioc (green). concentrations were used at which the r18 quenches the fluorescence signal emitted by the dioc. therefore, when allowed to bind to cells and viewed live by confocal microscopy, the labeled viruses were initially all red (fig. 3c, 0 min) . however, after about 60 min at 37uc, yellow and green intracellular spots became apparent increasing in numbers over time, because after fusion, the two lipids were diluted out and the green fluorescence of dioc was no longer quenched by r18 (fig. 3c) . some of the spots showed a ring-like fluorescence indicating that the dioc was localized in the limiting membrane of intracellular vacuoles. quantitative facs analysis showed that the dequenching of dioc became detectable already after 30 min at 37uc (fig. 3d ). it reached a half maximal level at 90 min, and plateaued after 240 min. treatment of cells with tb during facs analysis revealed that more than 90% of the fluorescent dioc failed to be quenched by this membrane impermeable agent confirming that the dioc was localized in intracellular organelles. from the time course, it was apparent that the fusion events occurred on the average 50 min after endocytosis. our interpretation of these results was that the virus particles and vlps that bound to the cell surface were endocytosed. endocytosis was rapid and efficient, and the internalized viruses accumulated in endocytic vacuoles. after a lag period, the viral envelopes underwent fusion with vacuolar membranes. to bring infection into the picture, cells with virus bound in cold were transferred to 37uc. at indicated times, they were placed on ice, incubated with trypsin to strip away surfaceattached rsv, re-plated and incubated for 10 additional hours to allow infection to proceed and gfp to be expressed. facs analysis demonstrated that in cultures that had been incubated at 37uc before trypsinization, the fraction of gfp-expressing cells increased with time. maximum infection was reached within 180 min, and the half time was around 30 min (fig. 3e ). that the time course coincided perfectly with the time course of virus endocytosis (fig. 3b) implied that productive infection depended on endocytosis. if rsv entry and infection depended on endocytosis as indicated by our experiments, we expected perturbants that inhibit endocytosis to block internalization and infection. in the experiments that followed, endocytosis of rsv was quantified by measuring the amount of the incoming n protein that was trypsin resistant using facs analysis 1 h after warming. infection was scored as a percentage of cells expressing gfp 6 hpi. it is important to mention that the dose-dependence (fig. s1 ) and toxicity (data not shown) of each inhibitor was carefully determined. for clarity for most of the inhibitors we will present data at a single concentration where we found a strong effect but low cytotoxicity. since rsv has been reported to enter cells by cme in hela cells [9] , we tested five cme inhibitors: chlorpromazine [15] , pitstop-2 [16] , and three inhibitors of dynamin-2 (dynasore, dyngo-4a and dynole-34-2) [17] . none of them influenced rsv endocytosis, although, internalization of the well-characterized cme cargo protein transferrin (trf) was efficiently inhibited by all (fig. 4a ). with the exception of pitstop-2, which was too toxic in the prolonged infection assay, none of the agents inhibited rsv infection (fig. 4b) . infection by semliki forest virus (sfv), a virus known to depend on cme, was efficiently blocked by all (fig. 4b ) [18] . rsv infection has been reported to be insensitive to an increase in endosomal ph [9] . this was confirmed by the lack of influence of bafilomycin a, ammonium chloride, and monensin on rsv infection (fig. 4c ). as expected all three agents blocked infection by sfv, which needs low endosomal ph to trigger fusion [19] . the small reduction in rsv infection observed for ammonium chloride and monensin may reflect the importance of a balanced vacuolar environment for productive rsv infection. taken together, the results indicated that rsv endocytosis and infection did not depend on cme nor did it require acidification. when rsv was bound to hela cells in the cold and the cells warmed to 37uc, rapid and dramatic changes in cell shape and actin distribution were observed (fig. 5a ). the number of actin stress fibers decreased, the cells rounded up, and transient blebs filled with actin formed on the cell surface (fig. 5a, 30 min) . these changes were clearly visualized by live cell imaging (movie s1). the cell morphology and actin distribution returned to normal within 2 hpi. when the ratio of g (globular) and f (fibrous) actin was determined, it was found that 30 min after addition of rsv the ratio of g to f actin was 2:1 compared to 1:2 in control cells . hela cells without (top) or with bound rsv (moi ,0.5) at 4uc (bottom) were transferred for 30 min to 37uc before fixation. cells were processed for confocal microscopy with anti-f-af488 (green), anti-n-af594 (red), and phalloidin-af647 (blue), and zstack image series acquired. the orthogonal views of z-stack projections (pseudocolored white) were generated with imagej. (b). rsv (moi ,1) was bound to hela cells at 4uc, unbound virus was removed and cells transferred to 37uc. at indicated times cells were placed on ice and treated with pbs or trypsin for 3 min (pbs wash vs. trypsin treated). samples were processed for confocal microscopy as in (a). (c). quantification of the image series represented in (b). analysis was performed with imaris to detect spots with anti-f staining only (left) or anti-f and -n staining (right) at different times after warming the cells to 37uc. doi:10.1371/journal.ppat.1003309.g002 disruption of actin filaments with cytochalasin d and latrunculin a as well as filament stabilization by jasplakinolide were found to strongly reduce rsv infection, whereas sfv infection was enhanced (fig. 5c ). rsv endocytosis was also significantly decreased (fig. 5d ). inhibition of cdc42 (pirl1), inhibited rsv infection effectively, while inhibitors of rac1 (nsc23766), rhoa (ct04) and its effector rock (y27632) had only a moderate effect (fig. 5e ). both infection and endocytic uptake were reduced when some of cdc42's downstream effectors were inhibited including pak1 (ipa-3), n-wasp (wiskostatin), and moderately when arp2/3 (ck-869) was targeted (fig. 5e , f). nocodazole and taxol that interfere with microtubules had no effect on rsv or sfv infection (fig. 5d) . these results demonstrated that actin and its regulators played a critical role during rsv endocytosis and infection. f-actin was transiently depolymerized, resulting in the formation of blebs. in addition, cdc42, pak1, and n-wasp were required for rsv internalization and infection. the formation of blebs, the involvement of actin, and the role of cdc42 and pak1 suggested that infectious entry of rsv occurred by macropinocytosis as recently shown for several other viruses [for reviews see [20, 21] ]. one of the characteristic features of macropinocytosis is an elevation in the uptake of extracellular fluid [22] . indeed, when serum-starved hela cells were exposed to rsv, we observed that the internalization of 10 kda dextran-af488, a soluble, fluorescent tracer added to the medium, increased by 50% and 120% at low and high moi, respectively, over mock treated cells (fig. 6a ). the elevation was significantly higher than in serum-stimulated cells. iif showed that majority of endocytosed viruses stained by anti-f and -p antibodies were localized in large, dextran-af488 filled, intracellular vacuoles ( fig. 6b ). macropinosome formation requires na + /h + exchanger (nhe) activity to modulate rho gtpases at the pm [23] . inhibition of nhe by eipa (an amiloride derivative) has become one of the diagnostic criteria for macropinocytosis. eipa inhibited rsv internalization and infection by 90% (fig. 6c ). in addition, pretreatment of cells with eipa blocked the increase in fluid phase uptake induced by rsv (fig. 6d ). taken together, these results demonstrated that rsv induces macropinocytosis and uses it for virus endocytosis and infection. macropinocytosis is usually initiated by activation of receptor tyrosine kinases (rtks) or integrins, followed by the activation of a spectrum of cellular signaling factors [24] . accordingly, we found that rsv infection was significantly decreased by two broad range protein kinase inhibitors; staurosporine (ser/thr kinases) and a multi-target protein tyrosine kinase inhibitor, genistein ( fig. 7c, d) . to test whether rtks were involved, we used a human phospho-rtk array comprising antibodies against 42 different phosphorylated rtks. lysates from cells exposed to rsv for 15 min, and lysates from mock-treated control cells were used as probes. the epidermal growth factor receptor (egfr) was the only rtk for which activation was detected; a five-fold increase in phosphorylation compared to control (fig. 7a ). when the egfr was depleted using sirna, greater than 50% reduction in infection was observed (fig. 7b ). we found, moreover, that egfr inhibitors (cas879127-07-8, iressa) significantly decreased rsv infection (fig. 7c ). inhibition of pi3k (wortmannin, ly294002, pi-103), a downstream effector of egfr, also reduced infection (fig. 7c ). egfr inhibitors had little effect on sfv. that the pi3k inhibitors boosted sfv infection was consistent with a distinct entry mechanism for this virus. in addition, inhibition of pkc (rottlerin, calphostin c) decreased rsv (fig. 7d ). although the effect on sfv was smaller, it suggested a role for pkc in the entry of both viruses. finally, since non-muscle myosin ii is thought to mediate closure of macropinosomes, we tested the effects of a myosin ii inhibitor (blebbistatin), and a myosin light chain kinase inhibitor, (ml-7) . serum starved hela cells were incubated with 20% fcs or purified rsv (moi ,10, 30), at 4uc for 1 h. the inoculum was replaced with medium containing 10 kda dextran-af488 and transferred for 15 min to 37uc before fixation. the mfi of af-488 measured by facs. (b). purified rsv (moi ,10) was bound at 4uc to serum-starved hela cells. the input virus was replaced with medium containing 10 kda dextran-af488 (green) and transferred to 37uc. at indicated times cells were fixed, permeabilized and stained with anti-f-af594 (red) and anti-p-af647 (blue) antibody. images represent a z-stack projection acquired with the same confocal microscope settings. (c). hela cells were pretreated with solvent (mock) or eipa at indicated concentration. (left) cells were infected with rsv (moi ,3) at 37uc for 6 hours before facs analysis of gfp expressing cells. (right) rsv (moi ,3) was bound to the cells at 4uc followed by 1 h of internalization at 37uc. cells were trypsinized, fixed and stained with anti-n-af488 antibody, and the mfi of af-488 measured by facs. (d). serum starved hela cells were pretreated with eipa at indicated concentration and incubated with purified rsv (moi , 30) or no virus control at 4uc. the inoculum was replaced with medium containing 10 kda dextran-af488 and eipa and transferred for 15 min to 37uc. cells were fixed and the mfi of af-488 measured by facs. doi:10.1371/journal.ppat.1003309.g006 [25] . both reduced rsv infection with little effect on sfv (fig. 7e) . all the inhibitors that decreased infection also reduced rsv endocytosis (fig. 7c-e bottom) . depending on the compound, rsv internalization was reduced by 60-90%. none of the inhibitors affected rsv cell binding (fig. s2) . thus, we concluded that infectious rsv cell entry and endocytosis were associated with activation of egfr and its downstream signaling partners including pi3k and pkc. combined with the requirement for myosin ii, these findings were consistent with productive rsv internalization by macropinocytosis. in addition, we performed a series of experiments in a549 cells (fig. s3 ). they revealed changes in actin morphology and polymerization after addition of rsv, and a role of egfr, nhe, cdc42, pak1, and other factors similar to hela cells in rsv infection. that internalization and infection were clearly dependent on the same cellular processes and factors in a549 cells indicated that entry by macropinocytosis was not hela cell specific. intracellular trafficking of macropinosomes is not well characterized, but it has been shown that like endosomes, they acidify and acquire rab5 followed by rab7 before fusing with endolysosomes [26] . wild type (wt) gfp-rab5 and gfp-rab7 as well as various constitutively active (c/a) and dominant negative (d/n) mutants of the rabs were transiently expressed in hela cells. after 15 min post warming, we observed that some of the incoming rsv colocalized with gfp-rab5 wt positive vacuoles (fig. 8a) . colocalization was even more evident in cells expressing the c/a gfp-rab5 mutant q79l that exhibits enlarged rab5-positive vacuoles that fail to undergo further maturation [27] . there was no detectable colocalization with rab7 at this time. after 120 min, some colocalization with gfp-rab5 wt was still observed. in cells expressing the d/n gfp-rab5 s34n, we noted accumulation of f-and p-stained particles inside large vacuoles in the perinuclear space. colocalization of rsv with gfp-rab7 wt was also detected. to determine whether rab5 and rab7 played a role in infection, we infected cells expressing gfp-tagged constructs of rab5, rab7, and their mutants with rsv-a2. to determine the fraction of infected cells among cells expressing gfp-tagged rabs, we stained the cells with anti-n-af647. facs analysis revealed that the d/n -gfp rab5 (s34n) was the only rab construct that caused a significant decrease in rsv infection when overexpressed (fig. 8b) . we confirmed this result by imaging of rrrsv expressing red fluorescent protein (fig. s4 ). together with our imaging data, these results indicated that rsv depends on rab5 gtp for infection but does not require rab7. infectious penetration is thus likely to be determined during early stages of macropinosome maturation. it is noteworthy that expression of the c/a rab5 (q71l), which is known to generate enlarged rab5-containing endosomes and prevent endosome maturation and trafficking to lysosomes [27] , did not affect infection. pretreatment of cells with pikfyve inhibitor (cas 371942-96-7) had no effect on the rsv infection while sfv infection was decreased by 50% (fig. 8c) . by generating ptdins(3,5)p2, pikfyve is involved in the maturation of endosomes and macropinosomes [28] . this suggested that full maturation of macropinosomes was not required for rsv. these results demonstrated that after pinching off from the pm, macropinosomes containing rsv acquired rab5 and later rab7. maturation of macropinosomes involving rab5 was evidently a critical step in infection, whereas later stages in maturation coordinated by rab7 and pikfyve were not essential. since acidification of macropinosomes was not needed for infection, we speculated that rsv required some other intracellular cue to trigger fusion. the f is unique among paramyxovirus fusion proteins in having two cleavage sites for furin-like proteases generating in addition to f1 and f2 (50 and 20 kda, respectively) a soluble 27 amino acid (aa) peptide (p27) [29, 30] . the p27 peptide is located between f2 and f1 n-terminal to the fusion peptide in f1 (fig. 9d) . we hypothesized that removal of this peptide after endocytosis might be required to activate the f protein. experiments in which cells were pretreated with a membrane permeable furin inhibitor, dec-rvkr-cmk, prior to addition of rsv indicated that a protease was indeed involved. dec-rvkr-cmk treatment reduced infection by about 80% (fig. 9a) . that a membrane impermeable furin inhibitor, a-pdx, had no effect on infection suggested that the activating proteolysis did not occur on the cell surface. a broad range protease inhibitor leupeptin (serine, cysteine, threonine proteases) caused only a slight increase in infection. that the inhibition of infection by dec-rvkr-cmk involved the f protein, was confirmed using a recombinant virus strain (rsvdshdg) that lacks the sh and g glycoproteins [5] . infection by this mutant virus was also blocked by dec-rvkr-cmk (fig. 9a) . when dec-rvkr-cmk inhibitor was applied 1 h after the virus inoculation, there was no inhibition indicating that the critical proteolytic step coincided with entry (fig. 9b ). fusion assays with r18/dioc labeled rsv and rsvdshdg revealed that dec-rvkr-cmk impaired viral fusion (fig. 9c ). its inhibitory effect was comparable to the effect of eipa, which blocked virus internalization (fig. 6c) . to confirm the presence of the p27 peptide in the purified virus, we used a targeted mass spectrometry approach based on selected reaction monitoring (srm) (fig. 9e) . as a negative control, we analyzed hep-2 cells extracts used to produce the virus. in trypsin digested virus preparations, we detected 2 peptides corresponding aa 113-131 and 109-123 of f protein both spanning p27 peptide (fig. 9d) . neither peptide was present in hep-2 control samples. srm transitions of the targeted peptides are included as supporting information in table s1 . finally, sds-page was used to monitor changes in the f protein itself during entry. blotting with an anti-f1 antibody revealed a protein band in the isolated virus migrating with a molecular weight of 50 kda, confirming that the f protein had been cleaved at least once already in the producer cells (fig. 9f) . when virus bound to cells in the cold were allowed cell enter for 1.5 h at 37uc, the mobility of the cell-associated f1 became faster (48 kda) indicating that further processing had occurred. the reduction in size of around 2 kda was consistent with the loss of p27 at the n terminus of f1. importantly, in cells pretreated with dec-rvkr-cmk, the processing of the 50 kda f1 protein did not occur. as expected, a-pdx and leupeptin did not influence the processing step. when the time course of f1 processing was followed, we observed that some processed f1 was detectable already 15 min after cell warming (fig. 9g) . it peaked at 60 min when the precursor was fully consumed. that the amount of f1 protein gradually decreased at later time points was probably due to lysosomal degradation explaining the decrease in band intensity in the endocytosis lanes in fig. 9f . . hela cells were pretreated with dec-rvkr-cmk, a -pdx or leupeptin at indicated concentration for 1 h before experiment and inhibitors were continuously present during following steps of the experiment. cells were infected with rsv or rsvdgdsh for 6 h before facs analysis of gfp expressing cells. (b). hela cells were infected with rsv or rsvdgdsh for 1 h. virus inoculum was replaced with medium containing 100 mm dec-rvkr-cmk and incubated for 6 before facs analysis of gfp expressing cells. (c). versene detached hela cells were pretreated with solvent (mock), dec-rvkr-cmk or eipa at indicated concentrations and inhibitors were continuously present during following steps of the experiment. rsv-r18/dioc or rsvdgdsh-r18/dioc (moi ,5) was bound to cells at 4uc. unbound virus was removed and cells were incubated at 37uc for 2 h in the presence of inhibitor. cells were fixed and the mfi of dioc fluorescence measured by facs and normalized to mock no inhibitor controls. (d). rsv f protein (574 aa) is proteolytically processed by furin like protease at the two sites (a aa-106 and b aa-136) to generate disulfide bonds linked f1+f2 and small peptide p27 (aa sequence depicted above). at the n-terminus of f1 is a fp (fusion peptide) and at the c-terminus tm (transmembrane domain), numbers indicate aa position and red underlines specify peptide sequences detected in mass spectrometry. (e). proteomic analysis of hep-2 cells and purified rsv particles. the n-terminal sequence of the p27 peptide (f protein) was quantified by a targeted mass spectrometry based on the selected reaction monitoring (srm). representative srm peaks of peptides (left) fmnytlnnakktnvtlsk 3+ and (right) elprfmnytlnnak 3+ peptides, corresponding to the aa 113-131 and 109-123 of f protein, respectively. different srm transitions for a peptide shown in different colors (see supporting information table s1 ). the bar graphs show the results of the targeted peptide quantitation, presented as the sum of the areas of all the srm peaks for a given peptide. where no peptide peak was detectable, noise values were reported as a reference. rt retention time, and cps counts per second. (f). hela cells were pretreated or mock treated with dec-rvkr-cmk, a-pdx, or leupeptin at indicated concentration. rsv (input control in the first line) was bound for 1 h in cold (b-binding) or after washaway unbound virus was internalized for 1.5 h at 37uc before processing (i-internalized). lysed cell samples were resolved by sds-page and blots were probed with anti-f1 antibody. (g). rsv was bound for 1 h in cold to hela cells; unbound virus wash away and cells were placed at 37uc for indicated times before, lysis, sds-page, and processing for western blot probed with anti-f1 antibody. doi:10.1371/journal.ppat.1003309.g009 these results indicated that to become fusion competent and infectious, the f1 protein underwent a second, highly efficient cleavage by a furin-like convertase in an endocytic compartment. the time course indicated that the processing of f1 occurred soon after endocytosis preceding fusion by about 30 min. to address whether our results applied to cell types infected by rsv in vivo, we tested polarized epithelial cells 16hbe14o obtained from human bronchial biopsies [31] . after 9 days in culture, the distribution of the tight junction marker, zo-1, showed that the cells had reached a polarized phenotype (fig. 10a) . after making certain that rsv could infect 16hbe14o cells from the apical side (fig. 10a) , we tested the effects of nine diagnostic inhibitors previously used in hela cell experiments (fig. 4b, 5ce , 6c, 7c, 9a). they inhibited dynamin, macropinocytosis, and furin proteases. rsv infection was quantified by an image-based approach that detected the fraction of gfp-expressing cells. in agreement with our findings in hela cells, inhibition of dynamin by dynasore had no effect on rsv infection in 16hbe14o cells; it even boosted infection. eipa and seven other inhibitors of macropinocytosis decreased infection in a dosedependent fashion indicating that macropinocytosis was involved in entry. the need for f1 processing was confirmed by dec-rvkr-cmk, which was found to decrease infection by as much as 95%. paramyxoviruses are generally thought to infect cells by fusing directly with the pm [32, 33] . that paramyxovirus particles can also be endocytosed is, however, also clear. this has most recently been documented for sendai, nipah, rsv, newcastle disease viruses and for a lentivirus vector pseudotyped with measles virus glycoproteins [9, 34, 35, 36, 37, 38] . which of the two pathways -fusion at pm or fusion after endocytosis -leads to infection is not clear. in our experiments, we found that intact rsv was rapidly and efficiently endocytosed with capsid, glycoproteins, and the lipid envelope intact. the rsv and capsid-free vlps accumulated within cytoplasmic vacuoles with a half time of about 30 min followed by fusion in the vacuoles with the half time of around 90 min. a sensitive fusion assay using r18/dioc-labeled fluorescent viruses showed that fusion occurred intracellularly. no fusion of viruses with the pm was detected, and no formation of syncytia by fusion-from-without was observed even after exposing cells to high moi (data not shown). the significant delay between rsv internalization and fusion could at least in part be explained by the requirement for post-endocytic cleavage of f protein. perturbations that inhibited endocytic uptake caused a dramatic reduction in infection confirming a role for endocytosis in infection. eipa inhibited both endocytosis and infection by 95%, and a similar level of inhibition was observed for agents that interfered with actin dynamics, a variety of kinases, and myosin ii. the inhibitory effect of rab5 d/n expression was also consistent with a role for endocytosis in infectious entry. proteolytic activation of the f protein necessary for fusion and infection occurred in intracellular compartments. we concluded that rsv and vlps were efficiently endocytosed, that penetration by membrane fusion occurred in endocytic vacuoles, and that at least 90% of infection was caused by endocytosed viruses. that the endocytic mechanism responsible for the entry was macropinocytic was demonstrated by the following observations: observations satisfied all the main criteria currently used to define macropinocytosis [20, 21] . when inhibitor studies were performed using polarized physiologically relevant epithelial cells (human bronchial epithelium cells, 16hbe14o), infectious entry of rsv was found to depend on the actin cytoskeleton, on cell signaling, and on a furinlike protease activity as also observed in hela cells. the results indicated that infection of these polarized epithelial cells monolayers derived from human bronchial tissue involved macropinocytosis and proteolytic activation of the f protein. macropinocytosis is a clathrin-independent mechanism for the uptake of fluid and cell-associated particles within large, uncoated vesicles formed at the pm [24] . in most cell types, it is transiently induced by the activation of rtks and downstream signaling factors [39] . in recent years, several viruses have been shown to use it for infectious cell entry. as recently reviewed [21] , the bestdescribed examples include large viruses such as vaccinia, ebola, adeno 35, and kaposi sarcoma-associated viruses. interestingly, nipah virus, a paramyxovirus of the henipavirus subfamily, also belongs to this group. it uses ephrinb2a as a receptor, the phosphorylation of which is required for macropinocytic internalization and infection in cho-k1 and veroe6 cells [38] . we found that egfr phosphorylation was activated by rsv, and that inhibitors such as iressa targeting this receptor blocked endocytosis and infection. it is noteworthy that iressa was only inhibitory when present during the first hour of virus cell contact confirming that its effect was entry-specific (data not shown). when the egfr was depleted using sirna, infection decreased only by 50% suggesting that other rtks may be able to compensate in long-term experiments. downstream effectors of egfr such as pi3k and pkc were also important for rsv endocytosis and infection. infection has previously been shown to promote cell survival mediated by pi3k/nfkb. in a549 cells, pi3k activation and phosphorylation of its effector akt occurs within 30 min after rsv addition [40] . interestingly, it has also been demonstrated that rsv binding to nheb cells induces pkc-a phosphorylation and translocation to the pm, while inhibition of pkc-a, as confirmed here, blocks rsv uptake and infection [41] . our results contradicted a previous report proposing that rsv entry in hela cells occurs by cme [9] . the authors based their interpretation on hits such as clathrin and associated proteins in a targeted sirna silencing screen against factors involved in endocytosis. (the hits also included actin modulators such as pak1 but their function in entry was not addressed). however, since the read-out was infection after 20 h, a role for cme in postendocytic steps in the rsv infectious cycle could not be excluded. in our experiments, we did not observe inhibition of rsv endocytosis or infection by five different agents that block cme: chlorpromazine, dynasore, pitstop-2, dyngo-4a, and dynol-34-2. importantly these agents efficiently inhibited sfv, a virus that enters via cme. that dynasore fails to inhibit rsv infection was also recently reported by others [10] . while macropinosomes are still poorly characterized, there is evidence that they undergo a maturation process similar to that of endosomes involving acidification, association with rab5 and rab7, and fusion with late endosomes or endolysosomes [42] . we noted that some of the vacuoles containing rsv were in fact rab5-and later rab7-positive. over-expression of a d/n rab5 mutant inhibited infection suggesting that rsv penetration required passage through 'early' macropinosomes that contained rab5. the lack of inhibition by rab7 mutants, a pikfyve inhibitor, and nocodazole, all known to inhibit vacuolar maturation, implied that macropinosome maturation beyond the rab5 positive stages was not necessary. finally, our results provided a likely molecular explanation for the endocytosis requirement exhibited by rsv. unlike other paramyxoviruses, the f protein in rsv has two activating cleavage sites [29] . our mass spectroscopy analysis and western blots showed that while f in the isolated virus had been cleaved in the a-site (rarr) generating f2 and f1, it had not been cleaved at the more c-terminal b site (kkrkr). the second cleavage occurred after endocytosis. inhibition of the second cleavage by dec-rvkr-cmk inhibited rsv fusion and infection. that dec-rvkr-cmk is a furin inhibitor suggested that the protease in question belonged to the furin family of convertases. the enzyme was evidently acid-independent, and active in early rab5 -positive macropinosomes. cleavage at the b site was most likely important because after removal the p27 peptide 'cap' from the n-terminus, the hydrophobic fusion peptide is rendered the most n-terminal sequence in f1. in other class i viral fusion proteins, including other paramyxovirus f proteins, the fusion sequence is invariably n-terminal [43] . in conclusion, we confirmed that rsv requires two cleavages in its f protein for infectivity and showed that the second cleavage occurs during cells entry. infectious entry depends on endocytosis, which the virus induces by transiently activating macropinocytosis. the virus most likely meets the enzyme that generates the second cleavage in rab5 positive macropinosomes, and fusion occurs after some delay in these vacuoles. in this respect, the virus resembles ebola and sars viruses, the fusion proteins of which are also activated within endocytic vacuoles by proteases [44, 45] . it is interesting to note that the f of nipah virus, which has a single monobasic cleavage site in its f, is activated after endocytic uptake by cathepsin [46, 47] . inhibitors of cathepsins block infection, and cathepsin double knock-out cells are not infected. the infectious entry of other paramyxoviruses (and other viruses that have phindependent membrane fusion) may thus be endocytosis-dependent and the mechanisms more complex than previously assumed. for rsv, it will now be important to analyze the molecular features of the entry process in more detail, to identify the protease(s), and to determine whether the intracellular route is relevant also in vivo. being inducible and highly regulated, the macropinocytic process may prove more amenable to inhibition than other endocytic mechanisms, and therefore more easily targeted by therapeutics. hela, a549 and hep-2 cells were obtained from the atcc and cultured in dmem supplemented with 10% fetal calf serum (fcs), 1 mm hepes, 1% glutamax (invitrogen). transformed bronchial epithelial 16hbe14o cells obtained from dr. d. gruenert [31, 48] , were grown in rpmi 1640 medium supplemented with 10% fcs, 1% l-glutamine and 1% nea for at least 9 days before infection. recombinant sfv-zsgreen stocks were kindly provided by dr. g. balistreri [49] . rsv-a2 was purchased from atcc. recombinant rsv strains expressing gfp (rgrsv, rgrsvdshdg) or rfp (rrrsv) were kindly provided by drs. m. peeples and p. collins [11, 50] . rsv was produced in hep-2 cells. virus was collected form cell culture supernatant. the inhibitors used included: pi-103 (alexis biochemicals), dynasore, dyngo-4a,dynol-31-2, pitstop-2 (ascent scientific), pirl1 (chembridge), wiskostatin (enzo), cas 879127-07-8, cas 371942-96-7, dec-rvkr-cmk, ly294002, nsc23766, staur-osporine, taxol, wortmannin, y27632, a-pdx (calbiochem), bafilomycin a, blebbistatin, calphostin c, chlorpromazine, cytochalasin d, eipa, genistein, ipa-3, jasplakinolide, latrunculin a, leupeptin, ml7, monensin, nh4cl, nocodazole, and rottlerin (sigma). antibody and fluorescent dyes that were used comprised: anti-n monoclonal mab858-3-5 and anti-rsv goat polyclonal ab1128 (millipore), anti-f monoclonal ab43812 (abcam), anti-p rabbit polyclonal (3-v biosciences, menlo park, usa), anti-zo-1, goat anti-mouse, goat anti-rabbit, donkey anti-goat af-conjugated, 10 kda dextran-af488, phalloidin af-conjugated, r18, dioc, and transferrin-af488 (molecular probes), goat anti-mouse, goat anti-rabbit hrp-conjugated (bio-rad). expression plasmids encoding gfp-tagged rab5, rab7 and its mutants were kindly provided by dr. m. zerial (max planck institute, dresden, germany). hep-2 cells (50-60% confluent) in t175 flasks were infected with rsv (moi 0.1) in 8 ml serum free dmem-hepes medium (dmem, 1 mm hepes) for 1 h at 37uc, and then inoculum was replaced with complete medium (dmem, 10% fcs, 1% glutamax, 1 mm hepes). after 48 h, the cell supernatant was collected, clarified by centrifugation, aliquoted, snap frozen, and stored in 280uc for experiments that did not require purified virus. for virus purification the method of ueba [51] was modified as follows. the pre-cleared cell supernatant was centrifuged (20.000 rpm, 90 min, 4uc, sw32 ti rotor, beckman optima 90-k ultracentrifuge) through 8 ml 20% w/v sucrose cushion in hbss-hepes buffer (hbss, 25 mm hepes). pellets were gently washed, reconstituted in 20% sucrose and centrifuged in a 35, 45, 60% sucrose step gradient (35.000 rpm, 90 min, 4uc, sw41 rotor, beckman optima 90-k ultracentrifuge). an opalescent virus band was collected from the 35 and 45% interface, overlaid over a 30-60% continuous sucrose gradient and centrifuged (35.000 rpm, 4 h, 4uc, sw41 rotor, beckman optima 90-k ultracentrifuge). the virus fraction at about 45% sucrose was harvested, diluted in the hbss-hepes and pelleted by additional centrifugation (20.000 rpm, 90 min, 4uc sw41 rotor, beckman optima 90-k ultracentrifuge). virus pellets were resuspended in hbss-hepes, snap frozen, and stored in 280uc. all rsv stocks were titered by infecting hep-2 cells with serial dilutions of the virus in 96 well plates. infection was allowed to proceed for 18-22 h at 37uc. fixed cells were assessed by microscopy for gfp expression, or stained with rsv anti-n antibody (af-488) to detect infected cells of rgrsv or rsv-a2 respectively. protein assay (bio-rad) was used to measure the amount of protein. when the influence of pharmacological inhibitors was tested, cells were preincubated with a medium containing inhibitors for 30 min at 37uc before virus binding or infection (except rho gtpase and protease inhibitors that required 1-5 h of preincubation, and pitstop-2 which was preincubated for 10 min). inhibitory compounds at indicated concentration were continuously present during all of the steps of infection and internalization experiments. dioc (6 mg) was added to freshly purified rsv (25 mg) in 1 ml of hbss-hepes buffer and incubated at room temperature while gently shaking for 1 h. to remove unincorporated dye, virus was filtered through 0.45 mm syringe filter (millipore) and snap frozen as 100 ml aliquots, stored in 280uc, and used within 2 weeks. labeled rsv-dioc was titered in hep-2 cells as described above. for the rsv-dioc endocytosis assay, cells were detached with versene solution (0.53 mm edta, ph 8.0), washed with pbs and chilled on ice. purified rsv-dioc, diluted in dmem-hepes was bound to the cells on ice for 1 h. the inoculum was washed away with pbs; the cells were resuspended in complete medium and transferred to 37uc. after desired times, the cells were fixed in 4% formaldehyde, washed, and resuspended in facs buffer (pbs, 0.2% fcs, 5 mm edta). for the facs data acquisition (bd biosciences, canto ii) samples were divided into two. one sample was acquired directly and the other after addition of trypan blue to a final concentration of 0.01% (invitrogen). alternatively, after internalization cells were treated with 0.5% trypsin for 10 min on ice. than washed with pbs, fixed in 4% formaldehyde, permeabilized with 0.01% triton x-100, stained with anti-f or anti-n antibody at 4uc over night followed by the af-647 secondary antibody staining. mean fluorescence intensity measured by flow cytometry (bd biosciences, canto ii) was normalized to the mock control without bound virus. purified rsv (25 mg) was resuspended in 0.5 ml of hbss-hepes buffer before adding of dioc (3.3 mm) and r18 (6.7 mm) mixture. labeling was performed for 1 h at room temperature while gently shaking. to remove unincorporated dye, virus was filtered through 0.45 mm syringe filter (millipore) and used freshly for the fusion assay. for the rsv-r18/dioc fusion assay, cells were detached with versene solution, washed with pbs and chilled on ice. purified rsv-r18/dioc, diluted in dmem-hepes was bound to the cells on ice for 1 h. the inoculum was washed away with pbs; the cells were resuspended in complete medium and transferred to 37uc. after desired times, the cells were fixed in 4% formaldehyde, washed, and resuspended in facs buffer. for the facs data acquisition (bd biosciences, canto ii) samples were divided into two. one sample was analyzed directly and the other after addition of trypan blue to a final concentration of 0.01% (invitrogen). rsv (moi ,3) was bound to subconfluent cells for 1 h at 4uc. than cells were washed with pbs, supplemented with medium containing 1 mm cycloheximide and incubated for 1 h at 37uc. cells were moved on ice and treated with ice cold 0.5% trypsin for 10-20 min, washed with pbs, fixed in 4% formaldehyde, permeabilized with 0.1% triton x-100 and stained with anti-n antibody over night at 4uc followed by the goat anti-mouse af staining. the mfi (mean fluorescence intensity) of af staining determined by facs (bd bioscience, canto ii) and analyzed by for the trf-af488 (2 mg/ml) internalization was performed in parallel in identical conditions with the exception that uptake was carried for 20 min. adherent cells were detached with versene solution, washed with pbs, chilled on ice, and pelleted. cell pellets were resuspended in dmem-hepes, containing the virus inoculum, and incubated 1 h at 4uc. when the effect of inhibitors was tested, cells were preincubated with media containing inhibitor for 30 min, and inhibitor was continuously present at each step of the experiments. cells were washed with pbs to remove unbound virus, fixed with 4% formaldehyde, permeabilized with 0.01% triton x-100, and stained with rsv anti-f over night at 4uc, followed by the secondary antibody labeling. samples were reconstituted in facs buffer and analyzed by flow cytometry (bd biosciences, canto ii). mean fluorescence intensity values were normalized to the mock control without bound virus. cells were detached with versene solution, washed with pbs, chilled on ice, and pelleted. virus input (purified virus stock (moi ,10) diluted in dmem-hepes) was divided in two portions. one was mixed with the cells and the other left for further analysis as virus input. virus was incubated with the cells for 1 h at 4uc. the cells were pelleted by centrifugation and the supernatant collected as an unbound viruses sample. the cell pellets were washed in pbs and resuspended in dmem-hepes of equal volume to the collected supernatant. the sds-page loading buffer was added to all samples, followed by denaturation for 10 min at 95uc. an equal volume of each sample was separated by the sds-page and subjected to western blot with anti-p or anti-n antibody. western blots were quantified by densitometry with quantityone software (bio-rad). subconfluent cells seeded in 24 well plates were starved over night with serum free dmem-hepes medium. purified rsv was bound to pre-chilled cells for 1 h at 4uc. virus inoculum was washed and cells were pulsed with the serum free dmem-hepes medium containing 10 kda dextran-af488 (0.5 mg/ml) for 15 or 60 min at 37uc. the cells were washed with 10 mm naoac, 50 mm nacl ph 5.5 followed by pbs wash. the cells were trypsinized, fixed in 4% formaldehyde and analyzed by flow cytometry (bd biosciences, canto ii). for confocal microscopy, cells were fixed in 4% formaldehyde, permeabilized with 0.1% triton x-100, and stained with rsv specific antibodies. images were acquired with zeiss lsm510 laser scanning confocal microscope. for transfection, cells were trypsinized, pelleted, and electroporated with 2 mg of plasmid dna using amaxa (nucleofactor solution r, program i-13). cells were seeded on 12 mm cover glass in 24 well plates for imaging or in the 6 well plates for facs analysis. experiments were performed after 12 h of transient expression. the experiment was performed according to the protocol provided by the f/g actin in vitro assay kit producer (cytoskeleton inc. cat # bk037). in brief, subconfluent cells in 3.5 cm dishes were inoculated with purified rgrsv (moi 30) for 30 or 120 min at 37uc. cells were washed, lysed, and clarified by centrifugation. supernatants were centrifuged (53000 rpm, 1 h, 37uc, tla120.2 rotor, beckman optima tlx ultracentrifuge), the resulting supernatants were collected (g-actin), and the pellets (f-actin) were resuspended in equal to supernatant volume of water containing actin depolymerizing reagent provided in the kit. equal volume of each sample was resolved by sds-page and western blots were developed with an anti-actin antibody. to measure ratio of the g and f actin western blots were analyzed by densitometry with quantityone software (bio-rad). for the infection assays cells were plated in optical bottom 96 well plates. after infection with rgrsv for 18-20 hours, the cells were fixed with 4% formaldehyde and counterstained with dapi. nine images per well were acquired with automated md2 microscope that autofocuses at each image acquisition (106 objective). the total cells number and the number of infected cells expressing gfp were scored by the matlab-based infection counter software described previously [52] . human phospho-rtk array kit was obtained from r&d systems (cat # ary001) and experimental procedure was followed according the manufacturers guidelines. in brief, serum-starved subconfluent cells in 3.5 cm dish were inoculated with purified rsv (moi 30) or null virus prep extract for 15 min at 37uc. then, cells were lysed and pre-clarified by centrifugation. the supernatant samples were incubated with the antibody array, and developed with provided reagents. developed arrays were analyzed by densitometry with quantityone software (bio-rad). subconfluent cells in 12 well plates were chilled on ice for 20 min. rsv (moi 10) in dmem-hepes was bound to cells for 1 h at 4uc. post binding samples were washed and lysed in ripa buffer (50 mm tris, 150 mm nacl, 2 mm edta, 1% np-40, 0.1% sds, ph 7.4). remaining samples were washed with pbs supplemented with complete medium and transferred to 37uc for 1 h; following cells were trypsinized, washed and lysed with ripa buffer. all samples were separated on the 10% bis-tris gels (invitrogen). western blots were probed with anti-f antibody (1:500), hrp conjugated goat ant-mouse (1:2000) and developed with super signal west pico (pierce). immunofluorescence staining and analysis 0.8610 5 cells were seeded on 12 mm coverslips in the 24 well plate 24 h prior experiment. plates were chilled on ice and then rsv was bound to cells in dmem-hepes for 1 h at 4uc. viruses were washed away and cells were supplemented with complete medium and transferred to 37uc. at desired times cells were fixed in 4% formaldehyde, permeabilized with 0.1% triton x-100 and incubated with 10% goat serum for 30 min. cells were then stained with appropriate primary and secondary antibody. coverslips were mounted to the glass slides with prolong gold anti-fade reagent (invitrogen). immunofluorescence images were captured with lsm zeiss 510 microscope with the confocal laser scanning set up (objectives 60 or 1006). per experiment, 10-15 images were captured and processed by imagej. for the virus particles detection imaris software was used set up to detect particles larger than 0.5 mm and quality greater than 15. live cell imaging was performed with olympus cellr, 206 objective with dic setting, equipped with 37uc incubator. hep2 cells or gradient purified rsv particles were lysed in denaturing buffer containing 8 m urea and 100 mm nh 4 hco 3 . lysates were briefly sonicated and proteins were reduced with 12 mm dtt for 30 min at 32uc and alkylated with 40 mm iodoacetamide for 45 min at 25uc. the samples were diluted 1:5 with 100 mm nh 4 hco 3 and digested with sequencing-grade porcine trypsin (promega) at an enzyme/substrate ratio of 1:100. the digestion was performed overnight and stopped with formic acid at final concentration of 2%. the peptide mixtures were desalted on sep-pak c18 cartridges (waters), eluted with 80% acetonitrile, vacuum centrifuged until dryness and resuspended in 0.15% formic acid. for each peptide, q1 and q3 masses, as well as collision energies (ce) for peptide fragmentation were calculated using skyline (v1.3, maccoss lab software). double and triple charged product ions from the y-and b-series and transitions in a mass range of 350-1250 da were considered (see full list of srm transitions in the supporting information table s1 ). the peptide samples were measured in srm mode on a triple-quadrupole/ion trap mass spectrometer (5500qtrap, absciex) equipped with a nano-electrospray ion source. for the chromatographic separation of the peptides, the instrument was coupled with an eksigent nano lc system (absciex) connected to a 15-cm fused silica column, 75 mm inner diameter (bgb analytik), packed in-house with magic c18 aq, 5 mm beads (michrom bioresources). the peptide mixtures were loaded from an autosampler cooled to 4uc (absciex) and separated with a linear gradient of acetonitrile/ water containing 0.1% formic acid from 5 to 35% acetonitrile in 30 min, with a flow rate of 350 nl/min. srm analysis was conducted with q1 and q3 operated at unit resolution (0.7 m/z half maximum peak width) with up to 70 transitions per run (dwell time, 30 ms; cycle time, 2.5 s). data were analyzed with the software skyline. peak area of the srm peaks was used for quantitation, after confirming co-elution and shape similarity of the transitions monitored for each peptide. outlier transitions (e.g., shouldered or noisy transition traces) were not considered in the calculations. results are presented as the sum of the areas of all srm peaks for a given peptide. all experiments were performed at least in triplicate, and presented as normalized values with +/2 standard deviation (sd). . rsv (moi ,0.5) was bound to a549 cells at 4uc followed by 30 min at 37uc. cells were by iif with anti-f-af488 (green), anti-n-af594 (red), and phalloidin-af647 (pseudocolored white) for confocal microscopy as, and z-stack image series acquired. the orthogonal views of image z-stacks (pseudo-colored white) were generated with imagej. (b). rsv (moi ,0.5) was bound to a549 cells at 4uc, virus inoculum was washed, cells warmed to 37uc, fixed at indicated times, and stained with phalloidin-af488 (pseudo colored white) and anti-f-af647 (red) antibody. images represent z-stack projections acquired with a confocal microscope. movie s1 rsv induces transient blebbing of hela cells. hela cells were inoculated with a purified rsv (moi 30) and immediately imaged with olympus cellr microscope with dic settings with the 206 objective, 1 frame per 10 sec speed at 37c. (avi) table s1 srm assays used to study f0 (uniprot accession number p03420, fus_hrsva). (docx) a review of palivizumab and emerging therapies for respiratory syncytial virus morphogenesis and ultrastructure of respiratory syncytial virus viral and host factors in human respiratory syncytial virus pathogenesis iduronic acidcontaining glycosaminoglycans on target cells are required for efficient respiratory syncytial virus infection functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene in vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo characteristics of fusion of respiratory syncytial virus with hep-2 cells as measured by r18 fluorescence dequenching assay recombinant vesicular stomatitis virus expressing respiratory syncytial virus (rsv) glycoproteins: rsv fusion protein can mediate infection and cell fusion small interfering rna profiling reveals key role of clathrin-mediated endocytosis and early endosome formation for infection by respiratory syncytial virus cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells respiratory syncytial virus engineered to express the cystic fibrosis transmembrane conductance regulator corrects the bioelectric phenotype of human cystic fibrosis airway epithelium in vitro protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein 90 in virus particle assembly a single-step, sensitive flow cytofluorometric assay for the simultaneous assessment of membrane-bound and ingested candida albicans in phagocytosing neutrophils dual wavelength imaging allows analysis of membrane fusion of influenza virus inside cells pharmacological inhibition of endocytic pathways: is it specific enough to be useful? role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition inhibition of dynamin by dynole 34-2 induces cell death following cytokinesis failure in cancer cells penetration of semliki forest virus from acidic prelysosomal vacuoles inhibition of semliki forest virus penetration by lysosomotropic weak bases virus entry by macropinocytosis gulping rather than sipping: macropinocytosis as a way of virus entry drinking a lot is good for dendritic cells amiloride inhibits macropinocytosis by lowering submembranous ph and preventing rac1 and cdc42 signaling shaping cups into phagosomes and macropinosomes myosin iib isoform plays an essential role in the formation of two distinct types of macropinosomes sequential activities of phosphoinositide 3-kinase, pkb/aakt, and rab7 during macropinosome formation in dictyostelium inhibition of rab5 gtpase activity stimulates membrane fusion in endocytosis visualisation of macropinosome maturation by the recruitment of sorting nexins cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion proteolytic activation of respiratory syncytial virus fusion protein. cleavage at two furin consensus sequences chemotactic antiviral cytokines promote infectious apical entry of human adenovirus into polarized epithelial cells paramyxovirus membrane fusion: lessons from the f and hn atomic structures paramyxovirus fusion and entry: multiple paths to a common end fusion of sendai virus and individual host cells and inhibition of fusion by lipophosphoglycan measured with image correlation spectroscopy role of endocytosis and cathepsinmediated activation in nipah virus entry newcastle disease virus may enter cells by caveolae-mediated endocytosis measles virus glycoprotein-pseudotyped lentiviral vector-mediated gene transfer into quiescent lymphocytes requires binding to both slam and cd46 entry receptors nipah virus entry can occur by macropinocytosis distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma a431 cells respiratory syncytial virus inhibits apoptosis and induces nf-kappa b activity through a phosphatidylinositol 3-kinase-dependent pathway protein kinase c-alpha activity is required for respiratory syncytial virus fusion to human bronchial epithelial cells macropinosome maturation and fusion with tubular lysosomes in macrophages virus membrane-fusion proteins: more than one way to make a hairpin endosomal proteolysis of the ebola virus glycoprotein is necessary for infection characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry activation of the nipah virus fusion protein in mdck cells is mediated by cathepsin b within the endosome-recycling compartment a mature and fusogenic form of the nipah virus fusion protein requires proteolytic processing by cathepsin l long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions phosphatidylinositol 3-kinase-, actin-, and microtubule-dependent transport of semliki forest virus replication complexes from the plasma membrane to modified lysosomes differential response of dendritic cells to human metapneumovirus and respiratory syncytial virus respiratory syncytial virus. i. concentration and purification of the infectious virus role of endosomes in simian virus 40 entry and infection we are grateful to drs. p. collins and m. peeples who provided us with recombinant rsv strains and dr. g. balistreri for the stocks of zsgreen-sfv and valuable advises. thanks to 3v-biosciences for anti-p serum. we also thank dr. t. hegar who made available the infectious counter software and m. bonvin for help with depletion experiments. finally, thanks to drs. p.y. lozach and j. mercer for helpful discussions. key: cord-280454-etf32afd authors: moustaqil, mehdi; ollivier, emma; chiu, hsin-ping; van tol, sarah; rudolffi-soto, paulina; stevens, christian; bhumkar, akshay; hunter, dominic j.b.; freiberg, alex; jacques, david; lee, benhur; sierecki, emma; gambin, yann title: sars-cov-2 proteases cleave irf3 and critical modulators of inflammatory pathways (nlrp12 and tab1): implications for disease presentation across species and the search for reservoir hosts date: 2020-06-05 journal: biorxiv doi: 10.1101/2020.06.05.135699 sha: doc_id: 280454 cord_uid: etf32afd the genome of sars-cov-2 (sars2) encodes for two viral proteases (nsp3/ papain-like protease and nsp5/ 3c-like protease or major protease) that are responsible for cleaving viral polyproteins for successful replication. nsp3 and nsp5 of sars-cov (sars1) are known interferon antagonists. here, we examined whether the protease function of sars2 nsp3 and nsp5 target proteins involved in the host innate immune response. we designed a fluorescent based cleavage assay to rapidly screen the protease activity of nsp3 and nsp5 on a library of 71 human innate immune proteins (hiips), covering most pathways involved in human innate immunity. by expressing each of these hiips with a genetically encoded fluorophore in a cell-free system and titrating in the recombinant protease domain of nsp3 or nsp5, we could readily detect cleavage of cognate hiips on sds-page gels. we identified 3 proteins that were specifically and selectively cleaved by nsp3 or nsp5: irf-3, and nlrp12 and tab1, respectively. direct cleavage of irf3 by nsp3 could explain the blunted typei ifn response seen during sars-cov-2 infections while nsp5 mediated cleavage of nlrp12 and tab1 point to a molecular mechanism for enhanced production of il-6 and inflammatory response observed in covid-19 patients. surprisingly, both nlrp12 and tab1 have each two distinct cleavage sites. we demonstrate that in mice, the second cleavage site of nlrp12 is absent. we pushed this comparative alignment of irf-3 and nlrp12 homologs and show that the lack or presence of cognate cleavage motifs in irf-3 and nlrp12 could contribute to the presentation of disease in cats and tigers, for example. our findings provide an explanatory framework for in-depth studies into the pathophysiology of covid-19 and should facilitate the search or development of more effective animal models for severe covid-19. finally, we discovered that one particular species of bats, david’s myotis, possesses the five cleavage sites found in humans for nlrp12, tab1 and irf3. these bats are endemic from the hubei province in china and we discuss its potential role as reservoir for the evolution of sars1 and sasr2. the ongoing pandemic of covid-19 (coronavirus disease-2019) has already had a deep health, economic and societal impact worldwide [1] . covid-19 is caused by a novel betacoronavirus, sars-cov-2. other members of coronaviridae family include the highly pathogenic sars-cov and mers-cov, responsible for widespread outbreaks in 2002 and 2012, respectively [2] . sars-cov-2 encodes a large (30 kb) single stranded, positive sense rna genome that contains multiple open reading frames (orfs). orf1a and 1ab produce two large replicase polyproteins precursors (450 kda for orf1a, 750 kda for orf1ab) which upon proteolytic cleavage generates 16 non-structural proteins (nsp), 1 to 16. other orfs encode the 4 main structural proteins of sars-cov2: spike (s), membrane (m), envelope (e) and nucleocapsid (n) proteins, as well as accessory proteins. processing of the polyprotein precursors relies on the two viral proteases, nsp3 and nsp5. as shown in figure 1a , nsp3 or papain-like protease (plpro) is responsible for the proteolytic cleavage of nsp 1-4. the protein nsp5, or 3c-like protease (3clpro), is responsible for the processing of other cleavage sites that results in nsp 5-16. nsp4 is uniquely cleaved by nsp3 on the n-terminus and nsp5 on the c-terminus [3] . as these two proteases are essential for viral replication, they are evident drug targets and considerable effort was spent by the structural biology community early in the sars-cov-2 outbreak. structures of the protease domains of nsp3 and nsp5 have already been reported, opening the door to the identification or development of inhibitors, using virtual or high-throughput screening (e.g. [4] [5] [6] [7] [8] ). viral proteins, especially those from rna viruses that have stricter constraints on their genome size, often perform multiple tasks. in addition to performing their intrinsic functions in the viral life cycle, many have evolved to interfere with innate immune responses or otherwise co-opt the host cell's machinery to facilitate optimal viral replication [9, 10] . the role of coronaviruses proteases in mediating virulence has been shown before [9, 11] . for example, plpro of both sars-cov [12, 13] and mers-cov [14] , as well as other coronaviruses [15, 16] , lead to inhibition of the type i interferon pathway. inactivation of different components of the pathway, including rig-i [12] , sting [12] traf3/ traf6 [17] , tbk1 [16] and irf3 [13, 14, 18, 19] , has been documented. these effects are mediated by partly mediated by the protease activity but mainly derive from the deubiquitinating and deigsgylating functions associated with fulllength nsp3 [20] [21] [22] . sars-cov plpro has also been reported to also activate tgf-β1 signalling [23] or down-regulate p53 [24] . similarly, 3clpro from the feline coronavirus, feline infectious peritonitis virus (fipv), inhibits type i interferon signalling through cleavage of nemo [25] , while the porcine deltacoronavirus (pdcov) 3clpro cleaves stat2. sars-cov 3clpro is responsible for virus-induced apoptosis [26] . therefore, we hypothesized that these proteases could cleave human innate immune pathway proteins (hiips), leading to interference with or dysregulation of the host response. to screen for hiips that might be targeted by sars-cov 2 plpro or 3clpro, we first leveraged the systems virology and systems biology tools present in relevant databases like innatedb [27] , pathbank [28] vipr [29] , virhostnet2.0, and virusmentha [30] to downselect a core set of hiips that covers almost all pathways involved in human innate immune responses. we then designed a fluorescent based in-vitro protease activity assay to screen this library of 71 full-length hiips (fig. 1b) . the protease domains of plpro and 3clpro of sars-cov2 were recombinantly purified and added to gfp-labelled target hiips, expressed in a eukaryotic cell-free system. proteolytic cleavage was assessed by sds-page gels. our screen of 71 hiips (fig. 1c ) revealed that only 3 proteins were directly cleaved by these two viral proteases. notably, we discovered that nsp3 directly cleaved irf3, while nsp5 cleaved nlrp12 and tab1. surprisingly, both nlrp12 and tab1 are cleaved at two different sites, creating three protein fragments. we identified the five cognate cleavage sites in these 3 hiips targeted by the plpro and 3clpro domains of sars2 nsp3 and nsp5, respectively. structure-function correlative analysis followed by comparative alignment of irf3 and nlrp12 homologs across relevant mammalian orders reveal the potential explanatory power of our findings. the cleavage of irf3 could explain the enigmatically blunted type-i ifn response that have been noted in early studies of sars-cov-2 infections [31, 32] , while the nsp5 mediated cleavage of nlrp12 might explain the hyperinflammatory response linked to severe covid-19 disease [33, 34] . indeed, the lack or presence of cognate cleavage motifs in irf3 and nlrp12 homologs presents interesting correlations with the presentation of disease in animal models; our results will enable the development of more effective animal models for severe covid-19. finally, we searched the available genomes of potential hosts, to determine whether sars2 could have evolved into an animal where the different cleavage sites would be present. we found that out of 11 species of bats, only one presents all five cleavage sites identical to humans for nlrp12, tab1 and irf3. as myotis davidii, is found endemically in hubei province of china, near the first epicentre of sars-cov-2 pandemic, we will discuss its potential role as reservoir host. an in-vitro protease assay identifies targets of sars-cov2 plpro and 3clpro the human target proteins to screen in the in-vitro assay were selected to contain major proteins associated with the signaling pathways of innate immunity and cell death [35] . relevant to infection by viruses, proteins downstream of the nucleic acid sensors mda-5 and rig-i have been selected (e.g. mavs, traf3, nfκb and irfs). also included are the effectors of the toll-like receptors, tlr3 and tlr7, such as trif, tram, traf6 or tab1. proteins involved in cell-death (e.g. traf2, caspases, bcl2, xiap) were also included. 71 human proteins were cloned for expression as gfp-fusions in a cell-free expression system based on the eukaryotic organism of leishmania tarentolae (lte). this system produces full-length proteins up to 200 kda with minimal truncations, minimal protein aggregation [36] and was previously used by our group to study the behaviour of various apoptotic proteins such as myd88 [37] , mal [38] or asc and nlrp3 [39] . the assay was designed as a one-pot reaction to rapidly identify proteolytic cleavage. purified recombinant protease domains (adjusted to a final concentration of 10 µm) were added to the lte during expression of the target proteins (see supplementary figure 1 ). the screening conditions were optimised to avoid offtarget effects and false positives. the human proteins targets were typically expressed at low concentration (reaching at most 1 μm), in a crowded environment (lte) that recapitulates the host cytosol. the proteases were allowed to react to target protein de novo synthesis for about 2 ½ hours, at 27°c (optimal temperature for protein expression using lte). under these conditions, it is probable that the activity of the proteases was greatly reduced. we used the gfp-tag on the target protein to directly visualize cleavage using reducing sds-page (fig. 1c ). as expected, partial denaturation (i.e. no thermal denaturation) maintained the fluorescence of gfp so that proteins could be imaged without any subsequent purification steps. comparing the protein migration patterns in the presence and absence of the protease identifies cleavable proteins. indeed, an intact protein would appear on the gel as a single fluorescent band. if the protein is cleaved, then the gel will show either a single band, at a lower molecular weight (in the event of a complete proteolysis of all target proteins), or multiple fluorescent bands, corresponding to the full-length protein and its cleavage product in the case of an incomplete cleavage process, as described in figure 1b . the use of a fluorescent tag also allows simple quantification of protein concentration based on fluorescence intensity [37, 39] . sds-page showed no difference of sizes in the presence or absence of viral proteases for most proteins, indicating that a large majority of human proteins were unaffected by the addition of plpro and 3clpro. this suggests that in our assay conditions, non-specific cleavage was not observed. however, 3 cases of proteolytic degradation were identified, giving confidence that the viral proteases are active. the fact that only specific members of the same family of proteins (e.g. irfs, fig. 1c ) were cleaved (irf3 cleavage by plpro) suggests high specificity and the recognition of specific sequences. there was no common reactivity between plpro and 3clpro reinforcing the idea that each viral protease did indeed recognize a specific consensus sequence. the screen results also revealed that protein expression levels were unchanged upon addition of the proteases, suggesting that none of the components required for cell-free expression were cleaved during the experiments. as shown in supplementary figure 2 , the cell-free lysate acts as a crowded environment made up of many different proteins. analysis of the coomassie stained gels shows that staining intensity and profile were similar, even at the highest plpro concentration, indicating that there was no significant cleavage of components of the cell-free reagent. to validate that plpro could cleave irf3, we titrated different concentrations of the protease in the reaction. as shown in figure 2a , a strong concentration-dependence was observed, as expected. when the same experiment was performed in the presence of 3clpro, no proteolysis was detected, validating that the cleavage is indeed specific to pl-pro (suppl. fig. 3 ). we then set out to identify the cleavage site on irf3. based on the proteolysis sites on orf1a and orf1ab [3] , and similarly to sars-cov plpro [40] and mers-cov plpro [41] , sars2-cov plpro recognises and cleaves after lxgg sequences (fig. 2b ). sequence analysis of the human irf3 shows the presence of a single lggg sequence at residues 268-271 of the canonical isoform. cleavage of the n-gfp tagged protein at this site would result in the formation of a gfp-tagged 57 kda fragment (30 kda +27 kda for the gfp) and a 17 kda untagged fragment, which corresponds well to the band obtained by sds-page (fig. 2c ). the identified cleavage site would be present on an exposed loop, based on previously solved structures (see fig. 2d , pdb: 1j2f [42] ) and therefore accessible to the protease. no such motif was found in any other member of the irf family (fig. 2e ), in agreement with our data. on the contrary, recognition motifs present in other proteins of our test panel (lagg in β-catenin, lvgg in stat5a and legg in nlrp12) did not get processed by plpro in our assay. in the case of stat5a, the cleavage motif is partially buried in the protein (see supplementary figure 4 ). however, the lagg motif in β-catenin is exposed at the surface in a structured a-helix (see supplementary figure 5 ); the legg motif in nlrp12 cannot be located on the only existing structure nlrp (nlrp3, pdb 6npy). it is possible in addition to local structures, the residue between l and gg would contribute to the selectivity of plpro cleavage. irf3 is a key mediator of type i interferon (ifn) response triggered by viral infections [43] . the c-terminal part of the protein is responsible for mediating interactions with upstream receptors and effectors sting, mavs and trif [44] . irf3 tail is also strongly targeted for post-translational modifications upon infection [45, 46] , leading to its homodimerization (pdb: 1qwt, figure 2f ), translocation into the nucleus and transcriptional activation [47] . therefore, we reasoned that plpro cleavage of irf3 would result in reduced ifn production, a feature that has been observed upon sars-cov2 infection [31] . similarly, we set out to validate 3clpro proteolysis of tab1 and nlrp12 ( fig. 3a and 4a ). as before, we observed a concentration-dependent cleavage of both proteins and verified that plpro did not have an effect, at any concentration (see supplementary figure 6 and 7). sds-page analysis reveals the presence of two cleavage sites on tab1, that can be more easily visualized when the gfp tag is placed at either the c-terminus (fig. 3a ) or the n-terminus (fig.3b ) of the protein. the recognition motif for 3cl-pro of coronaviruses sars-cov [48] , mers-cov [49] and sars2-cov [50] is often lq/(s,a,g) although the protease can also cuts after fq or vq motifs [3] . further, the presence of a combination of hydrophobic and positively charged residues at position p-3 and p-4 also seems to be preferred (fig. 4b ). in tab1, we identified a ltlqs motif at position 441 of the canonical form, that would give rise to a 48 kda n-terminal fragment and a 6 kda c-terminal fragment. another possible recognition motif (aslqs) is present at position 129 and that would give a 14 kda n-terminal fragment and a 40 kda c-terminal fragment (fig. 3c) . therefore, the two proteolytic fragments for c-gfp tab1 correspond to amino-acids 133-504 and 445-504 ( fig. 3a) whereas n-gfp tab1cleavage leads to the formation of proteolytic fragments corresponding to residues 1-132 and 1-444 (fig. 3b ). more details on calculation of sizes based on migration are included in supplementary figure 8 and 9. based on the reported structure of tab1 (fig. 3d , pdb: 2j4o, [51] ), the first cleavage site is on an exposed loop in the pseudo-phosphatase domain. the second cleavage site is in a disordered region of the protein that does not appear on the structure (see figure 3e for the full sequence of human tab1.) another target of 3cl-pro revealed by our screen is nlrp12. nlrp12 is an intracellular patternrecognition receptor, from the nucleotide-binding and oligomerization domain-like (nlr) receptor family, which regroups key mediators of the innate immune response and inflammation [52] . nlrp12 modulates the expression of inflammatory cytokines [53] through the regulation of the nfκb and mapk pathways [54] . formation of nlrp12 inflammasome can activate caspase 1 [54, 55] , which produces interleukin and leads to cell death. but nlrp12 can also form mixed inflammasomes with other nlrps, including nlrp3, and then play an inhibitory role [56, 57] . nlrp12 is also involved in adaptative immunity and controls mhc class i expression through a yet ill-defined mechanism [58] . unexpectedly, we also noted the presence of two additional bands when nlrp12 was submitted to 3clpro treatment, suggesting that two cleavage sites would also be found in nlrp12 (fig. 4a) . in figure 4b , we show the analysis of preferred residues for cleavage by 3clpro of sars-cov-2. sequence analysis identified a canonical lqa motif at residue 938, at the c-terminal of nlrp12, in the middle of the lr repeats (fig. 4c) . the cleavage at residue 938 would create a small c-terminal fragment (residues 938-1062), observed on the gel using the c-terminally gfp-tagged nlrp12 (fig. 4a ). there was no other canonical 3clpro recognition sequence (lqs) in nlrp12, to explain the second proteolytic event. when expanding the search to degenerated sequences (fq/vq), we identified a klfqg sequence at residue 238 (fig. 4c ). cleavage at this site would result in the formation of a 93kda c-terminal fragment (residues 241-1,062), observed in figure 4a . analysis of the sequences of other nlrps (nlrp1-nlrp14) shows that both motives are unique to nlrp12 (fig. 4d) . interestingly, the mouse homolog of nlrp12 possesses the first but not the second recognition motif (fig. 4d) . to further validate our data, we compared the effect of 3clpro on human vs. mouse nlrp12, each tagged at the n-and c-terminal position. figure 4e shows that human nrlp12 (left panel) is indeed cleaved twice, whereas mouse nlrp12 (right panel) is only processed once. only the smaller n-terminal fragment and corresponding larger c-terminal fragment are conserved, indicating that cleavage at residue 238 occurs for both species, while cleavage at residue 938 is specific to human. the calibration of migration of the fragments on the sds-page gels is further described in supplementary figure 10 . homology modelling of nlrp12, using the structure of its close relative nlrp3 (pdb: 6npy [59] ), shows that both sites are expected to be in an exposed, unstructured loops, readily accessible to a protease (fig. 4f ). similar to sars, sars2 utilizes ace2 and tmprss2 to achieve its entry step [60] [61] [62] . the cleavage of irf3, tab1, and nlrp12 was demonstrated by in vitro plpro or 3clpro cleavage at the previous figures. to validate the cleavage effects upon sars2 infection, we generated ace2 expressing and ace2/tmprss2 double expressing 293t cells by lentiviral expression system for enhancing the infectivity of sars2. 293t-ace2 and 293t-ace2/tmprss2 cells were then infected with sars2 and analyzed for viral and host protein levels. fig. 5 shows that the expression of irf3, nlrp12, and tab1 (panels a-c, respectively) was decreased in virus-infected cells (lanes 2-3 and 5-6) compared to the mockinfected controls (lanes 1). the lower expression of ace2 in 293t-ace2/tmprss2 cells compared with that in 293t-ace2 cells (lanes 4 and 1, respectively) might be caused by the proteolytic cleavage of ace2 due to tmprss2 overexpression [63] ; however, it did not affect the pattern of irf3, tab1, and nlrp12 during sars2 infection. combined with the results from in vitro assay, we found the proteases of sars2 (plpro and 3clpro) can degrade irf3, tab1, and nlrp12 probably through their protease activity, leading to the imbalance responses of host innate immunity experimental screens identify accessible cleavage sites in proteins and more importantly, detects noncanonical sequences. in this report, we show that the viral proteases plpro and 3clpro of sars-cov2 lead to the in-vitro proteolytic cleavage of three important proteins of the host immune response: irf3, tab1 and nlrp12 (fig. 1b) . these results first show the exquisite specificity of the viral proteases, with only 3 positive hits observed out of 142 experiments. in the case of plpro, the recognition sequence seems well defined and lxgg motifs are scarcely found in the proteins we studied. nevertheless, of the 4 proteins that harbor such motifs, only 1 (irf3) was cleaved in vitro. indeed, the motif is present on an unstructured loop of irf3 that made this site readily accessible. this shows that the presence of a recognition motif is required but not sufficient to predict biological activity. as such, experimental validation of potential targets, identified by bioinformatics remains critical. the identification of nlrp12 and tab1 as substrates of 3clpro further demonstrate the importance of this type of screening approaches. 3clpro recognition motif is not as well defined as plpro, and lq/(s,a,g) motifs are ubiquitously found in the proteome. in our protein list, multiple bona fide recognition motifs based on lq/s, lq/a and lq/g, respectively, can be found as well as several degenerated motifs. yet only two targets (and four cleavage sites) were recognized by 3clpro, emphasizing again the importance of site accessibility. more importantly, our data identify an unexpected cleavage site on nlrp12 (klfq/g) that does not resemble to the cleavage motifs present on the virus orf1a/1ab, further indicating that the determinants of selectivity for 3clpro are yet to be identified. viral proteases have probably evolved to efficiently process their own polypeptides but their ability to target host proteins, especially the ones involved in host defence, provide them with an evolutionary advantage. the three hiips identified in this screen (irf3, tab1 and nlrp12) are important contributors to the innate immune and inflammatory responses, either driving or dampening it. irf3 belongs to the interferon-regulatory factor (irf) family. all irfs possess a well-conserved dnabinding domain at the n-terminus and a variable c-terminal domain that mediates most of the interactions with the other irf proteins and other co-factors [64] . fine-tuning of the choice of partners rely on posttranslation modifications, such as phosphorylation and ubiquitination, in this region. as mentioned before, irfs, especially irf1, 3, 5 and 7, are major contributors to the production of and response to type i interferons, which stimulate macrophages and nk cells to elicit anti-viral responses [47] . therefore, irf3 has been found to be targeted by several different classes of viruses. paramyxoviruses, herpesviruses, reovirus and double stranded rna viruses, to cite some examples, have been shown to interfere with irf3 signalling through different strategies [65] . these include the control of protein expression, protein cellular localisation, modifications of the ptms, inhibition of protein-protein interactions or induced cellular degradation [10] . direct proteolytic cleavage of irfs by viral proteases has also been identified before. proteases of enteroviruses ev71 [66] and ev-d68 [67] have been shown to directly cleave irf7. papain-like protease domains of several coronaviruses including sars-cov and mers-cov have been implicated in the observed reduction of ifn signalling upon viral infection [68] . although this link seems well established, the molecular basis of this effect is still under debate. whether the enzymatic activity is required is also controversial. besides its proteolytic activity, plpro of coronaviridae also possess a deubiquitinase and deisgylation activity that contribute to inhibition of irf3 activity [13, 18, 19, 69] . further, sars-cov plpro has been shown to target proteins upstream of irf3. it can directly bind to sting, disrupt the formation of the sting-traf3-tbk1-ikkε complex or limit signalling by deubiquitinating sting, traf3, traf6 or tbk1 [44] . here we show that in-vitro, sars-cov2 plpro was able to cleave irf3 and validated that the effect could be observed in relevant virus infected cells ( figure 5 ). direct proteolytic cleavage of irf3 may contribute to the reduction of type i interferon production observed in covid19 patients [31, 32] . tab1 is part of the tab1/2/3/tak1 complex [70] that regulates the activity of tak1 (tgfβ-activated kinase 1), in response to different stimuli including tgfβ, il1, tnfα and upon viral and bacterial infection [71, 72] . tak1 can then activate the nfκb pathway or signal through the map kinases pathway [73, 74] . lei et al. showed that the 3c protease of enterovirus 71 (ev71) cleaved tab1 (along with tab2, tab3 and tak1) at two cleavage sites (q 414 s and q 451 s) to perturb the formation of the complex and inhibit cytokine release downstream of nfκb [66] . our identified cleavage site, at q 444 s, forms a protein that is reminiscent of the second isoform of tab1 (tab1β, which lacks the c-terminus) that loses its interaction with tak1 [75, 76] . further, the poly-ser region (452-457) is a substrate for p38 kinase [77] (whose binding site has been mapped to residues 408-414 on tab1) and phosphorylation controls cellular localisation and activity of the protein [70] . therefore, the loss of tab1 c-terminus through 3clpro cleavage would profoundly impact its ability to activate tak1 and result in decrease production of cytokine through nfκb signalling. finally, we identified nlrp12 as a substrate of 3clpro, and two cleavage sites could be identified. nlrp12, like other members of the nlrp family, possess a pyd domain, that binds the effector asc through homotypic interaction, followed by a nacht domain, which binds atp and mediates activation of the protein, and a series of lr repeats that gives specificity to each member by modulating proteinprotein interactions [78] . nlrp12, by similarity with nlrp3, is thought to be normally maintained in a monomeric, auto-inhibited conformation and release of this auto-inhibition is mediated by binding of a ligand (e.g. atp) to the nacht domain [79] . atp binding to nlrp12 plays a major role in regulating the protein's activity as it has been shown to not only induce self-oligomerisation, but also promote interaction with nfκb-induced kinase (nik) and subsequent degradation of nik [80] . mutations in the atp binding site are sufficient to increase production of proinflammatory cytokines and chemokines, mimicking loss of nlrp12 [80] . the first cleavage site in nlrp12, at residue 238, is located the nacht domain, in between the two walker motifs that mediate atp binding (walker a; residues 217-224 and walker b: residues 288-299, by analogy with nlrp3 [81] ). this cleavage breaks the nucleotide binding site and whether this leads to activation or repression of the protein activity is an open question. of note, this proteolytic cleavage also releases the pyd. pyd of asc, the nlrp3/12 adaptor, has been shown to drive polymerization of asc and formation of asc specks upon inflammasome formation [82] . whether nrlp12 pyd is able to polymerise on its own and induce downstream signalling and pyroptosis remains to be explored. the second cleavage site, at residue 938, releases 3 lrr motifs, and probably modifies protein-protein interactions. most relevant to covid-19, nlrp12 is known to negatively regulate the release of proinflammatory cytokines [83] . mutations in nlrp12 [84] have been linked to autoinflammatory disorders, in particular the familial cold autoinflammatory syndrome 2 (fcas2, omim: 611762). of note, truncation r284x, close to the identified cleavage site has been identified in patient suffering from hereditary periodic fever syndrome and leads in vitro to a dramatic increase in nf-κb activation [85] . up-regulation (and potentially over-activation) of nlrp12 has also been noted in patients with kawasaki disease [86] , a rare auto-inflammation of blood vessels in children. the emergence of kawasaki-like syndromes in children positive for sars-cov 2 [87, 88] could point to a molecular link between nlrp12 cleavage and inflammatory sur-activation. the recognition sequences on irf3 and nlrp12 are unique in their respective families of proteins, so we wanted to investigate how conserved these motifs were. to this end, we compared the protein sequences of irf3 and nlrp12 among species, specifically around these sequences. in both cases, the cleavage sites were located on well conserved portions of the proteins but small differences at or near the cleavage site were identified. figure 6 summarizes the conservation of the irf3 and nlrp12 sequences for 21 species that could be use as infection models. the plpro recognition sequence in irf3 is present in most species, except for rodents and ferrets, where the g at p2 is replaced by k, r or n, which would not be permissive to proteolytic cleavage. we then examined the sequences of nlrp12. the motif around the first cleavage motif is mainly conserved, but we noticed that the amino acid directly after the cleavage site varied significantly. in primates, this amino acid is a small neutral amino acid (g) that is replaced by a bulkier, charged residue d or h in other species. a similar trend is observed for the second cleavage site where the small amino acid a found in human is replaced by larger amino acids (v or i). such substitutions would likely affect the electrostatic environment and most likely inhibit the formation of active enzyme-substrate complexes. interestingly, apart from primates, only cats and tigers have 2/3 similar recognition sequences to humans, for both proteins. it has been reported that, besides primates, cats are amongst the few species can could not only be infected with sars-cov2, but develop covid-19 symptoms [89] . anecdotal evidence suggest that amur tigers (panthera tigris altaica) [90] and european minks (mustela lutreola) [91] could be infect/be infected by humans and develop symptoms. unfortunately, the genome annotation for european minks is incomplete and does not allow for a comparison of its nlrp12 sequence. this comparison once again highlights the difficulty of finding an animal model suitable for the study of sars-cov 2 infectivity and disease [92] . it has been rapidly established that mouse models of covid-19 were ill-adapted as murine ace2 the receptor for sars-cov2, is significantly different to the human isoform [93] . this led to efforts in developing transgenic mice models with humanized ace2 which was successfully used to study infection by sars-cov [94] and sars-cov 2 [95] . according to our analysis however, this model may not fully recapitulate the lack of interferon production if this is driven by irf3 cleavage. we have also shown that mouse nlrp12 is only cleaved once, compared to two cleavage sites for human nlrp12 (fig. 4d and 4e) , although the functional impact of these cleavages remains to be studied. ferrets are also a preferred model of respiratory viral infections and have been shown to be infected by sars-cov 2, but develop only mild symptoms [96] . so far, the most promising disease models amongst primates are rhesus macaques (macaca mulatta) that develop pneumonia [97] , although results of infection in capuchins (sapaju appella) are yet to be published. in non-primates, cats present the most symptoms with massive lung lesions [89] . we then extended our comparison to include wild animals that could have been host reservoirs of the virus, hypothesizing that the viral proteases had evolved to target innate immune proteins of their host. to date, the exact reservoir and intermediate hosts of the virus remain to be found. it is highly probable that the virus originates from a bat coronavirus (e.g. [98] [99] [100] ) and the malayan pangolins (manis javanica) might have acted as intermediate hosts [101, 102] to this end, we compared the irf3 and nlrp12 sequences in different bats, the malayan pangolin and the chinese tree shrew (tupaia chinensis), based on data availability (figure 7) . unfortunately, data for the masked palm civet (paguma larvata), the intermediate host for sars-cov, could not be easily retrieved. as before, the irf3 sequence around the cleavage site was remarkably conserved amongst species. on the contrary, the sequences for nlrp12 are highly divergent, especially at the c-terminus. interestingly, this (limited) analysis identified only one species (myotis davidii) that possess 3 similar cleavage sites compared to human. overall, the method presented here enables medium to high-throughput screen of the activity of viral protein or bacterial effectors and will help design new antiviral and antibiotic strategies. in this study, we presented the results on the first 71 hiips (human innate immune proteins), and the screen will be expanded to cover more potential targets and pathways in the future. our results show that in addition to the de-ubiquitinase activity of nsp3, sars-cov-2 uses its two proteases to further impact the host innate immune signalling. our results were validated in sars-cov-2 infected a293t-ace2 and 293t ace2/tmpssr2 cells, where decrease of irf3, nlrp12 and tab1 was demonstrated by western blotting. our findings of irf3 cleavage are consistent with the literature and the previous work showing an inhibition of interferon beta production in covid-19 patients. more importantly, the direct cleavage of nlrp12 by 3clpro could explain the hyper-inflammation observed in some patients. the fact that the two proteases of sars-cov-2 could have evolved to interfere with innate immunity is attractive. if one considers that most of evolution is driven by lucky side-effects, the additional targets irf3, tab1 and nlrp12 could give a selective advantage if their cleavage would either enhance transmission or delay the response to infection. the discovery that all five cleavage sites are present in a single species of bats gives support to this theory. the fact that myotis davidii can be found near the epicentre of the sars2 pandemic makes it a possible candidate for a previous reservoir host, even if it does not exclude other hosts for sars1 and sars2. further studies will be required to determine if these hosts display positive selection around the cleavage sites, and if zoonosis can be more precisely dated and determined. the authors would like to thank katherina michie, jack bennett and key personnel at the protein production facility of unsw for the purification of plpro and 3clpro of sars-cov-2. the authors would like to thank dr kate schroeder for the gift of the nlrp12 mouse plasmid in a previous collaboration, and prof. alexandrov for the cell-free plasmids compatible with lte protein production. (a) schematic of the organization of the genome of sars-cov-2, focusing on the non-structural proteins nsp1-16. as depicted, two proteases are encoded in orf1a: nsp3 or papain-like protease (plpro) and nsp5, or 3c-like protease (3clpro). plpro is responsible for three proteolytic cleavages, while the protein nsp5 cuts the large polyprotein at eleven different sites. (b) in our assay, the proteins are expressed using an eukaryotic cell-free system as a gfp fusion, and visualized using non-reducing sds-page using fluorescence. the proteases were added during protein expression, and the changes in banding pattern (protein size/integrity) were analysed to detect potential cleavage. here, the results obtained for the family of irf proteins are shown with plpro, and the additional band obtained for irf3 is indicated by a blue arrow (bottom panel, in the presence of nsp3/plpro). (c) overview of the proteins tested in this study and the proteolytic events detected: out of the 71 proteins tested, plpro cleaves only irf3 (indicated in blue) and 3clpro cleaves nlrp12 and tab1 (as shown in red). note that the first g is believed to be crucial for successful cleavage (c) representation of the domains found in irf3 and the position of the cleavage site (d) representation of irf3 structure (from pdb 1j2f). the cleavage sequence lggg is highlighted in blue; the site is presented in a flexible loop and seems fully exposed for cleavage by plpro. (e) alignment of the amino acids for human irf1-irf9, demonstrating that only irf3 would be predicted to be cleaved as observed. (f) structure of the irf3 homodimer (from pdb 1qwt), showing the two fragments (green, n terminal, green, and blue for c-terminal). the cleavage seems to affect the dimeric interface, but this would need to be demonstrated experimentally. the gel shows two additional bands upon cleavage, corresponding to the fragment 132-504 and to the fragment 444 to 504. these two fragments are visible as they carry the gfp tag; the fragments 1-132, 132-444 and 1-444 are not visible in this configuration. (b) same, but for the n-terminal gfp. in this case, the fragments 1-132 and 1-144 are fluorescent and can be detected on the gel, while the fragments 132-444, 444-504 and 132-504 are not fluorescent. (c) schematic representation of tab1 protein structure with the location of the identified cleavage sites on the primary sequence. (d) representation of tab1 structure (from pdb 2pom). the cleavage sequence aslqs is highlighted in red; the site is presented in a flexible loop in an helix-loop-helix motif, and seems fully exposed for cleavage by 3clpro. (e) full sequence of amino acids for human tab1, showing the two putative cleavage sites aslqs and ltlqs. the second cleavage site is not present in existing structures of tab1 but is predicted to be flexible and accessible. note that the removal of c-terminal tail of tab1, is mimicked by a natural isoform. (a): sds-page analysis of the cleavage of nlrp12 protein, with a c-terminal gfp tag. the protein was expressed alone or in the presence of increasing concentrations of the sars-cov-2 protease 3clpro. the gel shows that two separate cleavage sites produce two distinct fragments attached to the c-terminal gfp (b) logo analysis of the cleavage site predicted for 3clpro, from the polyprotein cleavage of sars-cov-2. note that the q residue in position p-1 is believed to be crucial for successful cleavage (c) representation of the domains found in nlrp12 and the position of the cleavage sites.(d) alignment of the amino acids for human nalps (nlrps), demonstrating that only nlrp12 would be predicted to be cleaved as observed. below, alignment of mouse nlrp12, showing that the first cleavage site is conserved, but the second site presents an a→v mutation that would disrupt cleavage. (e) sds-page analysis of human and mouse nlrp12, with different tag orientations, to demonstrate the differences between species. with an n-terminal gfp tag, the human nlrp12 appears as two fragments (1-238 and 1-938), as the fragments 238-938 and 938-1062 would be non-fluorescent. with a c-terminal tag, only the fragments 238-1062 and 938-1062 are fluorescent and are detected on the gel, and the fragments 1-238 and 238-938 are nonfluorescent. the banding patterns obtained in the presence of 3clpro are consistent with the predicted sizes. using the mouse nlrp12 constructs, only one cleaved fragment is observed in the n-term and cterm constructs. here, a myc-mcherry was used and the tag was detected by the red fluorescence of mcherry. in the n-term configuration, a single cleaved product is detected, corresponding to the fragment 1-238. in the c-term configuration, the fragment 238-1062 is detected; this confirms that the lqa→lqv mutation found in mice inhibits cleavage by 3clpro. (f) representation of nlrp12 structure (derived from the structure of nlrp3 from pdb 6npy). (top): the cleavage sequence lfqg (site 1, at residue 238) is highlighted in red; the site is presented in a flexible loop and seems fully exposed for cleavage by 3clpro. (a-c) stable 293t-ace2 and 293t-ace2/tmprss2 cells generated by lentiviral transduction were infected with sars-cov-2 at 0.01 or 0.1 moi. uninfected cells plated and treated identically served as mock-infected controls. 72 hpi, cell lysates were collected, and the indicated proteins were detected by western blot using the relevant antibodies as specified in methods. coxiv serves as the loading control. the relative amounts of irf3, tab1 and nlrp12 was quantified by densitometry and presented as a ratio of coxiv with the ratio obtained from mock infected 293t-ace2 cells set at 1. all primates tested presented both irf3 and nlrp12 cleavage sequences, except for the rhesus monkey and cynomolgus ("crab-eating") monkey where the second nlrp12 cleavage site is mutated or missing. in rodents, most of the typical species used for testing in laboratories (rat, hamsters, and mouse) have a mutation in the most important residue for plpro cleavage. cats, tigers (and most feline, not shown) present both irf3 and nlrp12 cleavage sites and we would predict that both proteins would be cleaved during sars-cov-2 infection. ferrets have variations in these sites, and it is unlikely that the proteins would be affected. cats and tigers can present respiratory symptoms, but dogs are not affected. unfortunately, the genome of the european mink (mustela lutreola) could not be found at this stage. european minks can present the disease and transmit sars-cov-2. horses, pigs and camels possess the irf3 cleavage site but not the nlrp12 sites; on contrary, the rabbit has a mutation in irf3, but we would predict that the second nlrp12 site would be cleaved. both cleavage sites of tab1 are exactly conserved across all these species. (*) the protein sequences for cotton rats (sigmodon hispidus) and minks (neovison vison) were found using tblastn against shotgun genomes, with the query aah7172.1 for irf3 protein [(isoform 1) homo sapiens] and np_653288.1 for nacht, lrr and pyd domains-containing protein 12 [(isoform 2) homo sapiens]. most "exotic" species that would be relevant for sars-cov, mers or sars-cov-2 present the correct cleavage site for irf3. we found that one species of bats, davids' myotis, presents the three cleavage sites in irf3 and nlrp12 (and also the two cleavage sites in tab1). this species of bats is endemic to the province of hubei, where the sars-cov-2 pandemic originated. another small animal found in the same province, the chinese tree shrew, displays at least two cleavage sites for irf3 and nlrp12; the first cleavage site in nlrp12 (klfrg) may potentially be cleaved. the species of pangolin described in china (manis javanica) does not possess the nlrp12 cleavage sites (note that 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nlrp3, nlrp12, and ifi16 inflammasomes induction and caspase-1 activation triggered by virulent hsv-1 strains are associated with severe corneal inflammatory herpetic disease. front immunol beyond the inflammasome: regulatory nod-like receptor modulation of the host immune response following virus exposure nlrp12 regulates anti-viral rig-i activation via interaction with trim25 cutting edge: monarch-1: a pyrin/nucleotide-binding domain/leucine-rich repeat protein that controls classical and nonclassical mhc class i genes structural mechanism for nek7-licensed activation of nlrp3 inflammasome angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor tmprss2 and adam17 cleave ace2 differentially and only proteolysis by tmprss2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein irf and stat transcription factors -from basic biology to roles in infection, protective immunity, and primary immunodeficiencies the molecular basis of viral inhibition of irf-and stat-dependent immune responses. frontiers in immunology enterovirus 71 3c inhibits cytokine expression through cleavage of the tak1/tab1/tab2/tab3 complex 3c protease of enterovirus d68 inhibits cellular defense mediated by interferon regulatory factor 7 viral innate immune evasion and the pathogenesis of emerging rna virus infections. viruses plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production post-translational modifications of the tak1-tab complex tak1, more than just innate immunity. iubmb life pan-optosis). frontiers in cellular and infection microbiology the kinase tak1 can activate the nik-iκb as well as the map kinase cascade in the il-1 signalling pathway irak and tak1 are required for il-18-mediated signaling tab1β (transforming growth factor-β-activated protein kinase 1-binding protein 1β), a novel splicing variant of tab1 that interacts with p38α but not tak1 transforming growth factor-β-activated protein kinase 1-binding protein (tαβ)-1α, but not ταβ1β, mediates cytokine-induced p38 mitogen-activated protein kinase phosphorylation and cell death in insulin-producing cells identification and functional characterization of novel phosphorylation sites in tak1-binding protein (tab) 1. plos one nlr proteins: integral members of innate immunity and mediators of inflammatory diseases the inflammasomes. cell atp binding by monarch-1/nlrp12 is critical for its inhibitory function cryopyrin/nalp3 binds atp/datp, is an atpase, and requires atp binding to mediate inflammatory signaling unified polymerization mechanism for the assembly of asc-dependent inflammasomes the multifaceted nature of nlrp12 nlrp12 in autoimmune diseases mutations in 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covid-19 a genomic perspective on the origin and emergence of sars-cov-2. cell genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a novel bat coronavirus closely related to sars-cov-2 contains natural insertions at the s1/s2 cleavage site of the spike protein probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak isolation of sars-cov-2-related coronavirus from malayan pangolins gateway-compatible vectors for high-throughput protein expression in proand eukaryotic cell-free systems species-independent translational leaders facilitate cell-free expression cell-free gene expression: an expanded repertoire of applications a cell-free approach to accelerate the study of protein-protein interactions in vitro. interface focus leishmania cell-free protein expression system the controls and protease-treated lte reactions were then mixed with lds (bolt lds sample buffer, thermofisher) and loaded onto sds-page gels (4-12% bis-tris plus gels, thermofisher); the proteins were detected by scanning the gel for green (gfp) or red (mcherry) fluorescence using a chemidoc mp system (biorad) and proteolytic cleavage was assessed from the changes in banding patterns, as shown in figure 1b . note that in this protocol, the proteins are not treated at high temperature with the lds and not fully denatured, to avoid destruction of the gfp/mcherry fluorescence. as proteins would retain some folding, the apparent migration on the sds-page gels may differ slightly from the expected migration calculated from their molecular weight. we have calibrated our sds-page gels and ladders using a range of proteins, as shown in supplementary information. leishmania tarentolae extracts were prepared in house using the protocol described previously [104, 107] briefly, leishmania tarentolae parrot strain was obtained as lexsy host p10 from jena bioscience gmbh, jena, germany and cultured in tbgg medium containing 0.2% v/v penicillin/streptomycin (life technologies) and 0.05% w/v hemin (mp biomedical). cells were harvested by centrifugation at 2500 x g, washed twice by resuspension in 45mm hepes, ph 7.6, containing 250mm sucrose, 100mm potassium acetate and 3mm magnesium acetate and resuspended to 0.25g cells/g suspension. cells were placed in a cell disruption vessel (parr instruments, usa) and incubated under 7000 kpa nitrogen for 45 minutes, then lysed by rapid release of pressure. the lysate was clarified by sequential centrifugation at 10 000 x g and 30 000 x g and anti-splice leader dna leader oligonucleotide was added to 10μm. the lysate was then desalted into 45mm hepes, ph 7.6, containing, 100mm potassium acetate and 3mm magnesium acetate, supplemented with a coupled translation/transcription feeding solution and snap-frozen until required. we verified that the expression patterns and cleavage of the proteins in this study was independent of the batch of lte used. cells were lysed with ripa lysis buffer (pierce) containing a cocktail of protease inhibitors (cell signaling). equivalent amounts of proteins determined by the bradford protein assay (bio-rad) were separated by sds-page and transferred through the trans-blot turbo transfer system (bio-rad). to avoid the nonspecific antibody reaction, the membranes were blocked with intercept blocking buffer (li-cor) prior to the addition of primary antibodies. after primary antibodies incubation, the blots were then treated with alexa fluor 647 conjugated secondary antibodies (invitrogen) and developed signals using the chemidoc mp image system (bio-rad). the following commercial antibodies were used: anti-irf3 (ab68481) and anti-ace2 (ab108252) from abcam, anti-nlrp12 (pa5-21027) from invitrogen, anti-tab1 (#3226) from cell signaling, anti-sars-cov-2 n (gtx632269) from genetex, anti-tmprss2 (sc-515727) from santa cruz biotechnology, anti-coxiv (11242-1-ap) from proteintech. sars-cov-2 proteases cleave irf3 and critical modulators of inflammatory pathways (nlrp12 and tab1): implications for disease presentation across species and the search for reservoir hosts. purification of plpro (nsp3) and 3clpro (nsp5):e. coli c41(de3) cells were transformed with the sars-cov-2 pet-28a-nsp5 plasmid (idt dna). single colonies were used to inoculate lb media supplemented with kanamycin (50 µg/ml). 500 ml cultures were grown at 37°c until an od600 of 0.6 was reached, cooled to 28°c and induced for 4 hours with 1 mm iptg. following growth, the cells were pelleted by centrifugation (5000 x g, 20 min, 4°c) and washed with 1x pbs before storage at -80°c. frozen cell pellets were resuspended in buffer a (20 mm sodium phosphate buffer ph 8, 500 mm nacl, 10 mm imidazole) and lysed on ice via sonication using a branson sfx250 sonifier (8 minutes at 50% amplitude, 2 s pulse on, 2 s pulse off). cell debris was removed from the lysate by centrifugation (10,000 x g, 30 min, 4°c) and subsequent filtration of the supernatant through a 0.22 µm syringe filter. 20 ml of the clarified lysate was loaded onto a 1 ml hitrap imac sepharose ff column (ge healthcare, illinois) charged with ni2+ and preequilibrated with buffer a. unbound proteins were removed from the column through washing with 10 column volumes (cv) of buffer a. bound proteins were eluted with a stepwise gradient of buffer b (20 mm sodium phosphate buffer ph 8, 500 mm nacl, 500 mm imidazole) as follows: 0-5% b, 2 cv; 5% b, 5 cv hold; 5-25% b, 2 cv; 25%b, 5 cv hold; 25-100% b, 2 cv; 100% b, 5 cv hold. fractions containing nsp5 were exchanged and concentrated into buffer c (20 mm tris, ph 7.4, 10% glycerol), flash frozen and then stored at −80°c. all purification steps were performed on ice or at 4°c. protein concentrations were determined using a linearized bradford protein assay. the 71 human innate immune proteins (hiips) listed in figure 1c were cloned as gfp or mcherry fusions into dedicated gateway vectors for cell-free expression. open reading frames (orfs) were sourced from the human orfeome collections, versions 1.1, 5.1 and 8.1 and transferred into gateway destination vectors that include nterminal or c-terminal fluorescent proteins [103] . most proteins screened were expressed as n-terminal enhanced gfp fusions (vector pcellfree g03); for tab1 and nlrp12, c-terminal gfp were also used to validate the cleavage sites. the specific gateway vectors were created by the laboratory of pr. alexandrov and sourced from addgene (addgene plasmid # 67137; http://n2t.net/addgene:67137; rrid:addgene_67137). mouse nlrp12 constructs were sourced from the laboratory of dr kate schroeder (imb, university of queensland).the hiips were expressed in vitro using a cell-free expression system derived from leishmania tarentolae [104, 105] . this eukaryotic system enables expression of full-length proteins with minimal truncations and non-specific aggregation, for proteins up to 150kda in size []. this system has been used recently to study the folding and oligomerisation of nlrp3 proteins and the polymerization of asc [ref jmb] or the formation of higher-order assemblies of myd88 [ref bostjan ref bmc] . the expression is simply set-up as a one-pot reaction where the plasmid encoding the protein of interest is added to the leishmania tarentolae extracts (lte); expression occurs within 3h at 27℃ and expression yields can be evaluated by the fluorescence intensity of the gfp/mcherry tags [106] . the 71 hiips proteins were expressed individually in 10 µl reactions (1µl dna plasmid at concentrations ranging from 400ng/µl to 2000ng/µl added to 9 µl of lte reagent). the mixture was incubated for 30 minutes at 27℃ to allow the efficient conversion of dna into rna. the samples were then split into controls and protease-containing reactions. the proteases plpro (nsp3) and 3clpro (nsp5) were added at various concentrations, and the reactions were allowed to proceed for another 2.5h at 27℃ before analysis. the cell-free expression system was loaded onto the sds-page gels following the classic protocol and stained by coomassie to reveal the proteins present in the lte system. the sars-cov-2 protease plpro (nsp3) was added at concentration ranging from 60 µm to 1µm (same as in figure 2a ). the gel shows on the right that the banding pattern of lte is not affected by plpro. (left): the density of bands across the control (lte without plpro added) was compared to the one obtained when 60µm of purified plpro was added in the expression system. no significant differences were noted, confirming that plpro doesn't have a non-specific cleavage activity on the proteins of lte. also, the intensity of the gfp-tagged protein bands on the gels did not vary significantly when plpro or 3clpro were added, suggesting that the expression levels were unaffected, and that the components of the cell-free systems essential for expression were not cleaved. with different molecular weights, ranging from 15 to 140 kda; the hiips were expressed in lte, mixed with lds after 3h of expression, and separated on the sds-page gel. in this gel, two different ladders were used to calibrate migration of 12 proteins of interest. the page ruler prestained ladder has been developed specifically for the 4-12% bis-tris gels (thermofisher, black •) and seems more accurate to predict sizes of gfp-tagged proteins (in white diamonds ◊). the trendline indicated on the graph (right) will be used to estimate the size of the gfp-tagged fragments of tab1 and nlrp12. probably due to the non-denaturation of the protein tab1 in our lds loading protocol, the full-length protein human tab1 migrates slower than expected on the sds-page gel (estimated size 64 kda from the calibration, vs 54.4 kda expected). nevertheless, the variations in size due to 3clpro cleavage are consistent with the two sites at position 132 and 444. on the gel, the fragments are indicated, and the migration was analysed on the master curve to estimate the size of the gfp-tagged fragments. the 27kda of the gfp were taken into account in the table (right) to compare the predicted and observed sizes of the proteins and protein fragments. the full-length human nlrp12 proteins, tagged in n-term and c-term with gfp, were mixed with 3clpro and analysed by sds-page gels. the five bands obtained for the full-length nlrp12 and the four fluorescent cleavage products were analysed on the predictive size/migration plot. the variations in size are perfectly consistent with the two cleavages sites at position 238 and 938. on the sds-page gel on the left, the fragments are indicated, and the migration was analysed on the master curve to estimate the size of the gfp-tagged fragments. the sizes obtained (after removing the 27kda contribution of the gfp) were compared to the predicted sizes of the proteins and protein fragments. key: cord-275307-d7htyfcl authors: gaglia, marta maria; rycroft, chris h.; glaunsinger, britt a. title: transcriptome-wide cleavage site mapping on cellular mrnas reveals features underlying sequence-specific cleavage by the viral ribonuclease sox date: 2015-12-08 journal: plos pathog doi: 10.1371/journal.ppat.1005305 sha: doc_id: 275307 cord_uid: d7htyfcl many viruses express factors that reduce host gene expression through widespread degradation of cellular mrna. an example of this class of proteins is the mrna-targeting endoribonuclease sox from the gamma-herpesvirus kaposi’s sarcoma-associated herpesvirus (kshv). previous studies indicated that cleavage of messenger rnas (mrna) by sox occurs at specific locations defined by the sequence of the target rna, which is at odds with the down-regulation of a large portion of cellular transcripts. in this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify sox cleavage sites across the mrna transcriptome. these data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. this degenerate element is well represented in both human and kshv mrna, and its presence correlates with rna destabilization by sox. this represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables rna cleavage at specific locations without restricting the range of targets. furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity. triggering wide-spread rna degradation is a common strategy that viruses use to decrease host gene expression, also known as host shutoff [1, 2] . viral factors from many different families including herpesviruses, coronaviruses and orthomyxoviruses either directly cut rnas or indirectly stimulate rna cleavages in an endonucleolytic fashion [3, 4] . cellular rna exonucleases are then recruited to degrade the fragments, resulting in a reduction in rna and consequently protein levels [3] . despite the fact that the proposed role of most of these host shutoff ribonucleases (rnases) is to modulate immune responses, they are generally thought to have little or no specificity and to affect host messenger rnas (mrnas) indiscriminately. however, increasing evidence suggests that this view may be overly simplistic, and that some of the rnases display selectivity for or against specific targets [5] [6] [7] [8] [9] [10] [11] [12] . this type of specificity may provide an additional level of regulation in viral control of the host transcriptome. how this selectivity is achieved and how it is balanced with the widespread shutoff phenotype remain open questions. the sox family of proteins from gamma-herpesviruses is an example of a viral rnase that displays both broad targeting of rnas and a poorly understood level of selectivity. gammaherpesviruses include the human pathogens kaposi's sarcoma-associated herpesvirus (kshv), which causes kaposi's sarcoma as well as lymphomas in immunocompromised individuals and remains a leading cause of cancer-linked death in sub-saharan africa. the sox (orf37) protein is expressed early during the lytic cycle of kshv infection and its expression triggers rna degradation, which is recapitulated by expression of the protein alone [13] . homologs of sox in the other human gamma-herpesvirus, epstein barr virus (ebv bglf5), and in the model murine pathogen murine herpesvirus 68 (mhv68 musox) also degrade rna in cells [14, 15] . studies in mhv68 suggest that host shutoff by the sox family of proteins is crucial for viral replication in specific cell types and for systemic spread of the virus and establishment of a latent infection [16] . transcriptomic studies of mrna levels during kshv or mhv68 infection and in cells overexpressing sox demonstrate that this family of proteins triggers the degradation of a majority of both host and viral transcripts [6, 7, 17] . however, in-depth mechanistic studies of sox reveal a more complex picture. sox targets mrnas, as opposed to non-coding rna species, a specificity that is related to the association of sox with polyribosomes [5] . moreover, selected transcripts, like the cytokine interleukin 6 (il-6) [6] and apoptosis enhancing nuclease (aen) [7] , are spared from sox-mediated decay. in the case of il-6, protection is conferred by the presence of a protective sequence in the 3' untranslated region (utr) [8] , but aen appears to be intrinsically resistant to sox mediated degradation [7] , without a clear protective element in its sequence. the most unexpected observation, however, is that kshv sox and ebv bglf5 cut rnas at specific locations that appear to be determined by an unknown targeting element [3, 5] . these specific cleavages become apparent upon knockdown of the human rnase xrn1, the major 5'-3'-directed rnase in eukaryotic cells, which is responsible for clearing the 3' rna fragments generated by these rnases [5] . this ability of sox to cut at specific locations within mrna yet target the majority of transcripts argues for a degenerate targeting motif. in general, the principles guiding the positioning of rna cleavages by cellular and viral mrna endonucleases are not well understood. in the case of endonucleases, the term "sequence specificity" is sometimes used to refer to preferential cutting at specific dimers, often inferred from in vitro studies (for example in datta et al. [18] ). however, this specificity cannot explain cutting of rnas at single locations in mrnas. additional specificity can be conferred by localization of the target mrna to a specific site in the cells [19] or proximity to a "landmark" feature on the rna, such as the 5' of the transcript [20, 21] or the stop codon location [22] . the sox targeting system is unprecedented because the sequence of the targeting element alone appears to direct cleavages in mrna, and because the targeting element is longer than 25 nt [5] . to address how sox specificity is mediated, we applied a degradome sequencing technique called parallel analysis of rna ends (pare) [23] to map cleavage sites of the sox protein across the human transcriptome. development of a stringent python-based analysis algorithm, which we term pydegradome, allowed identification of sox-dependent cuts at specific locations across the mrna transcriptome. the sequences surrounding these sites contained no strong consensus sequence, but rather a degenerate sequence pattern that nonetheless conferred specificity when analyzed experimentally in endogenous mrna targets. the presence of a more complex targeting motif explains how sox achieves cleavage specificity without sacrificing target breadth, and offers a framework for understanding how additional viral and cellular endonucleases may operate. development of a novel bioinformatics pipeline to detect highconfidence sox cleavage sites across the transcriptome following pare prior analyses of individual mrnas indicated that the kshv rnase sox cuts at specific locations within the rna, in a manner dependent on the sequence surrounding the cleavage site [5] . by performing 5' rapid amplification of cdna ends (5' race) on the gfp reporter mrna, we found that the gfp mrna was cleaved in the same location regardless of whether sox was transiently expressed in 293t cells or expressed from the kshv genome in lyticallyreactivated islk.219 cells (s1 fig) . this is in agreement with the fact that sox activity does not rely on any additional viral proteins, and that its cleavage activity can be studied in transfected cells [5, 7, 13] . to dissect the specificity of sox cleavage across the mrna transcriptome, we designed an approach to map sox cleavage sites in endogenous mrnas using pare [23, 24] . pare is an rna-seq based methodology that allows mapping of the 5' ends of uncapped, phosphorylated rna species, such as mrna degradation fragments ( fig 1a) . as we previously found that the sox degradation intermediates are cleared by the host 5'-3' exonuclease xrn1 [5] , we stabilized degradation intermediates in 293t cells expressing a gfp-sox fusion [7] by knocking down xrn1 (s2a fig) . cells expressing gfp alone were used as controls to filter out rna fragments generated by cellular rnases or other processing events. this was important because multiple studies have shown that pare and similar techniques detect a large number of rna fragments in human cells, many of which are of unknown origin [25] [26] [27] . we prepared and sequenced pare experimental procedure and peak finding analysis pipeline. a) diagram of the pare procedure. b) schematic of pydegradome analysis approach, which uses read counts in a control sample to generate a table of thresholds to compare test sample counts to. the table lists thresholds for a particular user-defined confidence level and for a range of ratios between control and test samples. the applicable ratio for each position is computed by multiplying a user-defined multiplicative factor by the ratio of total read counts for the exon in test vs. control samples, thus accounting for variation in rna levels and total mapped reads. read counts in test sample at each position must exceed the threshold to be identified as part of a peak. c) example of plot of read counts (5' end only) for 200 nt surrounding a sox cut site identified by pydegradome within the 3'-most exon of the limd1 nm_014240 transcript in the four samples. note that y-axis has a logarithmic scale. this example shows the expected distribution for a cut site followed by exonucleolytic degradation due pare libraries from two replicates of sox-expressing or gfp control cells and extracted the 5' end of each mapped read, which represents the cleavage site (s1 table) . conventional analysis of pare and similar degradome datasets relies on detecting cut sites in each condition and then comparing conditions to each other a posteriori, but initial attempts at detecting and validating cut sites indicated that this approach did not provide sufficient discriminatory power to identify sox-specific cleavage sites. in previous studies using pare or similar approaches [26, 27] , additional information about the pathways, such as the mirna sequences for mirna cut site studies or the proximity of the site to a stop codon for studies of smg6 and nonsense mediated decay, was used to further select "true" cut sites. however, such contextual information does not exist for sox cleavage specificity. therefore, we devised a python-based analysis approach that would directly use our control dataset as a baseline, which we termed pydegradome ( fig 1b) . the analysis uses a bayesian probability framework to determine whether the read counts at a given location differ significantly between control and test samples, taking into account random variations in the number of reads. using bayes' theorem, we determine for each location whether the underlying rate of fragment production in the test sample is a multiplicative factor larger than the control rate at a user-defined confidence level. the user also chooses the multiplicative factor. for a given control read count, we thus compute a threshold that the read counts in the test samples have to exceed to be considered part of a "peak" (fig 1b) . to improve efficiency when testing thousands of locations, the software builds a reference table of the thresholds for the entire dataset. this approach allowed the identification of locations within the transcriptome where the read counts were statistically higher in the samples from sox-expressing cells than in control samples (peaks). in order to correct for up-or down-regulation of the rnas and for the total number of reads obtained for each sample, the ratio used to determine thresholds was computed from the user-defined multiplicative factor and the ratio of the total number of reads mapping to each exon in test vs. control samples (fig 1b) . to prevent isolated high read counts from skewing our analysis, we integrated read counts within small windows (4 nt) rather than single nucleotides. adjacent windows that passed the cutoff were then combined into a single peak. we optimized the userdefined confidence level and ratio by determining how many peaks were detected when comparing each sox replicate sample to its gfp control to detect sox-specific peaks, or performing the opposite comparison to detect gfp-specific peaks. in addition, we also ran the program to detect peaks specific to only one biological repeat, by comparing the two sox or gfp replicates to each other, as these peaks may represent experimental noise. although we consistently detected more sox-specific peaks than gfp-specific peaks, varying the parameters improved discrimination of sox-specific peaks (s2b fig) and reduced detection of "noise" peaks specific to one repeat. based on this optimization, we empirically set the final iteration of the program to detect 4 nt windows with read counts in the test samples that are four fold higher than read counts in the control samples within a confidence level of 99.99%. because these parameters are conservative, we expect that the sox cut sites we detected do not represent a comprehensive list of all sox cut sites, but rather only the highest confidence sites. within each peak, we also determined the position where the read count was highest, and we considered this position the location of putative cut site (with the cleavage occurring 5' of this position) (fig 1b and 1c ). with similar optimization, this program could be used to identify the ends of degradation fragments in other degradome datasets that contain matching test and control samples. sox cuts sites are abundant and not positioned relative to landmark features of mrna using the approach detailed above, we detected a higher number of peaks specific to sox-containing samples relative to control samples, consistent with broad mrna targeting by sox (fig 2a; s2 table) . even when varying the allowed distance between peaks from 0-15 nt in sample replicates, the sox samples contained~3-5 times the number of reproducible ("shared") sox-specific peaks (s2c fig). up to 20% of the sox-specific peaks but fewer than 9% of the control gfp-specific peaks were shared between the replicates, indicating that many of the sox cleavages reproducibly occur at a given site (fig 2b) . the read counts at the putative cut site in the two sox replicates were highly correlated ( fig 2c, spearman's ρ = 0.892, p value < 0.0001), further demonstrating that these peaks correspond to specific sox-mediated cleavages. for downstream analyses, we focused on cut sites that were detected in both the replicates using the 99.99% confidence level and were 0-5 nt apart (s3 table) . example plots of the read counts around identified cut sites are shown in figs 1c and s3a-s3f. several virally encoded host shutoff factors that trigger rna degradation, including herpes simplex virus 1 vhs and sars coronavirus nsp1, are thought to induce sequence-independent cuts near the 5' end of the message [20, 21] . to examine whether sox cleavage sites in endogenous mrnas are position-specific, we compared the location of the sox-specific cut sites to those found only in control samples using the human transcript annotation from ensembl grch37. in both sox and gfp control samples, more cut sites occur towards the ends of the transcripts, most frequently corresponding to the 5' and 3' untranslated regions (utrs) of the mrna (fig 2d and 2e ). it remains unclear whether this non-specific end bias is due to a general preference for cleavage in non-translated regions or a consequence of the pare approach. we also computed the position of the cuts relative to landmarks such as the transcript start site, start codon, stop codon or annotated 3' end. only a fraction of the peaks was located within 50 nt of any of these landmarks in either case (fig 2f) . although a greater percentage of the sox cut sites occurred within 50 nt of start codons, this still only accounted for 15% of the cut sites. furthermore, the 15-20% of both sox and gfp peaks near annotated transcription start sites may represent the beginning of full-length decapped mrnas rather than endonuclease cleavage fragments. collectively, these analyses indicate that sox cut sites are not restricted to a particular region of the mrna, although cleavage sites in both sox and control gfp samples may be enriched in areas of the transcripts that are not covered by ribosomes. these findings are consistent with our previous reporter mrna data [5] . we next selected seven sox cut sites for independent experimental validation ( fig 3a) . the selection was based on three criteria: 1) position more than 50 nt from the annotated 5' end of the transcript in order to eliminate potential transcription start sites, 2) high number of mapped reads, and 3) clear sox-specific peaks in a visual inspections of the read plots (figs 1c and s3a-s3f). we used two approaches to validate the cut sites, targeted 5' race and insertion in reporter constructs, and found that all sites validated in at least one of the two assays. we detected a 5' race fragment that appeared specifically in sox-expressing cells (s3g fig) and whose size corresponded to the predicted sox cleavage location for four of the transcripts. (we were unable to detect the rnas for bloc1s4 and srsf3 using control primers). our second validation approach was designed to test the hypothesis that specific rna sequences or structures flanking endogenous cut sites direct cleavage by sox even when removed from their native context, as we had seen for reporter mrnas [5] . we inserted 200 nt pare analysis identifies sox-specific cut sites in endogenous rnas. a) number of peaks/cut sites identified specifically in sox or gfp samples. b) fraction of the peaks identified in sox or gfp samples that were detected in both biological replicates ("shared peaks"), relative to the maximum distance allowed between the peaks. c) correlation plot of the heights of peaks found in both sox+ samples. peak height is defined as the highest read count within the peak window, at the position defined as the putative cut site. d) position of the cut sites found in both replicates ("shared cut sites") within the mrnas relative to the length of the transcript. e) position of the shared cut sites relative to the coding region of the mrna (in all samples > 90% of surrounding the cleavage sites from the mrna targets identified by pydegradome into a gfp reporter ( fig 3b) . we then co-expressed these constructs with sox in xrn1-depleted cells and tested whether the inserted sequence caused a sox-specific cut in the mrna. the gfp reporter we used is normally cut by sox at~nt 140 of the coding region [5] , generating a degradation intermediate that is~250 nt shorter than the full length mrna. we found that the insertion of the sequences from six out of seven of the candidate sox cleavage sites resulted in the appearance of a second rna fragment in sox-expressing cells (fig 3c and 3d ). interestingly, the intensity of the additional cleavage products varied between the insertions, suggesting some sequences are better sox targets than others. in particular, in the gfp reporters with insertions from the limd1 mrna (fig 3d) , we found that the original cleavage site in the gfp coding region was almost completely abolished in favor of the new cleavage site, as evidenced by the disappearance of the longer degradation intermediate. moreover, insertion of the limd1 200 nt sequence in a different position in the gfp mrna also elicited sox cleavage ( fig 3e) , further demonstrating functionality of the targeting sequence regardless of its broader context. taken together, these data indicate that we have identified bona fide sox cleavage sites in endogenous mrnas, and that these sites contain specific elements that lead to sox targeting. the sox cleavage site likely occurs in an unstructured region of the mrna and is characterized by an a-rich sequence just upstream of the cleavage to identify features that define a sox cleavage site, we searched the sequences surrounding the sox cut sites detected in both biological repeats for structural or sequence similarities, using the cut sites shared by the two gfp samples as a comparison set. first, localfold [28] was used to determine the likelihood that the nucleotides around the cut are located in unpaired regions (i.e. accessibility). this program is a variation on the vienna algorithm rnafold and is based on the assumption that potential structures are formed locally, which is consistent with the success of our insertion experiments. we found that the nucleotides 5' of the sox cut were more accessible (thus presumably unstructured) than surrounding sequences ( fig 4a) . this pattern was different from the sequences surrounding the gfp sites, suggesting that it is feature specific to sox cleavage sequences. we also computed the log likelihood for different nucleotides at positions around the sox cleavage ( fig 4b and 4c ). although no strong consensus sequence emerged, two features stood out from this analysis. first, the position right after the cut site (position 0) was preferentially c or t. when we computed the frequency of different nucleotides at the cut site for both soxspecific and gfp-specific cleavages, we found that the pyrimidines c or t were found at 85% of the sox cut sites, whereas c or a were the most frequent bases at position 0 in cuts specific to gfp control samples ( fig 4d) . this distribution is not due to a bias in library preparation, as a was the most frequent base at the beginning of both aligned (s4a we also found that there were more as and fewer cs in the 10 nt 5' to the cleavage site. we computed the fraction of putative sox cut sites that had a dimers or trimers in the 6 nt preceding the cut and found that a stretches were found before~60% of our mapped cut sites (fig the peaks fall within coding transcripts). nd = not determined, because multiple transcript isoforms are present in the annotation and the cut site position would differ between isoforms. na = no coding sequence annotated. f) position of shared cut sites relative to annotated landmarks on transcripts. for all panels in this figure, a scanning window of 4 nt, a multiplicative factor of 4, a confidence level of 99.99%, were used to predict cut sites. all cuts sites 5 nt apart in the two replicates were used for the analyses in panel c-f (sox-specific peaks: n = 456, gfp-specific peaks: n = 84). doi:10.1371/journal.ppat.1005305.g002 . the gfp-based reporters were then co-expressed with sox ("+" or "+sox") or an empty vector control ("-") in shxrn1-treated 4e). this was not a general feature of the sequences that produce rna fragments, as only five of the cut sites found solely in the control samples were preceded by an a dimer and none by longer a stretches. these analyses suggest that although sox cut sites are defined by a degenerate sequence pattern, this sequence is enriched for pyrimidines at a cut site adjacent to an unstructured stretch of a residues. experimental analysis of an endogenous sox cleavage sequence confirms role of oligo a sequence and potential structural element to probe the sox targeting element further we examined more in detail the sox targeting element in the validated endogenous mrnas (fig 3a) . the structure prediction program rnafold [29] predicted that the 50-54 nt surrounding the cut sites in six out of seven of the rnas fold in hairpin structures with oligo-a stretches and the cleavage sites in unpaired loops (figs 5a and s5a). the only exception was mapk8ip3, which also lacked the oligo-a sequence. these structure predictions mirror the accessibility results from localfold analysis (fig 4a) , which indicated that the positions from -10 to 0 relative to sox cut sites are more likely to be unpaired. similarly, we predicted the structures of all 50 nt sequences surrounding the soxspecific cut sites in our dataset and determined how many of the sequences presented either the cut site, an a dimer or both the cut site and an a dimer in an unstructured region. for 90% of the sox cut sites, at least one of these two features was predicted to be in an unpaired region, with over 30% of the sequences predicted to have both ( fig 5b) . we reasoned that if these structural features are important for cleavage, the efficiency of the cleavage could vary depending on the length of the inserted endogenous sequence. fragments of different sizes may not be able to fold into the native structure equally well and the stability of the resulting structures may vary. consistent with this idea, we found that changing the length of the inserted fragments for two different rnas (limd1 and srsf3) changed the efficiency of sox cleavage, measured by the intensity of the degradation intermediate (figs 5c, 5d, s5b and s5c), although sequences of 33 nt were sufficient to elicit sox cleavage. in particular, when we shortened the limd1 inserted sequence from 200 nt to 54 nt, 39 nt and 33 nt, the efficiency of cleavage progressively diminished (fig 5c and 5d ), consistent with limd1 sequences adopting a stem-loop structure that becomes destabilized upon sequential deletions of the putative stem region. surprisingly, shortening the inserted sequence for srsf3 had the opposite effect and increased the efficiency of the cleavage (s5b and s5c fig). because the same sequences are present in the 200 nt and the 33 nt srsf3 insertion, this observation cannot be explained by the presence of a targeting sequence alone. instead, we hypothesize that the shorter insertion folds more stably into an autonomous element that is required for targeting by sox. we previously found that mutating a tgaagt sequence 5 nt before the gfp cut site to tgagtg could abolish the cleavage site in gfp (s6a fig). the limd1 site is also preceded by a similar tgaaag sequence predicted to be in an unpaired loop. to test directly whether the a trimer in the limd1 sequence was required for the positioning of the cleavage, we mutated the tgaaag sequence in our insertion reporter to tgcccg, tggggg or tgtttg. as predicted by our data analysis, we found that mutation of the a trimer prevented the limd1 sequence from eliciting sox cleavage, indicating that the aaa is an integral part of the sox recognition site (fig 5e) . moreover, deletion of one of the three as in the 39 nt insertion cells and the gfp mrna from these cells was detected using northern blotting. the arrowheads point to the additional cleavage fragments resulting from the insertions. images are representative of results from at least three experimental replicates. [42] representation of the frequency of each base in the 20 nt surrounding the cut sites found in both sox samples (n = 267). the position of the cut site is reduced the efficiency of cleavage dramatically (fig 5c and 5d) , while rnafold structure prediction suggested that this deletion is unlikely to substantially alter the structure of the rna (s6b fig). similarly, mutating an a dimer just upstream of the cut site in a srsf3 insertion construct reduces sox-mediated cleavage (fig 5g) . while the upstream a dimer likely contributes to sox targeting, we note that it is not always required, as a similar mutation in a pgam1 insertion construct did not abolish cleavage (s6c fig). overall, these data are consistent with the idea that the a-stretch is an important feature of the sox cleavage specificity. lastly, our analysis indicated that the nucleotide g is under-represented at the position immediately following the sox cut site (position 0, fig 4b-4d ). we found that mutating the nucleotide at position 0 from an a to a g prevented sox-mediated cleavage in two out of three of the rna we tested (limd1 and srsf3, but not the pgam1) (figs 5f, 5g and s6d). these data suggest that sox activity is inhibited by the presence of a g nucleotide as the residue 3' of the cleavage. collectively these data experimentally validate our computational finding and strongly suggest that sox cut sites are defined by a combination of sequence and structural features. a conserved sequence pattern is specific to sox-dependent cut sites although sequences flanking the sox cleavage sites lacked a strong consensus motif, our analysis showed that the frequency of the bases around the cut site diverged from the expected distribution for human rna sequences (fig 4b and 4c ). this suggested to us that there is a conserved sequence pattern among sox target sequences. in order to be able to search rna sequences for this variable motif, we derived a position weight matrix (pwm) for the positions -10 to +9 from the sox cleavage site (0 being the nucleotide 3' of the cut site, fig 4c) . the pwm is a matrix that lists the probabilities (transformed into log likelihood to correct for the background frequencies of the nucleotides) for each of the four bases at each of the positions of the putative motif. the log likelihoods for our pwm were derived from 129 very high confidence sox cut sites identified in both of our experimental repeats using a very stringent confidence level of 99.999% and located at least 50 nt away from a transcriptional start site. the logo in s7a fig is a pictorial representation of the pwm. we then used the pwm to confirm that sox cut sites specifically matched the motif by scoring several subsets of potential sox target or control sequences using the pwm. the presence of preferred nucleotides in positions -10 to +9 from the cut site results in higher (positive) log likelihood scores, whereas a poorer match to the motif produces a lower (negative) log likelihood score. indeed, the sequences flanking the set of reproducible sox cut sites (identified with a confidence level of 99.99%) were a closer match to the motif compared to those surrounding gfp-specific fragment ends, as shown by the distribution of the log likelihood scores (fig 6a) . a similar difference was seen when analyzing the sequences surrounding all potential sox and control (gfp) cut sites from each of the two biological repeats. sequences around sox cut sites were a good match (positive log likelihood score) to the motif more frequently than control sequences (fig 6b) . in both analyses (fig 6a and 6b) , we removed the sequences of the 129 high confidence cut sites used to derive the pwm from the analyzed sets. the results of these analyses indicate that although the indicated. d) percentage of sequences with each of the four nucleotides at the cut site (position 0) among the gfp or sox specific peaks. e) percentage of sequences surrounding putative cut sites that contain at least one oligo-a stretch within the 6 nt upstream of the cut (for d and e, sox: n = 267, gfp: n = 53). for all panels in this figure, the shared cut sites were determined based on a scanning window of 4 nt, a multiplicative factor of 4 and a confidence level of 99.99%. all cut sites with the same exact position in both sox or both gfp samples that were >50 nt away from an annotated transcription start site and had sufficient surrounding sequences within the same annotated exon (300 nt in a, 50 nt in b and 20 nt in c and d) were included in these analyses. the varying number of sequences used for the analyses in the different panels is a result of the requirement for sufficient flanking sequences in the same annotated exon, but as many sequences as possible were analyzed in each case. doi:10.1371/journal.ppat.1005305.g004 fig 5. the sequence features at the sox cut site in limd1 and srsf3, as well as a structural element around the site, are required for sox cleavage. a) predicted structure of the 54 nt surrounding the sox cut site in limd1, highlighting the a trimer (asterisk) and the cut site (arrow), as well as the ends of the 39 nt, and 33 nt insertions used in d. b) rnafold was used to predict the structure of all 50-nt sequences surrounding sox-specific and gfpspecific cut sites (based on a scanning window of 4 nt, a multiplicative factor of 4 and a confidence level of 99.99%). the proportion of cut site where the location of the cut was predicted to be unpaired, that had an unpaired a dimer within 5 nt of the cut or both is plotted. c-g) gfp reporters were co-expressed with sox ("+") or an empty vector control ("-") in shxrn1-treated cells. the gfp mrna was detected using northern blotting. the empty arrowheads point to the additional cleavage fragment resulting from insertions, whereas the filled arrowheads point to the normal gfp cleavage fragment. c-d) 54-33 nt surrounding the sox cleavage site in the limd1 mrna were inserted into the gfp reporter at nt 715. in the 39 nt δa construct, one of the three as found at positions -7 to -5 from the cut site was deleted. a representative blot is shown (c), as well as the quantified intensity of the signal from the different rna species (d), plotted relative to the intensity of the bands from the 54 nt insertion construct. error bars = standard deviation, ** p < 0.001, *p < 0.05 (student's t-test). e) the a trimer preceding the sox cut site in limd1 was mutated to a c, g or t trimer in the limd1 100 nt insertion construct. images are representative of results from at least three experimental replicates. f) the a immediately 3' of the sox cut site in limd1 was mutated to a g in the 100 nt insertion construct. g) the a immediately 3' of the sox cut site in srsf3 was mutated to a g (a ! g) and the a dimer preceding the sox cut site was mutated to a c dimer (aa ! cc) in the 33 nt insertion construct. fig 6. a degenerate motif defines sox cut sites. a position weight matrix (pwm) for nucleotide likelihood in the 20 nt surrounding the sox cut sites was generated from the 129 sequences that contained sox-specific sites with a confidence level of 99.999%. sequences were scored using this matrix, after removing the 129 "parent" sequences where applicable. a) frequency distribution histogram of log likelihood scores for the 20 nt surrounding the gfp-or sox-specific cut sites. gfp: n = 61; sox: n = 178. b) frequency distribution histogram of log likelihood scores for the 20 nt surrounding all cut sites found in the two gfp and sox samples. gfp rep1: n = 693; gfp rep2: n = 409; sox rep1: n = 849; sox rep2: n = 1160. c) all human and kshv annotated precise sequence composition may vary, there is a specific element marking sox cut sites that is not observed in control samples. furthermore, our stringent analysis parameters have likely resulted in an underestimation of the number of true sox targeting sites, because even sites detected in only one of our two replicates were generally a good match to the sox targeting motif. we next used the pwm to test whether the prevalence of the targeting element differed between the human and viral mrna transcriptomes. we analyzed annotated transcripts with a 20 nt scanning window, computed a log likelihood score for every possible 20 nt sequence, determined the highest possible motif score for each transcript and plotted the distribution of the scores. in agreement with the widespread mrna cleavage by sox, most of the annotated human transcripts have at least one sequence that is a good match to the motif (log likelihood score > 0, fig 6c) . the prevalence of high scoring sequences may explain how sox is able to degrade most transcripts. moreover, we found that kshv transcripts also contained sequences that matched the motif (fig 6c) , which suggests that sequence specificity is not used by sox to discriminate between host and viral mrnas. this results is consistent with findings from the related gamma-herpesvirus mhv68 [16] that show degradation of viral transcripts by proteins of the sox family. we have listed examples of human and kshv rnas with high log likelihood scores in s4 table. interestingly, within human transcripts, coding rnas tended to contain sequences that were slightly better matches to the motif than non-coding rnas (ncrnas, fig 6d) , as did spliced rna in comparison to non-spliced rnas (s7b fig). the meaning of these small but statistically significant differences is unclear, particularly since the log likelihood scores for all groups were generally high, indicating the presence of good sequence matches to the motif. we also wanted to test whether the sequences around the experimentally determined sox cleavage sites were more likely to match the motif than other locations on the same transcripts. we analyzed the transcripts containing sox cuts sites identified in the pare data using a 20-nt scanning window as described above. we then ranked the log likelihood scores for all possible 20-mers in the transcript, and asked how highly ranked the score for the actual cut site was. 70% of the experimentally observed cut sites ranked within the top 10% of scores for their rna, indicating that the surrounding sequences were a very good match to the motif compared to other sites in the same mrna ( fig 6e) . finally, we tested whether the relative degradation of different human transcripts by sox was correlated with how well their sequences matched our degenerate motif. human transcripts were classified as down-regulated by sox or sox escapees based on the data from an rnaseq experiment comparing human mrna levels in cells overexpressing gfp-sox or gfp alone [7] the best motif score for each rna detected in the rnaseq experiment was then computed using a 20-nt scanning window as described above. we found that the down-transcripts were scored using the pwm. the frequency distribution histogram for the top scores for each transcript is plotted. d) human transcripts were divided into coding and non-coding based on the annotation in ensembl. the frequency distribution histogram for the top scores for each transcript in the two sets of human rnas is shown. p value (kolgorov-smirnoff test) < 0.001. e) all possible scores for mrnas carrying an observed sox cut site far from the transcription start site (n = 271, confidence level = 99.99%) were computed and ranked in comparison to all the possible scores for the transcript containing the cut. 10 out of 271 (4%) of the cuts were found at the site with the best score. the cumulative frequency distribution for the percentile of the score at the cut was plotted. f) human rnas were classified into sox targets ("down-regulated rnas") or sox escapees ("escapees rna") based on the results from clyde and glaunsinger [7] . the frequency distribution histogram for the top scores for each transcript in the two sets is shown. p value (kolgorov-smirnoff test) < 0.001. regulated rnas (fold change < 0.75) had better motif scores than the escapee rnas (fig 6f) . similarly, when we plotted the fold change for each gene against the best motif score for that gene, we found that there was a modest but significant inverse correlation between the fold change in mrna levels and the motif scores (spearman's ρ = -0.21. p value < 0.0001, s7c fig) . these analyses suggest that the level of down-regulation of mrnas by sox is in part determined by the degree to which their sequence is a good match for the sox targeting motif. we have identified key sequence features of the targeting element that directs the rna endonuclease sox to cleave a significant fraction of the mrna transcriptome. as we had hypothesized from the analysis of individual reporter mrnas [5] , sox cleavages in endogenous mrnas occur in a sequence-specific manner. although surprisingly large, this element is not defined by strong sequence consensus, but instead contains a small number of conserved residues. structural features may also contribute to motif-driven sox targeting. our data resolve how a sequence-specific nuclease can target such a breadth of targets. sox presents a model of rna targeting in which cleavages are at the same time sequence specific and highly promiscuous. this is achieved through the use of a degenerate sequence/structure pattern that is anchored by key residues to define specific rnase targeting locations. good matches for a loosely defined sequence pattern can be found in all viral and host rnas, enabling cleavage to be simultaneously specific and widespread. although this approach may be less efficient than the location-driven targeting of the cap-proximal region reported for other host shutoff factors that promote mrna cleavage [20, 21] , such a mechanism may provide more regulatory opportunities. also, it may explain why sox has less dramatic effects on rna than other viral rnases [3] . many cellular endonucleases have few described targets and transcriptome-wide targeting analyses of other cellular and viral rnases are limited. it will thus be of interest to apply high-throughput sequencing approaches to isolate degradation fragments in other systems and investigate whether any other viral or cellular rnases use principles similar to those employed by sox to achieve target specificity. notably, recent studies of specificity of rnase l, a host rnase that is activated in response to viral infection and cleaves viral rnas and host rrnas, have also suggested that a combination of sequence and structure is important for targeting [30, 31] . however, the requirements for rnase l targeting appear less stringent than those for sox, as the preferred cleavages sites occur at uu/ua dinucleotides in unpaired regions of structured rnas [30, 31] . the rna motif underlying sox cleavage specificity could be used for target selectivity, enabling a subset of viral and cellular mrnas to escape cleavage. our observation that the majority of both viral and cellular transcripts contain the sox targeting motif is in agreement with the fact that in mhv68, viral mrnas are broadly susceptible to degradation by the sox homolog musox [16] . however, subsets of viral and cellular transcripts are not susceptible to host shutoff [6, 7, 16, 17] , and it is likely that at least some of these escape due to the absence of a robust targeting element. indeed, the correlation between the scores of matches to the motif and the level of degradation of host mrnas suggest that sequences within the motif can influence rates of degradation of different rnas. this may result in a more nuanced effect of soxmediated degradation. we also note that the level of degradation seen by steady-state level measurements is likely influenced by additional variables that are unrelated to the efficiency of sox cleavage, including the efficiency of removal of different sequences by xrn1 and a reduction of transcriptional rate due to a feedback loop triggered by the rna degradation [32] . therefore the relationship we see may be an underestimation of the contribution of targeting preference. an additional level of regulation for transcript selectivity is also provided by the presence of dominant protective elements like the sox-resistance element we have identified in the 3' utr of the il-6 gene [8, 9] , which prevents cleavage of the gfp rna despite the presence of a strong targeting sequence. we expect that both dominant and passive mechanisms of escape from sox-mediated targeting ultimately shape the landscape of host gene expression during sox-mediated host shutoff. a limitation of our analyses is that we are unable to readily explore, both computationally and experimentally, the contribution of structural elements to the sox cleavage site. computational analysis of shared structures is difficult when the sequences involved are not evolutionarily related. moreover, even in well-characterized examples of the same protein binding different rnas, for example in the case of bacterial ribosomal proteins binding both ribosomal rnas and their own 5' utrs, the features that are recognized are highly variable [33] , and the remainder of the structure may serve as a scaffold. nonetheless, our identification of endogenous cut sites makes experimental analysis of a putative sox target structure possible in the future. a major outstanding question is how sox recognizes the targeting motif. in vitro studies indicate that the binding affinity of sox for rna is much lower than its affinity for dna [34] , which is also processed by sox during viral genome replication in the nucleus. this suggests that sox may not recognize rna targets directly but may instead be recruited by a protein partner. this model is supported by the observation that point mutations that abolish sox host shutoff activity in cells do not affect its rnase activity in vitro [34, 35] , pointing at a likely protein-protein interaction. a sox partner protein would have to be a fundamental factor in rna metabolism and/or a rna binding protein with promiscuous specificity, as it must bind a large portion of the cellular rnas. another possibility is that sox directly recognizes its target sequence, and that the apparent low affinity for rna in vitro is due to the fact that a noncognate sequence was used for the binding assay. however, the fact that sox cleaves the gfp rna sequence when gfp is expressed from an rna polymerase ii promoter, but not when it is expressed from an rna polymerase i or iii promoter [5] argues against this scenario. how this motif potentiates sox targeting, as well as whether it is used as a protein-binding scaffold for other purposes in the cell related to mrna fate remain important questions for the future. cells and xrn1 knockdown hek293t, hek293t shxrn1 and kshv-infected islk-219 [36] and islk-219 shxrn1 cells were maintained in high-glucose dmem (gibco) supplemented with 10% fetal bovine serum (hyclone). shxrn1 cells were generated using ptripz-shxrn1 (thermoscientific, clone v2ths_89028/rhs4696-99704634, targeting sequence: tatggtgagatatactatg). to induce expression of the shxrn1, cells were treated with 1 μg/ml doxycycline (fisher) for 4-5 days prior to harvesting. lytic induction of kshv was induced in islk-219 cells [36] by treating cells with 1 μg/ml doxycycline and 1 mm sodium butyrate for 4-5 days prior to harvesting. the same induction also led to anti-xrn1 shrna expression in the islk-219 shxrn1 cells. for the second biological repeat of the pare experiment, cells were transfected twice with the sirna against xrn1 as previously described [5] . plasmids pd2egfp-n1 was purchased from clontech. pcdna3.1-gfp-sox was previously described [7] . pcdef3-sox was previously described [13] . pd2egfp-δtgaag was previously described [5] . to test sox-mediated cleavage of endogenous mrna sequences, 33-200 nt surrounding putative cleavage sites in the human mrnas to be tested were cloned from hek293t cdna using vent polymerase (neb). they were then inserted into the bsrgi site at nt 715 of the gfp coding region in pd2egfp-n1, as previously done to test gfp sequences [5] , either by restriction enzyme digest or through a modified version of quikchange mutagenesis [37] . an ecorv site was also generated at nt 459 of the gfp coding region using quikchange mutagenesis (agilent) and the 200 nt surrounding the cleavage site in limd1 were inserted at this location. quikchange mutagenesis (agilent) was used to insert out the following mutations: 1) mutate the aaa sequence preceding the limd1 cleavage site to ccc, ggg and ttt in the pd2egfp construct containing the 100 nt limd1 fragment; 2) mutate the aa sequence preceding the cleavage site to cc in the pd2egfp constructs containing either the 100 nt pgam1 or the 33 nt srsf3 fragments; 3) mutate the cleavage site from a to g in the pd2egfp constructs containing either the 100 nt limd1 fragment, the 100 nt pgam1 fragment or the 33 nt srsf3 fragment; 4) mutate the gfp tgaagt to tgagtg. all primers used for cloning are listed in s5 table. pare library preparation and sequencing hek293t cells treated with sirnas against xrn1 (repeat 1) or expressing shrnas against xrn1 (repeat 2) were transfected either with pcdna3.1-gfp-sox or pd2egfp-n1. in both cases >75% transfection efficiency was observed. one day after transfection total rna was harvested and purified using rnabee (teltest). rna was then treated as described in zhai et al. [24] to generate pare libraries. briefly, poly(a)+ rna was purified, and rna adapters were ligated to free 5' phosphate-bearing rna ends. a second poly(a) purification was used to remove unligated adapter. cdna was synthesized using oligodt directed primers, and the cdna was then amplified 15 times. as the adapter includes an mmei restriction endonuclease site, mmei was used to cut the double stranded amplicons 20 bp downstream of the adapters. 3' dsdna adapters were then ligated to the 3' end of the amplicons. this created libraries of 20 bp tags corresponding to the 5' end of rna fragments flanked by adapters, similar to small rna libraries. libraries were checked on an agilent bioanalyzer and sequenced at the vincent j. coates genomics sequencing laboratory at uc berkeley using a hiseq2000 illumina sequencer. raw data are available on the ncbi gene expression omnibus database as study gse70373. reads flagged by the casava 1.8 program were eliminated and cutadapt [38] was used to trim away the adapter sequence at the read 3' end (sequence: tggaattctcgggtgc-caaggaactccagt). because the pare protocol should produce 20-21 nt sequence tags from the 5' ends of phosphorylated rna fragments, trimmed reads that were longer than 22 nt or shorter than 19 nt were discarded. the resulting sequences were aligned using tophat 2.0.10 [39] using bowtie1 as recommended for short sequences. no mismatches were allowed (-n 0 option), and only alignments that uniquely mapped to the annotated portion of the genome (-t -x 1 options) were retained, to simplify downstream analysis. for the alignment and subsequent analyses, grch37 and the ensembl annotation for this genome build were used. these and other analysis were carried out on an imac computer (mid 2011 model, 3.4 ghz intel core i7, 16gb ram). a bayesian probability framework was used to find peaks that were specific to test samples compared to control samples, which takes into account random variations in the observed number of reads. at a given location and a given experiment, we assume that there is an underlying rate at which reads are produced, and the observed count follows a poisson distribution with mean equal to this rate. in both the control and test data sets, we find that the frequency of reads per location follows a power-law distribution, as is typical for gene expression and deep sequencing data [40, 41] , and we therefore assume that the prior distributions for the underlying rates follow this powerlaw distribution, where the power is fitted from the data. at a given location, we then use bayes' theorem to construct posterior distributions for the rates, given the observations of the read counts. we then deem that there is a significant difference between the control and test at that location, if the posterior probability of the test rate being a multiplicative factor larger than the control rate exceeds some confidence level. the multiplicative factor (ratio) and confidence level are chosen by the user. the observed counts vary over a large range, from single digits up to values in the millions, and a key feature of the method is that is can effectively deal with these variations within a unified theoretical framework. in practice, for a given control read count, we can compute a threshold for the test read count, beyond which the difference in underlying rates is significant. the software builds a table of the thresholds using bicubic splines so that many locations can be tested efficiently. the peak finding python scripts are attached as s1 files. parameters were empirically optimized for the analysis so that a scanning window of 4 nt, a multiplicative factor between test/control read counts of 4 and a confidence level of 99.99% or 99.999% were used to output specific peaks. parameters used in the different analyses are specified in the figure legends. after identifying peaks in single test/control comparisons, the peaks found in the biological repeats were compared. for subsequent bioinformatics analysis of sequences only peaks that were found in both biological repeats were used (figs 1d, 2b, s2c ). sequences surrounding cleavage sites as defined by chromosomal positions were extracted, using the human genome assembly grch37 build as a reference. as many sequences as possible were used for each analysis. however, because the short reads do not provide information about mrna isoform and splicing, for all sequence analyses only cut sites that had sufficient flanking sequences within the same annotated exon were used. for motif analysis (figs 4b, 4c and s7a) and rnafold structure prediction (fig 5b) 25 nt or 10 nt on either side of the cut site were used. for the accessibility computation (fig 4a) , localfold.pl [28] , a modification of the rnaplfold algorithm within the vienna rna package (v2.1.6) [29] was used using default settings (window = 200 nt and maxspan = 150 nt) and sequences of 300 nt on each side of the cut site were analyzed. the log likelihood of each base at each position was calculated using background frequencies of nucleotides derived from the human cdna list from the ensembl grch37 build. weblogo3 [42] was used to generate a graphical representation of the sequence motif from aligned sequences. to score matches to the motif, a position weight matrix was generated using log likelihoods for positions -10 to +9 relative to the cut site using 129 cut sites that were deemed high confidence in our analysis (i.e. identified using a 99.999% confidence level and position at least 50 nt away from an annotated transcription start site). the log likelihood score was then calculated for all sequences surrounding cut sites that were identified in different subsets of the data. the 129 cut sites used to generate the matrix were always eliminated from the sets that were analyzed. to compute the score matches in human and kshv mrna, the log likelihood score for each 20 nt sequence was calculated in all sequences longer than 20 nt listed in the human cdna fasta repository associated with the ensembl grch37 build or in a kshv mrna fasta-formatted list (compiled using data from arias et al. [43] ). the highest score was recorded for each mrna. to separate the human rnas into coding and non-coding their ensembl annotation was used. rnas annotated as "protein coding", "nonsense mediated decay" and "non stop decay" were considered coding, whereas rnas annotated as "antisense", "lincrna", "mirna", "snorna", "processed transcript", "unprocessed pseudogene", "pseudogene", "transcribed unprocessed pseudogene", "transcribed processed pseudogene", "processed pseudogene", and "unitary_ pseudogene" were considered non-coding. the ensembl annotation was also used to determine whether the transcripts were spliced. the motif scores for human mrnas detected in clyde and glaunsinger [7] were also compared to the level of degradation, that is the fold change in steady-state mrna levels between gfpexpressing and gfp-sox-expressing samples in the cited study. for the analysis in fig 6f, the transcripts were categorized into "down-regulated" (fold change in sox vs. gfp < 0.75) and "escapees" (fold change in sox vs. gfp > 0.75). the structure of the sequences surrounding the validated cut sites was predicted using the rnafold webserver (vienna rna package [29] ). rnafold v. 2.1.6 [29] was used to predict structures around all candidate cut sites, and the results were analyzed to determine whether either of the nucleotides at position -1 and 0 was predicted to be unpaired. they were also analyzed to determine whether they had an a dimer within 5 nucleotides 5' of the cut site that was also predicted to be unpaired. custom python2.7 scripts (s2 files) were used unless otherwise noted. where noted in the figure legends, the kolgorov-smirnoff test was used to determine whether the distribution of scores were significantly different. total cellular rna was isolated for northern blotting using trizol (life technologies). rna was separated on formaldehyde gels (1x mops buffer, 1.8% agarose, 12.3 m formaldehyde) in mops buffer (40 mm mops (sigma), 10 mm sodium acetate, 1 mm edta, ph 7.0) and transferred by capillary blotting onto nitrocellulose membrane (bio-rad) using 10x ssc buffer (1.5 m nacl, 0.15 m sodium citrate, ph 7.0). northern blots were probed with 32 p-labeled dna probes made using decaprime ii (ambion), against the 3' utr of the gfp reporters. blots were imaged using a fujifilm scanner fla-9000. quantification of the blots was carried out using imagej [44] . 5' rapid amplification of cdna ends (race) was carried out on 5 μg of total rna using the first choice rlm-race kit following manufacturer's protocol (life technologies). 5' race primers are listed in s5 table. protein harvesting and western blotting protein was isolated for western blots in protein lysis buffer (10 mm tris ph 8.0, 150 mm nacl, 1% triton x-100) containing complete edta-free protease inhibitors (roche), separated on sds-page gels run in tris-glycine buffer and transferred onto pvdf membranes (emd millipore). western blots were performed with mouse anti-xrn1 antibodies (bethyl laboratories or santa cruz biotechnology, 1:1000) or mouse anti-tubulin antibodies (1:3000, sigma aldrich). secondary antibodies were used at 1:5000 dilution and purchased from southern biotech. a) the tgaagt sequence at positions -6 to -1 relative to the cut site in the gfp rna (see s1b fig) was either partially deleted (δtgaag) or mutated to tgagtg. the wild-type gfp and the two mutated reporters were co-expressed with sox ("+") or an empty vector control ("-") and the gfp mrna was detected using northern blotting. the arrowhead points at the position of the normal gfp cleavage fragment. b) predicted structures (with rnafold) of the 39 nt surrounding the sox cut site in limd1, showing the wild-type sequence on the left ("39 nt") and the mutated sequence lacking one of the as on the right ("39 nt δa"). c-d) gfp reporters were co-expressed with sox ("+") or an empty vector control ("-") in shxrn1-treated cells. the gfp mrna was detected using northern blotting. the empty arrowheads point to the additional cleavage fragment resulting from insertions, whereas the filled arrowhead in e points to the normal gfp cleavage fragment. the a dimer preceding the sox cut site in pgam1 was mutated to a c dimer (c) or the a at position 0 was mutated to g (d) in the pgam1 100 nt insertion construct. (eps) s7 fig. additional analyses of prevalence of the degenerate sox targeting motif. a) weblogo3 [42] representation of the frequency of each base in the 20 nt surrounding the cut sites found in both sox samples using confidence level setting of 99.999% and excluding sites near an annotated transcription start site (n = 129). b) human transcripts were divided into "spliced" and "not spliced" based on the annotation in ensembl. the frequency distribution histogram for the top scores for each transcript in the two sets of human rnas is shown. p value (kolgorov-smirnoff test) < 0.001. c) the fold change in mrna levels in sox-expressing vs. control cells from clyde and glaunsinger [7] is plotted against the best motif score for that gene. spearman's ρ = -0.21, p < 0.001. (eps) s1 table. number of reads obtained from pare. % mapping (no restrictions) indicates the percentage of reads that map to the human genome if the requirement for unique mapping to a previously annotated region of the genome is removed. (docx) s2 table. number of peaks detected. the number of peaks detected by using each of the samples as test or control in the pydegradome program (and plotted in fig 2a) is listed. parameters used for this analysis were a scanning window of 4 nt, a multiplicative factor of 4, a confidence level of 99.99%. (docx) s3 table. sox cut sites identified by our analysis. this table lists sox cut sites identified in both replicates with confidence level of 99.99% or in one with confidence level 99.99% and in the second with confidence level 99.9%, and that were 5 nt apart in the two replicates. the table includes the chromosomal position of the cut site, the read count at the cut site in each replicate, the gene name and the confidence level setting used for the identification. it also indicates whether the cut site could be a transcriptional start site (tss) and whether the cut site was used for the analyses in figs 2 and 4 or to generate the pwm for analyses in fig 6. only cut sites identified in both replicates with confidence level 99.99% were used for the analyses shown in figs 2 and 4. (xlsx) s4 table. list of human and kshv transcripts with highest log-likelihood scores of a match to sox targeting motif. (docx) s5 table. primers used for cloning and 5' race analysis. (docx) s1 files. compressed archive of scripts required for pydegradome analysis. "readme.txt" file with instructions on how to run the analysis, as well as the two scripts required for the analysis are included in the archive. (zip) s2 files. compressed archive of scripts used for analyses in the paper. "readme.txt" file with instructions on how to run the analyses, and several scripts used to analyze the data. (zip) emerging roles for rna degradation in viral replication and antiviral defense kshv and the pathogenesis of kaposi sarcoma: listening to human biology and medicine a common strategy for host rna degradation by divergent viruses an overlapping protein-coding region in influenza a virus segment 3 modulates the host response coordinated destruction of cellular messages in translation complexes by the gammaherpesvirus host shutoff factor and the mammalian exonuclease xrn1. renne r, editor highly selective escape from kshv-mediated host mrna shutoff and its implications for viral pathogenesis deep sequencing reveals direct targets of gammaherpesvirus-induced mrna decay and suggests that multiple mechanisms govern cellular transcript escape an rna element in human interleukin 6 confers escape from degradation by the gammaherpesvirus sox a ribonucleoprotein complex protects the interleukin-6 mrna from degradation by distinct herpesviral endonucleases sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage the ul41 protein of herpes simplex virus mediates selective stabilization or degradation of cellular mrnas the herpes simplex virus 1 ul41 gene-dependent destabilization of cellular rnas is selective and may be sequence-specific lytic kshv infection inhibits host gene expression by accelerating global mrna turnover host shutoff is a conserved phenotype of gammaherpesvirus infection and is orchestrated exclusively from the cytoplasm host shutoff during productive epstein-barr virus infection is mediated by bglf5 and may contribute to immune evasion gammaherpesviral gene expression and virion composition are broadly controlled by accelerated mrna degradation host transcript accumulation during lytic kshv infection reveals several classes of host responses characterization of pa-n terminal domain of influenza a polymerase reveals sequence specific rna cleavage decay of endoplasmic reticulum-localized mrnas during the unfolded protein response the herpes simplex virus vhs protein induces endoribonucleolytic cleavage of target rnas in cell extracts a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein smg6 promotes endonucleolytic cleavage of nonsense mrna in human cells construction of parallel analysis of rna ends (pare) libraries for the study of cleaved mirna targets and the rna degradome rapid construction of parallel analysis of rna end (pare) libraries for illumina sequencing diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon micrornas, drosha, and additional nucleases human nonsense-mediated rna decay initiates widely by endonucleolysis and targets snorna host genes identification of smg6 cleavage sites and a preferred rna cleavage motif by global analysis of endogenous nmd targets in human cells global or local? predicting secondary structure and accessibility in mrnas viennarna package 2.0 ribonuclease l and metal-ion-independent endoribonuclease cleavage sites in host and viral rnas rnase l targets distinct sites in influenza a virus rnas viral nucleases induce an mrna degradation-transcription feedback loop in mammalian cells bacterial rna motif in the 5' utr of rpsf interacts with an s6:s18 complex crystal structure of a kshv-sox-dna complex: insights into the molecular mechanisms underlying dnase activity and host shutoff the exonuclease and host shutoff functions of the sox protein of kaposi's sarcoma-associated herpesvirus are genetically separable generation of a doxycycline-inducible kshv producer cell line of endothelial origin: maintenance of tight latency with efficient reactivation upon induction integration of pcr fragments at any specific site within cloning vectors without the use of restriction enzymes and dna ligase cutadapt removes adapter sequences from high-throughput sequencing reads tophat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions zipf's law in gene expression methods for analyzing deep sequencing expression data: constructing the human and mouse promoterome with deepcage data weblogo: a sequence logo generator kshv 2.0: a comprehensive annotation of the kaposi's sarcoma-associated herpesvirus genome using next-generation sequencing reveals novel genomic and functional features nih image to imagej: 25 years of image analysis we thank albert tai at the tufts genomics core facility for discussion on the analysis, rachel brem and members of the brem lab (buck institute of aging research) and glaunsinger lab for helpful discussions. we thank alicia bicknell and members of the glaunsinger lab and gaglia lab for critical reading of the manuscript. conceived and designed the experiments: mmg. performed the experiments: mmg. analyzed the data: mmg chr bag. contributed reagents/materials/analysis tools: chr. wrote the paper: mmg bag. key: cord-315483-l6dm82pp authors: santhakumar, diwakar; rohaim, mohammed abdel mohsen shahaat; hussein, hussein a.; hawes, pippa; ferreira, helena lage; behboudi, shahriar; iqbal, munir; nair, venugopal; arns, clarice w.; munir, muhammad title: chicken interferon-induced protein with tetratricopeptide repeats 5 antagonizes replication of rna viruses date: 2018-05-01 journal: sci rep doi: 10.1038/s41598-018-24905-y sha: doc_id: 315483 cord_uid: l6dm82pp the intracellular actions of interferon (ifn)-regulated proteins, including ifn-induced proteins with tetratricopeptide repeats (ifits), attribute a major component of the protective antiviral host defense. here we applied genomics approaches to annotate the chicken ifit locus and currently identified a single ifit (chifit5) gene. the profound transcriptional level of this effector of innate immunity was mapped within its unique cis-acting elements. this highly virusand ifn-responsive chifit5 protein interacted with negative sense viral rna structures that carried a triphosphate group on its 5′ terminus (ppp-rna). this interaction reduced the replication of rna viruses in lentivirus-mediated ifit5-stable chicken fibroblasts whereas crispr/cas9-edited chifit5 gene knockout fibroblasts supported the replication of rna viruses. finally, we generated mosaic transgenic chicken embryos stably expressing chifit5 protein or knocked-down for endogenous chifit5 gene. replication kinetics of rna viruses in these transgenic chicken embryos demonstrated the antiviral potential of chifit5 in ovo. taken together, these findings propose that ifit5 specifically antagonize rna viruses by sequestering viral nucleic acids in chickens, which are unique in innate immune sensing and responses to viruses of both poultry and human health significance. innate immune responses, primarily triggered by interferons (ifns) and their antiviral effectors, can establish an extremely potent antiviral state to efficiently restrict virus replication and virus-induced pathologies in the susceptible host 1 . to initiate these innate responses, viruses are detected by the host pathogen recognition receptors (prrs) through recognition of viral molecular signatures known as pathogen associated molecular patterns (pamps) 1 . pamps associated with most viruses include viral double-stranded rna (dsrna), which is produced as replicative intermediate and single-stranded adenosine/uridine (au)-rich regions 2 . these pamps effectively activate cascades of signalling events that culminate in the production of ifns in virus-infected cells. the released ifns transcriptionally activate hundreds of ifn-stimulated genes (isgs) in uninfected neighbouring cells that instigate direct or indirect antiviral activities 3, 4 . well-studied examples of these antiviral effectors are dsrna-activated protein kinase r and 2′-5′ oligoadenylate synthetase which bind to dsrna, and interferon induced proteins with tetratricopeptide repeats (ifit)-1 and -5 that bind to 5′-triphosphate containing rna 1, 5 . ifit genes are evolutionary conserved and are originated possibly by gene duplication 6 . all members of the ifit family consist of multiple tetratricopeptide repeats (tpr) throughout the length of the protein that are mainly responsible for the protein-protein interaction and assembly of larger protein complexes 7, 8 . most mammals encode several ifit genes including ifit1/isg56, ifit2/isg54, ifit3/isg60 and ifit5/isg58, however, mice and rats lack ifit5 and horses lack ifit1 6 . amongst all known members of the family, ifit1 and ifit5 are highly responsive both at the transcription and translation levels to diverse cellular stresses including those induced by dsrna, virus infections and lipopolysaccharides 1, 5 . the ifit proteins family is responsible for diverse array of cellular activities including nucleic acid sensing and direct antiviral effects 9 . specifically, ifit1 is proposed to be a negative-feedback regulator of virus-triggered induction of type i ifns and cellular antiviral responses 10 whereas ifit5 potentiate antiviral signalling 11 . additionally, mammalian ifit5 can sense and bind to numerous short cellular rnas such as initiator trna, and these interactions are mapped across the protein surface 12 . the most prominent features of ifit5 proteins are attributed to their involvements in the inhibition of virus replication through nucleic acid sensing and leading to possible inhibition of translation 8, 9 . ifit5 protein, in addition to ifit1, discriminates the cellular and viral mrna for initiating downstream antiviral activities by recognition of discrete features at the 5′ termini 7, 13 . most eukaryotic cellular ribosomal and transfer rnas (rrnas and trnas) carry monophosphate at 5′-termini whereas messenger rnas (mrnas) bear n7-methlguanosine cap (cap0) attached to the first base through 5′-5′ triphosphate bridge that recruits cellular factors involved in rna processing and translation initiation 14, 15 . additionally, in most higher eukaryotes methylation occurs at the 2′-o position of the first or second base yielding the cap1 (m 7 gpppnmn) or cap2 (m 7 gpppnmnm) structures, respectively 14 . although cap1 and cap2 are not crucial for mrna translation, human and murine ifit1 protein can inhibit translation of cap1-lacking mrna 16, 17 . these features of cellular rnas are also mimicked by several viruses as countermeasure strategies 17 . however, viral genomic and subgenomic rna of negative sense single-stranded viruses such as influenza a viruses (iav) and newcastle disease viruses (ndv) bear triphosphate at the 5′-termini 14 . these pamps are sensed by cellular prrs and initiate innate immune responses, which ultimately restrict virus growth 1, 5 . significant genetic, functional and structural features have been recently attributed to the mammalian ifit genes and proteins [16] [17] [18] [19] . there is limited information available on the repertoires of cellular proteins that recognizes different populations of viral nucleic acid in avian species especially in chickens which differ significantly in mounting innate immune responses and are infected by pathogens that continuously pose zoonotic threats to public health 5 . here, we have genetically characterized chicken ifit and revealed that chickens, in contrast to other vertebrates, encode only one ifit gene (chifit5). the chifit5 gene was transcriptionally highly responsive to both type i ifns and rna viruses (iav and ndv) and interestingly this responsiveness was mapped to isre motif in the cis-acting elements. we also demonstrated that chifit5 specifically interacts with ssrna carrying 5′-ppp moiety. through this interaction, chifit5 applies strong antiviral activities in lentivirus-transduced stable cell lines whereas crispr/cas9-mediated chifit5 knockout promoted virus replication. finally, employing the rcas-based retroviral gene transfer vector system 20 , we generated transgenic chicken embryos expressing chi-fit5 and demonstrated its antiviral potential in ovo. these findings were further evaluated by rcas-mediated gene silencing in developing transgenic chicken embryos by assessing the replication kinetics of rna viruses. these analyses provide evidence of the presence of a functional homologue of ifit5 and expand our understanding on the breaths and dynamics of nucleic acid sensing in chicken. genomic annotation of ifit locus revealed that chicken genome encodes ifit5 gene. the genome of all major mammalian species (including human, mouse, dog and horse) encodes for ifit genes, however, these genes are only genetically and functionally characterized in a limited number of species 9, 19 . the human genome encodes four ifit genes (ifit1, ifit2, ifit3 and ifit5) whereas rats and mice lack ifit5 and horses lack ifit1 (fig. 1a ). in addition, several pseudogenes have been identified in different animal species including ifit1b (in human, mice and rabbits), ifit1c (in mice), ifit3-like (in dogs and mice) and ifit5-like gene (in dogs and rabbits). to identify corresponding ifit homologues in chicken, initially several ifit genes from human and mouse were used in the blast algorithm in the ensembl database. based on high genetic similarity with huifit5, only a single gene (ensgalt00000010311) was identified in the chicken genome (fig. 1a) . the ifit locus, encoding all ifit genes, is primarily mapped in between the lysosomal acid lipase/cholesteryl ester hydrolase (lipa) and pantothenate kinase 1 (pank1) genes. since only a single ifit gene (ifit5) was identified in the chicken genome, we next examined an approximately 9.0 kb genomic sequence spanning aadn04000218.1 contigs in the chicken chromosome 6 (chromosome 6: 19,342,835-19,429,041). immediately adjacent to the lipa gene, a sequence gap was identified whose estimated length was 400 bps in the ensembl chicken genome build (ensembl release 85 -july 2016) ( supplementary fig. 1a ). long primers (supplementary table 1 ) flanking each end of the gap were used to amplify genomic fragments and subsequent sequencing of the products was used to cover the genomic gap. we used the sequenced fragments as input in webaugustus and predicted any possible genes in the entire ifit locus. only one gene (ifit5) was predicted in any strand of the input dna using chicken as species parameters ( supplementary fig. 1a ). the genomic gap has now been filled with the latest ensembl chicken genome (ensembl release 87 -dec 2016) and no gene other than ifit5 has been identified (until 17 april 2018). to confirm the sequence of the cdna and to identify the genomic structure of the identified ifit gene, the transcript was amplified from rna extracted from ndv-infected cefs. complete sequence analysis of the gene revealed an open reading frame (orf) of 1440 bps (479 amino acids excluding stop codon) with high sequence identity (48% and 67%) with the human and duck ifit5 proteins, respectively ( supplementary fig. 1b) . the identified gene showed a characteristic exon/intron organization where two exons were separated by a few kilobases (kb) long intron (fig. 1b) . the first exon encodes barely a kozak sequence (catg) and 5′ untranslated region (utr). the second exon codes the rest of the orf (1435 bps) and 3′ untranslated regions (utr) for chicken ifit5 gene. based on the transcriptomic data from marek's disease virus (mdv, strain rb1b)-infected cefs (fig. 1b) and cdna sequencing data, a complete ifit5 gene was annotated. phylogenetic analysis of characterized chicken (ch) ifit5 gene with all known human, mouse and duck ifit genes clustered the chicken gene with scientific reports | (2018) 8:6794 | doi:10.1038/s41598-018-24905-y duck and human ifit5 (fig. 1c) . one of the structural hallmarks of all ifit proteins is the presence of multiple trp motifs dispersed throughout the length of the protein 6, 21 . the consensus chicken, duck and human ifit5 sequences were used to predict trps using ncbi's conserved domain database. both duck and human ifit5 proteins structurally carried ten trp motifs and two multi-domains whereas chicken ifit5 encoded eight predicted and ten structure-based tprs (fig. 1d) . taken together, the gene synteny, genetic similarity, genomic architecture and annotation indicate that chickens encode only one ifit gene compared to at least four in mammals. based on genetic clustering, it is highly similar to ifit5 genes of other avian (chickens and ducks) and mammalian species. moreover, no ortholog for ifit1, ifit2, ifit3 and no pseudogenes were identified in the current ensembl chicken genome build (ensembl release 87 -dec 2016). mouse ifit1 protein is predominantly expressed in the cytoplasm and upon stimulation with the ifn, it accumulates in the cytoplasm (>1 × 10 6 molecules per cell), and this abundance is identified to be crucial for its antiviral function 7 . beside the distribution pattern of mouse ifit1, no other ifit proteins have been investigated for their subcellular distributions. to delineate the expression dynamics and sub-cellular distribution, df-1 cells were transfected with v5-tagged chifit5 and transient expression of chifit5 was compared in ndv-stimulated or mock-treated cells. confocal microscopy figure 1 . genomic architecture along with relative loci around ifit genes in human, mouse, dog, horse and chicken, and gene annotation in chicken. (a) the ifit locus in compared species is flanked upstream with lipa gene and downstream with pank1 gene. direct syntenic analysis identified a single ifit5 gene in chicken compared to four in other species aslong with other pseudogenes. (b) transcriptomics profiling of chicken primary fibroblasts which were mock-infected or infected with rb1b strain of mdv. blue bar represents the transcript in the current chicken genome assembly whereas the red bar represents the mappability of the transcript to the chicken genome. a final transcript from mdv-infected data and gene characterization is shown at the bottom. (c) phylogenetic analysis of ifit genes in different species. based on the clustering patterns and sequence homologies, the single identified gene clustered closer to ifit5 of duck and human. (d) putative tetratricopeptide repeats (tpr) showing characteristic features of ifit proteins. (e) expression and subcellular distribution of chifit5 in chicken embryo fibroblasts. chicken cells were transfected with 500 ng of mammalian expression vectors encoding v5-tagged chifit5 for 24 hours and were left untreated (ndv-) or were treated with 1 moi of ndv-gfp (ndv+) for another 24 hours before fixation, staining for nucleus (blue), chifit5 (red) and gfp marker (ndv). scientific reports | (2018) 8:6794 | doi:10.1038/s41598-018-24905-y using anti-v5 antibodies showed that chifit5 was exclusively cytoplasmic and was expressed throughout the cytoplasm; however, this expression was concentrated at the cell surface (fig. 1e, upper panel) . we were unable to demonstrate the distribution pattern of chifit5 under ndv infection (fig. 1e , lower panel) probably due to the profound antiviral state induced by chifit5 so that infection of ndv in the chifit5-expressing cells could not be achieved. it has previously been demonstrated that human ifit5 enhances the innate immune responses by interacting with rig-i (not identified in chicken) and mavs and this interaction occurs at the mitochondria 11 . since mavs localizes on both mitochondria and endoplasmic reticulum (er)-derived membranes (mem), we labelled mitochondria and er in presence of chifit5 to assess the distribution and localization of chifit5 with mavs. in df-1 cells, chifit5 localized in close proximity to the mitochondria and no co-localization was observed between er and chifit5 (data not shown). owing to lack of cross-reactivity of human ifit5 antibodies and specificity of anti-sera which we have raised against chifit5, these experiments were performed using tagged-chifit5. therefore, future studies are required to assess the expression patterns of the endogenous chi-fit5 under virus or non-virus stimuli. transcriptional profiling of chifit5 in vitro and ex vivo. we next assessed the nature of ligands that can transcriptionally induce the expression of chifit5. different ligands that can either directly induce the transcription of ifit5 such as ifn-β or stimuli that result in the expression of ifns, which in turn induce the ifit5 transcription, were assessed ( fig. 2a) . tlr4 (lipopolysaccharide, lps) and tlr3 (poly i:c, synthetic dsrna) ligands significantly induced the expression of chifit5 gene by 25 and 1500 folds, respectively (fig. 2b) . the induction is likely through the activation of ifns since the chifn-β stimulated cells profoundly increased the transcription of chifit5 by 4000 folds compared to untreated or mock-treated cells. there are evidences that negative sense single stranded rna viruses (e.g. influenza and ndv) produce dsrna as intermediate by-product during the virus replication cycle 2,22-24 . therefore, it is plausible that induction of chifit5 expression in chicken cells infected with ndv is mediated by virus-generated dsrna (fig. 2b ). to further assess the temporal effect of ndv infection on the induction of chifit5, a time course profiling ifn and ifn-regulated genes was evaluated. the virus-induced expression of chifit5 was profoundly observed as early as 2 hours post-infection (fig. 2c ) and the expression was maintained for 4 hours. a slight reduction in ifit5 expression was observed at 8 hours post-infection (hpi) was reconstituted at 24 hpi (fig. 2c ). this biphasic expression of ifit5 after virus infection was repeatedly observed in chicken cells. however, the pattern of expression of myxovirus-resistance protein (mx) gene, which is another well-characterized isg 25 , shows a steady up-regulation and peak expression was observed at the latest time post-infection (fig. 2e ). the levels of chifn-β gene induction was proportional to the ndv replication (fig. 2d) , therefore, it can be inferred that the virus-induced expression of chicken ifit5 is ifn-dependent and that chifit5 is an early-isg with capacity to modulate initial steps of virus life cycle. to investigate transcriptional activation of ift5 following virus infection in chicken, we analysed a panel of rna transcripts derived from selected tissues (liver, kidney, spleen, beak, trachea, lungs and duodenum) of chickens (3-weeks-old rhode island red) which were mock-infected or infected with h9n2 avian influenza virus strain a/chicken/pakistan/udl01/08 (udl08/h9n2). all tissues from infected birds contained relatively higher levels of chifit5 compared to the corresponding tissue samples derived from mock-infected chickens (fig. 2g) . these results conclude that virus infection positively regulates the transcriptional dynamics of chifit5 in chickens. promoter structures contributing to the ifn, dsrna or virus-mediated transcriptional activation of chifit5 gene. most studied-ifit genes respond to ifn or ifn stimuli through one to four ifn-stimulated response elements (isre), which are mainly present within 200 base pair (bp) of the transcriptional start site in the orientation of the encoded gene or in the reverse complement order 6 . on the other hand, several ifit genes lack isre motifs in their promoters including ifit2 from chimpanzee, ifit2b from human, chimpanzee and dog, and ifit5 from horse, chimpanzee and dog 6 . since our data and previously published transcriptomics studies 26 support the profound expression of ifit5 gene against a wide range of stimuli, we analysed the 5′ flanking region of chifit5 gene for motifs that regulate the expression of chifit5. in addition to the gene-encoding sequences, approximately 1.2 kb sequence including the putative promoter (supplementary fig. 2a ) was isolated and sequenced. inspection of the promoter region revealed the presence of two consecutive isre motifs within 85 bps from the transcriptional start site ( fig. 3a and supplementary fig. 2b ). these elements were preceded by tata element, and the binding motif for specificity protein 1 (sp1) transcription factor. a putative and weak ifn-gamma-activated site (gas) was identified at the distal end of the promoter and six consecutive gaaann elements were predicted between gas and sp1 motifs (fig. 3a) . gas is essential for ifn-gamma-mediated transcription of the genes whereas gaaann elements have been demonstrated to regulate the virus-induced and type i ifn-regulated genes via the binding of interferon regulatory factors 27, 28 . to determine the role of these cis-acting regulatory elements in the responsiveness of different ifn ligands and stimuli, the entire promoter region (1122 bp) was cloned upstream to the luciferase gene in a promoter-less pgl3.1-basic vector (fig. 3a) . additionally, five reporter constructs were generated each containing either all of the gaaann elements, sp1, tata box, double or single isre promoter sequences (fig. 3a,b) . luciferase reporter assays were performed to assess whether the upstream region of the chifit5 gene can mediate responses to stimuli from chifn-β, dsrna or virus infection. df-1 cells were transiently transfected with these reporter plasmids and subsequent luciferase activity was monitored after stimulation with either chifn-β (fig. 3c) , dsrna ( fig. 3d ) or with ndv (fig. 3e ). similar to ifn-induced promoter activation of chmx gene 25 , the chifn-β induced 13-fold higher luciferase activities in full-length promoter (fig. 3c ). however, further deletion of gas, gaaann, sp1 and tata box resulted in an approximately 4-fold reduction in basal transcription activity. additional deletion of the distal isre reduced the ifn-responsiveness of the promoter. however, repetitive luciferase assays demonstrated that isre motif just proximal to the transcriptional start site was exceedingly responsive to the ifn-treatment with a highest of 19-fold induction in luciferase activity as compared to the control vector without a promoter sequence (fig. 3c ). in the dsrna-dependent promoter induction (fig. 3e ), results demonstrated that the above elements and motifs (gaaann, spi and tata box) are least important in controlling the transcriptional activation of luciferase gene and a non-significant difference was observed in the absence or presence of these motifs. however, promoter sequence containing dual isre motifs severely compromised the responsiveness of dsrna on the ifit5 promoter and the dsrna-dependent promoter induction was restored when distal-isre was deleted, further suggesting the importance of this isre motif (fig. 3e ). similar to dsrna induction, the construct containing only one isre motif was highly inducible by ndv compared to the construct carrying both isre motifs (fig. 3e) . as was observed in the ndv-induced chmx promoter activation, all other constructs with variable lengths and motifs were also responsive to virus infections. comparison of these three stimuli indicated lower luciferase activities in ndv and dsrna-treated chicken cells compared to chifn-β induced promoter activation, presumably due to indirect activation of ifit5 promoter by inducing endogenous ifns. following the demonstration of robust reporter function of the chifit5 promoter-containing proximal isre motif by the chifn-β, dsrna and ndv, we compared the sequence of cis-acting regulatory elements with the previously characterized chmx promoter 25 . in silico analysis of highly responsive 122 bp sequence indicated that the isre motif (5′-gctttcactttct-3′ at position −29 to −13 in pchifit5 −101/+123 construct) was identical to the consensus isre (g/a/t)g/ctttcn 1-2 tttc(a/t/c) found in most of the ifn-inducible genes including chmx promoter sequence (fig. 4a ). for further absolute delineation of importance of this motif, we mutated both triplet thymidines (ttt), the core residues for the ifn-inducible activation of genes ( fig. 4b and supplementary fig. 2c ). responsiveness of the wild type (wt) and mutated isre motifs was assessed in chicken df-1 cells with or without stimulation by chifn-β, dsrna or ndv using luciferase assays. while both pchifit5-101/+123 and pchifit5-101/+123-mut promoters were not auto-stimulatory, stimulation with either of the stimuli positively regulated the luciferase genes in pchifit5-101/+123. interestingly, stimulation of mutated promoter with chifn-β ( . . chicken and human ifit5 restricts the replication of rna virus in stably expressing chicken fibroblasts. to assess the antiviral potential of chifit5 protein, we generated a lentivirus construct which bicistronically expresses chifit5 and a red fluorescent protein tagrfp under the control of emcv (encephalomyocarditis virus) internal ribosome entry sequence (ires) 3 . cef cells were transduced with the appropriate vsv-g pseudotyped lentiviral particles and infected with gfp expressing ndv. after 24 hours, the virus infectivity (gfp+) was quantified in lentivirus transduced (rfp+) cell population using fluorescence-activated cell sorting (facs) (fig. 5a ). lentivirus expressing firefly luciferase (ffluc), not expected to affect the virus replication, was used as a negative control. human irf1 (huirf1), a potent virus restriction factor 3 , was used as a positive control ( fig. 5b and supplementary fig. 3a ). compared to ffluc control, both huirf1 and chifit5 significantly inhibited ndv replication (fig. 5b,c) , while the antiviral effect of chifit5 was greater than that of huirf1 in cefs. since two populations of gfp+ cells were observed, further gating of low gfp+ and high gfp+ indicated a profound and cumulative antiviral effect compared to ffluc control ( supplementary fig. 3b ). these results showed that the chifit5-mediated antiviral effect is not profound at individual cell levels but it attenuates replication of the virus after initial infection. to compare anti-viral effects of chifit5 (only ifit gene identified in chickens so far) with its orthologous and homologous human proteins, huifit1, huifit2, huifit3 and huifit5 were expressed in chicken cells and antiviral affect was monitored (fig. 5a ). similar to chifit5, both huirf1 and huifit5 showed significant inhibition of ndv replication whereas no antiviral activities were observed by other huifit proteins in chickens (fig. 5d ). to further demonstrate the effect of ifit5-induced inhibition of ndv replication, we observed an exclusive replication of either ndv (gfp+) or transduction of lentivirus expressing ifit5 (rfp+) in 85% cells compared to ffluc expressing cells (n = 200) (fig. 5e, upper panel) . it is possible that transduction of lentivirus particles may interfere with the virus entry and/or ndv replication. to examine the lentivirus-mediated restriction of ndv replication, we used ffluc-transduced cells in the control group. the analysis of several microscopic fields showed simultaneous expression of lentiviruses-transduced ffluc protein and ndv infection (fig. 5e , lower panel and fig. 5b ), demonstrating that the transduction of lentivirus particles doesn't interfere with the ndv replication. collectively, these results demonstrate that ifit5 proteins of both human and chicken are potent cellular restriction factors against rna virus infection in chicken primary cells. to further evaluate the antiviral effect of chifit5 on virus replication, we applied a loss-of-function approach using crispr/cas9 genome editing technology. using synthetic grna and cas9 expression plasmid (fig. 6a) , the chifit5 was targeted for editing. df-1 cells were co-transfected with hybridized crrna and tracr-rna as well as a vector expressing hspcas9 and puromycin resistance marker (puro r ) gene. for fast and efficient enrichment of genetic modification, a population of stably-integrated cells was selected with puromycin, and was further enriched using facs (fig. 6a) . the relative frequency of gene editing in the puromycin-resistant and facs-enriched cell was estimated from a dna mismatch detection assay using t7 endonuclease i (t7e1) (fig. 6b) . t7e1 assay showed a mutation frequency of 40% within the chifit5 gene, however, t7e1 assays are likely to underestimate the fold enrichment 29 . we subsequently sequenced the mutated sites and confirmed the in-frame or out-of-frame gene editing (fig. 6c) . results of the t7e1 assay and sequencing showed that sgrna2 sufficiently edited the gene and puromycin selection greatly improved the enrichment of the cells. additionally, most of the mutations in the chifit5 gene appeared to be deletions, which introduce a pre-mature stop codon to the beginning of exon 2 of the chifit5 gene. the chifit5 deletion-confirmed df-1 cells (chifit5 ko df-1) were isolated, expanded and assessed for virus replication. both wt df-1 and chifit5 ko df-1 were infected with ndv and vsv, and replication of viruses was monitored for 24 hours. both ndv (fig. 6d) and vsv (fig. 6e ) replicated efficiently in wt df-1 cells over the time course of infection (2 to 24 hours). however, deletion of chifit5 by crispr/cas9 further supported the replication of ndv (fig. 6d) and vsv (fig. 6e) , confirming that chifit5 is a crucial antiviral effector and elimination of such factors weakens the hostile barriers of the host. these results also highlight the possible exploitation of innate immune genes in promoting virus replication for vaccine production. additionally, we applied facs to quantify the percentage infectivity of the wt df-1 and chifit5 ko df-1 cells for gfp-expressing recombinant ndv. data demonstrated that the wtdf-1 cells infectivity by ndv (45.6%) could further be enhanced (63.2%) in the absence of chifit5 (supplementary fig. 4) . cumulative quantitative measurement of ndv-infectivity was significantly enhanced in chifit5 ko df-1 cells compared to wt df-1 cells (fig. 6f ). next, in order to counter-confirm the antiviral potential of chifit5, the level of vsv replication was assessed in conventional plague assay in df1 cells either overexpressing or knocked out with chifit5 (fig. 6g) . the vsv replicated effectively in wt df-1 cells and overexpression of chifit5 suppressed the vsv whereas crispr/cas9 knockout df-1 cells substantially supported the virus replication (fig. 6g) . together, these results confirm the potential of chifit5 as an important host restriction factor, at least against evaluated rna viruses. chicken ifit5 interacts with 5′ppp-containing rna. different human and mouse ifit proteins (ifit1, ifit2, ifit3 and ifit5) interact with rna carrying multiple genetic modifications at their 5′ ends 9,19,30-32 . since ifit5 was the only identified ifit protein in chickens, we aimed to explore whether chifit5 interacts with rna in a similar mechanism reported for huifit5 or chifit5-rna interaction is redundant to other members of ifit family. in order to explore the molecular mechanisms of chifit5 recognition of rna, we used 378 bp rna without any known similarity to viral rna sequences and structure, and generated (i) rna bearing terminal 5′ hydroxyl group (5′ohrna), and (ii) rna bearing 5′ triphosphate (5′ppprna) (fig. 7a) . these populations of rna, mimicking viral rna ends, were biotinylated and coupled with agarose beads. the beads were then incubated with chicken df-1 cells expressing v5-tagged chifit5, the ribonucleoproteins were purified (fig. 7b ) and the interaction of chifit5 was determined by staining chifit5. while neither huifit5 nor chifit5 interacted with the 5′ohrna, both proteins recognized rna carrying ppp at the 5′ end (fig. 7c) . overexpression of chicken chifit5 in transgenic chicken embryos restricts the replication of rna virus. we next asked whether the in vitro demonstrated antiviral activities of chifit5 are translatable in ovo in developing chicken embryos. for this purpose, we constructed rcasbp(a)-chifit5 recombinant viruses to generate mosaic-transgenic chicken embryos that are constitutively expressing chifit5, and monitored its impact on the replication of ndv (fig. 8a) . in addition, a reporter virus, rcasbp(a)-egfp, was generated to monitor the rescue of the virus and to investigate the effect of retroviruses on host responses. rcasbp(a)-chifn-β, expressing chifn-β which is a known antiviral cytokine 5 , was generated as a positive control (fig. 8a) . expression of this transgene did not compromise the replication of retroviruses, and the induction of innate immune responses was not significant (data not shown), confirming previous reports that rcasbp is a safe in ovo gene transferring system 33, 34 . stable cells lines expressing retrovirus-mediated chifit5 and chifn-β were used to monitor virus replication. while mock-infected or rcasa(bp)-wt infected df-1 cells did not interfere with the replication of ndv (fig. 8b) and vsv (fig. 8c) , stable expression of chifit5 and chifn-β established a profound antiviral state against both viruses over the time course of infection. these functionally validated infectious df-1 cells were used to generate transgenic chicken embryos. three-day-old embryonated chicken eggs were inoculated with wt retroviruses or viruses that were expressing chifit5 or chifn-β to generate mosaic transgenic chicken embryos (fig. 8d) . additionally, embryos were either mock-inoculated with pbs or with non-infectious df-1 cells to exclude the possibility of antiviral effects by the retrovirus infected df-1 cells. these five groups of embryonated eggs were either mock-challenged with pbs or virus-challenged with ndv, and were incubated for another five days (fig. 8d ). compared to controls, retroviral expression of ifit5 negatively affected the development of healthy embryos until day 9 post-embryonation before challenge with ndv (fig. 8e) . quantitative analysis of ndv in allantoic fluids showed that the wt retroviruses or non-infectious df-1 cells alone were unable to interfere with the replication of ndv, however, transgenic embryos stably expressing chifit5 or chifn-β significantly restricted the virus replication compared to the mock-treated control (fig. 8f) , indicating that chifit5 can restrict virus replication in developing transgenic chicken embryos. collectively, these results further confirm the antiviral activity of chifit5 in ovo. ablation of chicken chifit5 in transgenic chicken embryos ameliorates the replication of rna viruses. next, we asked whether silencing of endogenous chifit5 in developing embryos would facilitate the replication of ndv. for this purpose, we streamlined two individual gene delivery protocols for specific and optimal gene silencing in chicken cells and embryos. first, a total of three short hairpin rnas (shrna) targeting the chifit5 and a scrambled shrna with a highly confident predicted score were cloned in the prf-prnaic as described before 35 (fig. 9a) . transfection of chicken df-1 cells with these vectors expressing shrna under a chicken u6 promoter showed a reliable level of expression of the rfp marker gene indicating the functional integrity of the system (fig. 9b) . although all shrna were effective in silencing, quantitative analysis of ndv-induced chifit5 mrna showed that shrna#3 was the most effective (>50%) (fig. 9d) . next, the validated shrna cassette that included chicken u6 promoter, shrna, leader and trailer sequences was transferred to the compatible rcasbp(a) vectors for silencing of chifit5 in ovo (fig. 9c) . additionally, we used shrna silencing-resistant rcasbp(a)-chifit5 retroviruses ( supplementary fig. 5) to stably express the chifit5 in developing embryos in the presence of shrna against endogenous chifit5 gene. the replication competency of shrna expressing retroviruses was compared to the wt retroviruses (fig. 9e ) before using them to generate transgenic chicken embryos. using the experimental strategy depicted in fig. 9f , we monitored the replication of ndv in transgenic embryos that were either stably expressing chifit5 or were expressing chifit5 in the endogenously silenced ifit5 gene. the retroviral overexpression of chifit5 or silencing of chifit5 were normalized to non-infectious df-1 cells or wt rcaspb(a) that did not express the transgene. quantitative analysis of ndv showed an expected reduction in replication of the virus in ifit5-overexpressing embryos, whereas silencing of endogenous chifit5 favoured the replication of ndv compared to the control df-1 cells (fig. 9g) . a moderate reduction in the virus replication was observed in transgenic embryos that were simultaneously silenced for endogenous chifit5 and were stably expressing silence-resistant chifit5 (fig. 9g) . taken together, these results not only confirm the antiviral activities of chifit5 in different experimental settings but also highlight the potential use of rcaspb(a) in enhancing the replication of avian viruses in embryonated eggs. innate immune responses are key to the success of host resistance to virus infection and isgs provide a front-line role in these defences by acting at multiple stages of the virus replication cycle [2] [3] [4] [5] . among the myriad of isgs, members of the ifit family have attracted recent attention for both immunological as well as virological reasons wild type and chifit5 ko -confirmed df-1 cells were infected with ndv or were left uninfected for 24 hours before processing them for facs. cumulative mfi of gfp positivity in wild type and chifit5 ko df-1 cells based on at least 3 independent experiments. (g) df-1 cells were either mock transfected (wt df-1 cells) or were transfected with 2 ug of chifit5 expression plasmid (chifit5 oe df-1 cells). these cells and crispr/cas9 knockout df1 (chifit5 ko ) were either left un-infected or were infected with vsv for 48 hours before staining with crystal violet. the plagues developments were imaged using hand-held camera. scientific reports | (2018) 8:6794 | doi:10.1038/s41598-018-24905-y due to their abundant and profound transcription and translation responses to diverse stimuli such as ifns and viruses 9, 19 . significant advances have been made during studies of the mechanisms of both human and mouse ifit proteins 19 . however, the knowledge on the breadth and plasticity of the antiviral properties of ifit proteins are currently inadequate for non-mammalian hosts. characterizing the repertoire of ifit proteins and investigating their functions in chicken are of special interest because of the unique features of the chicken immune system including the absence of essential components of innate immune induction and signalling such as rig-i, irf3, and irf9 5 in chickens. intriguingly, while chickens are lacking these components, they still mount profound innate responses against virus infections. understanding the alternative means of innate immune regulation and antiviral defences in chicken could establish the foundation to control chicken-mediated emergence of zoonotic infections such as influenza viruses 36 . in order to explore the ifit genes in chicken, we began with genetics and functional genomic approaches and revealed that chicken encodes only a single ifit gene compared to 4 genes in mammals (human and mice). based on its sequence and structure similarities, and phylogenetic associations, this single chicken ifit gene is classified as ifit5 (chifit5). currently the chicken genome is about 90% annotated and all chromosomes are correctly characterized 37 , however, the possibility of orthlogous and pseudogenes in the non-annotated part (10%) of the chicken genome cannot be excluded at this stage. with regard to the complex ifn pathways and a number of major missing genes in chicken, the innate immune genes, as general sensors of self and non-self, are under continues evolutionary selection pressure compared to the pathogen-specific adaptive immune responses. therefore, it is plausible that ifit proteins are generated by paralogue expansions and/or gene deletions in chicken 6 . ifit5 is the only gene that is missing in the mice and rat genomes 6 whereas it is the only gene identified in chicken, as this study shows. it remains to be determined in future if chifit5 also plays additional antiviral functions of the redundant ifit1 and ifit3. we observed a profound transcription of chifit5 gene by diverse stimuli that acted upon the ifn induction or ifn signalling pathways, which was further confirmed by the transcriptomics profiling of the mdv-infected chicken fibroblasts. interestingly, structural and functional characterization of the chifit5 promoter, required for effective transcriptional regulation of chifit5, was mapped within 100 bp of the transcriptional start site and carried a single isre motif. this minimum essential requirement of the promoter justifies several folds induction of chifit5 gene as early as 2 hours of post ndv-infection. although transcription of ifit5 was generally proportional to the virus replication and ifn induction, an ifn-independent regulation of chifit5 is also possible, especially when the chifit5 gene up-regulation was observed in earlier time points of virus-infection when the ifn gene was barely detectable. it is also plausible that transcriptional activation of chifit5 is directly regulated by irf7 or related transcription factors and thus warrants future investigations. nevertheless, these transcriptional patterns augment essential roles of the chifit5 protein during the early stages of virus replication such as interaction with viral genetic material (viral rna/dna) and viral protein translation. previous rna-protein analysis revealed that all ifit proteins assemble into multimeric complexes (except ifit5) and that these interactions are crucial for co-functionalities of these proteins 7 . while all ifit proteins can make multimers, ifit5 exists as a poorly characterized monomer 31 . probably due to these structural flexibilities, reports on ifit5 functionalities are inconsistent 7,30-32 . however, ifit5 protein can arguably sense a broad range of rna structures including single stranded rna with mono-(p) and tri-phosphates (ppp) at the 5′ ends, double stranded dna and rna with cap0 modifications 8, 13, 18 . in order to understand the binding potential of chifit5 to modified rna that either interacts specifically with human ifit5 or with human ifit1, we coupled modified rna-coated beads with quantitative binding assays for chifit5. our results indicated that chifit5 specifically interacted with rna that carried 5′-ppp modifications and failed to interact with rna in which 5′-ppp was replaced with oh. these rna-protein interaction studies highlighted the principal roles of chifit5 for direct recognition of foreign ppp-rna and to subsequently exert downstream antiviral activities. since ppp-rna is found within the genome of most viruses carrying negative sense single stranded rna genomes such as influenza, ndv, vsv 38 , and ppp-rna is produced as an intermediate product during the replication of viruses with positive sense rna genomes such as coronaviruses 39 , it is plausible that chifit5 sense foreign rna (bearing ppp-rna) while ignoring self rna (bearing cap in the case of mrna and monophosphate in the case of rrna and trna). compared to four essential ssrna cellular sensors including rig-i, tlr7, tlr8 and ifit5 in mammals 1,13 , rig-1 is missing and tlr8 is disrupted due to insertion of retroviruses in the tlr locus in chickens 5, 39, 40 . because of these fundamental differences in innate immune genes between avian (e.g. chicken) and mammals, future studies are needed to investigate if the interaction of ifit5 with 5′-ppp-ssrna can induce downstream ifn signalling 11 , and if so, does this interaction compensate for the deficiency of rig-i and tlr8 in chickens. this specific interaction with ppp-rna could lead to attenuation of virus replication by sequestering viral rna for transcription and translation. to investigate this possibility, we applied both gain-in-function and loss-in-function methodologies and evaluated the antiviral potential of ifit5 against negative sense single stranded rna viruses, including a poultry specific (ndv) and model rna virus (vsv). lentivirus-delivered stable expression of chifit5 or huifit5 compromised the replication of viruses, whereas crispr/cas9 mediated knocking-out of the chifit5 gene supported virus replication in chicken fibroblasts. intriguingly, overexpression of human ifit1, ifit2 and ifit3 failed to establish an antiviral state in chicken fibroblasts, suggesting that chickens have opted exclusively for the antiviral activities of ifit5. our attempts to monitor virus replication in mosaic transgenic chickens overexpressing chifit5, or silenced for endogenous chifit5 yielded strong evidence that this cytokine possesses antiviral activities in developing embryos. thus rcas-mediated gene delivering and silencing approaches can be exploited to study gene functionalities in ovo at the early embryonic developmental stage and may establish the basis for evaluation of genetic resistance against pathogens. taken together, we characterized the ifit locus in chicken and systemically analysed the functional rationale for antiviral activities of chifit5 against rna viruses using both functional genomics and molecular biological approaches. the foundations built in this study warrant future investigations to assess the potential of chifit5 in sensing the nucleic acid of many diverse viruses and bacteria (which also generate ppprna), and the impact of these interactions on host innate immunity. data mining and sequence analysis. chicken genome (ensembl) and expressed sequence tags (est) databases were screened for the homologues of ifit family gene members using the basic local alignment search tool (blast) algorithm. a single transcript showing high sequence-similarity to human ifit5 was identified in the putative ifit locus. using sequences from public databases and transcriptomics data from marek's disease virus (mdv)-infected chicken embryo fibroblasts (cef), an open reading frame (orf) was revealed and extracted. the chicken ifit5 (chifit5) coding region was amplified from ndv-infected primary cefs, whereas sequence-covering gap in the ifit locus was amplified from genomic dna using primers mentioned in supplementary table 1 . the genomic nucleotide sequence of chifit5 promoter region was amplified using primers provided in supplementary table 1 . the orf and homology searches for chifit5 were carried out in the orf finder programme (http://www.ncbi.nlm.nih.gov/projects/gorf) and blast tool (http://www.ncbi.nlm.nih.gov/ blast/) integrated in the ncbi database. possible gene transcription start sites were identified using promoter predictor programme (http://www.fruitfly.org/seq_tools/promoter.html) whereas potential transcription factor binding sites were identified using the matinspector server (http://www.genomatix.de). gene synteny and tetratricopeptide repeats (tpr) were predicted using ensemble as well as conserved domain databases, respectively. the ifit5 sequences from aves and non-aves were acquired from ncbi and aligned using clustalw programme. the phylogenetic analysis was performed using neighbour-joining method with bootstrap value of n = 2,000 in the mega programme version 6 41 . cells culture, media and antibodies. cefs were prepared from 9-day-old embryonated eggs at the pirbright institute as described previously 42 . immortalized chicken fibroblasts (df-1), human embryonic kidney cells 293t (hek-293t) and madin-darby canine kidney (mdck) cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% foetal bovine serum (fbs), 1% penicillin and streptomycin (p/s) at 37 °c in 5% co 2 incubator. amv-3c2-s (gag) antibodies were purchased from hybridoma bank of iowa, university of iowa. α-v5 and j2 antibodies for the detection of v5 tag and dsrna were from genetex, and scicons, respectively. alexa-flour 568 secondary antibodies were purchased from invitrogen carlsbad, ca, usa and irdye 800cw α-mouse secondary antibodies were acquired from li-cor, nebraska usa. poly i:c scientific reports | (2018) 8:6794 | doi:10.1038/s41598-018-24905-y (a synthetic analogoue of dsrna), dimethyl sulfoxide (dmso) and lipopolysaccharide (lps), were purchased from invivogen and sigma whereas chicken ifn-β was produced in hek-293t cells 43 . (ndv-gfp) was generated using reverse genetics system as described before 44 and rescued virus particles were propagated in embryonated chicken eggs 45 . the ndv-gfp strain was quantified using immunostaining and expressed as focus-forming units (ffu). vesicular stomatitis virus (vsv) expressing gfp (vsv-gfp) was kindly provided by dennis rubbenstroth (institute for virology, medical centre -university of freiburg, germany). vsv-gfp was propagated and quantified in df-1 cells and was represented in ffu or images showing plaques. allantoic fluids and infectious viruses from cell culture supernatants were titrated by plaque assays on mdck cells. briefly, mdck cells were inoculated with 10-fold serially diluted samples and overlaid with 0.6% agarose (oxoid, hampshire, uk) in overlay dmem (1× mem, 0.21% bsa v, 1 mm l-glutamate, 0.15% sodium bicarbonate, 10 mm hepes, 1× penicillin/streptomycin (gibco, carlsbad, ca, usa) and 0.01% dextran deae, with 2 µg ml −1 tpck trypsin (sigma-aldrich, dorset, uk). plates were incubated at 37 °c for 72 h and plaques were developed using crystal violet stain containing methanol. plasmids construction and mutagenesis. the full-length chicken and human ifit5 was pcr-amplified using primers that were tailed with 5′ bamhi site, 3′ ecori/spei site and a consensus kozak translation sequence (ccaccatg) (supplementary table 1 ). the bamhi and ecori/spei digested amplicons were sub-cloned in the mammalian expression vector, pefplink-v5 (kindly provided by steve goodbourn, st. george's university of london), which contains an n-terminal v5 tag. for identification of cis-acting elements in the chifit5 promoter, a 1.2 kb genomic sequence was amplified and cloned into the kpni and xhol sites in the promoter-less vector, pgl3.1 basic (promega) and named as pchifit5-1053/+123. subsequent five truncated versions of the promoter were amplified from full-length pchifit5-1053/+123 using primers mentioned in the supplementary table 1 and cloned between kpni and xhol sites in the pgl3.1 basic vector. for production of the chifn-β, the orf for chifn-β (accession number, nm_001024836) was cloned in pcdna3.1+ and final constructs were labelled as pcdna3.1-chifn-β 43 . the reporter plasmid pgl3-p-chmx-luc was kindly provided by nicolas ruggli, switzerland and renilla luciferase plasmid (phrl-sv40) was purchased from promega, madison, wi, usa. mitochondrial (dsred2-mito-7, #55838)) and er (mcherry-er-3, #55041) markers were obtained from addgene. triple thymidine duplex (tttnnnttt) in pchifit5-101/+123-wt construct was mutated into tatnnntat using quikchange lightning site-directed mutagenesis kit (agilent technologies) and was named as pchifit5-101/+123-mut. all mutagenesis oligonucleotides were designed in the quikchange primer design tool and these primers are provided in the supplementary table 1. all clones were sequenced from both ends for correct frame and orientation or were digested with unique cleavage sites to confirm the gene inserts. confocal microscopy. chicken cells were transfected with individual or combined plasmids for indicated time points using lipofectamine 2000 (invitrogen) at a ratio of 1:3 or were infected with lentiviruses, retroviruses or ndv-gfp for indicated time points. these cells were then fixed for 1 h in 4% paraformaldehyde and permeabilized using 0.1% triton-x100 before incubation with primary antibodies raised against v5 tag, dsrna (j2), or gag protein of retroviruses. additionally, depending upon the experimental needs, different fluorescent markers (rfp, gfp) were used. afterwards, cells were incubated with corresponding secondary antibodies at 37 °c for 2 h. after brief staining with 4′,6-diamidino-2-phenylindole (dap1) (nuclear), slides were visualized using a leica sp5 confocal laser scanning microscope. western blotting. all the transfections for subsequent western blot analysis were performed following the same protocol as described for immunofluorescence unless otherwise indicated. cells were lysed in a hypotonic buffer and protease inhibitor cocktail (sigma). proteins were separated by sds-page under reducing conditions and analysed by western blotting using anti-v5 (gentek) and irdye-labelled secondary antibodies (li-cor biosciences). the signals were acquired and quantified using the odyssey infrared imaging system (li-cor biosciences). ifn bioassay. ifn-induced protection against vsv-gfp was used to identify ifn-producing stable clones and to quantify ifn preparations, as described before 46 . briefly, df-1 cells were seeded in 96-well plates until they are 90% confluent and treated with serial dilutions of supernatants containing interferons for 24 hours. these interferon stimulated cells were inoculated with vsv-gfp (moi of 1). at 24 hours post-infection, vsv-gfp replication was correlated with the change in gfp fluorescence signal intensities using luminometer (promega, madison, wi, usa). the percentage antiviral activity of ifns were determined by comparing the percentage reduction of corrected gfp signal intensity (gfp signal intensity of ifn treated and virus infected wells minus background fluorescence signal intensity of uninfected control) with the mock treated and vsv-gfp-infected control wells. one unit (u) of ifn in the tested ifn preparations was defined as the volume containing 50% inhibitory activity against vsv-gfp. a total of 1000 us of ifns were used for stimulation of cef or df-1 cells. tronic expression and gateway-compatible destination vector (ptrip.cmv.ivsb.gene.ires.tagrfp) for lentiviruses was kindly provided by charles rice, the rockefeller university, usa. to generate chifit5 entry clone, gene encoding chifit5 was pcr amplified with oligonucleotides (supplementary table 1 ) containing attb sites flanking gene-specific sequences. pcr products were purified over qiagen columns (qiagen) and cloned into pdonr (invitrogen) with bp clonase. bp clonase reactions were transformed into escherichia coli (invitrogen), and colonies were screened by restriction digestion and sequencing. the gene sequences from pentr clones scientific reports | (2018) 8:6794 | doi:10.1038/s41598-018-24905-y were moved into ptrip.cmv.ivsb.gene.ires.tagrfp using lr clonase ii (invitrogen) according to the manufacturer's instructions. after lr reaction products transformation, one or two colonies for each construct were grown in 3 ml luria-bertani (lb) broth with ampicillin, and transfection-quality plasmid dna was purified over anion-exchange columns (qiagen). lentivirus constructs expressing human ifit1, ifit2, ifit3, ifit5, irf1 (positive control) and ffluc (negative control) were kindly provided by charles rice, the rockefeller university, usa. all constructs were sequenced using primers provided in supplementary table 1 to confirm the gene insertion before rescuing lentiviruses. poly-lysine pre-coated plates with seeding density of 4 × 10 5 per well of 6-wells plates and were co-transfected with gene expressing proviral dna (huifit1, huifit2, huifit3, huifit5, chifit5, huirf1, or ffluc), hiv-i gag-pol and vsv-g in a ratio of 1:0.8:0.2 using lipofectamine 2000 (invitrogen). supernatants collected at 48 h and 72 h post-transfection were cleared by centrifugation (1500 rpm for 5 min) and were pooled and supplemented with 20 mm hepes and 4 μg/ul polybrene (sigma). for titration of lentiviral pseudoparticles, cef cells (1 × 10 6 ) were transduced with serial dilutions of individually rescued pseudoparticles for 24 hours. trypsinized cells were fixed with 4% paraformaldehyde and processed for facs for quantification of percentage rfp+ cells. the volume of the lentiviral pseudoparticles that infected 50% of cef cells was used to transduce cefs. titrated lentiviral pseudoparticles were stored at −80 °c before use and the same stock of the virus was used for all experimentation. duced with 10 moi of lentivirus-expressing specific gene in dmem media containing 5% fbs, 20 mm hepes and 4 μg/ml polybrene. transduction was facilitated by centrifugation (1000 g for 1 h at 37 °c) and cells were incubated at 37 °c. a day later, cells were infected with gfp-tagged virus (ndv-gfp) at 0.5 moi and the infection was stopped after 1 hr and replaced with fresh dmem containing 5% fbs. after 24 hours of infections, cells were trypsinised and the cell suspensions were incubated with live dead marker (near ir cat no: l10119) according to the manufacturer's protocol. the cells were then fixed with 2% paraformaldehyde (pfa) for 30 min before analysing the cells by flow cytometry. as described before 47 , live and singlet cells were gated based on forward and side scatter, and four-quadrant plots were generated using the untransduced and uninfected (rfp negative and gfp negative), uninfected (rfp positive and gfp negative), and untransduced (rfp negative and gfp positive) cells. analysis was carried out using flowjo software applying the same gating and analysis strategies for all samples. construction of ifit5 knockout cell line using crispr/cas9. a synthetic gene-targeting approach was applied to specifically knockout the chicken ifit5 from the chicken embryo fibroblasts. for this purpose, two components (crrna and tracrrna) of single guide rna (sgrna) which are crucial for targeting specificity and scaffolding/binding ability for crispr associated protein 9 (cas9) nuclease were synthesized by dharmacon. targeting the beginning of the second exon of chicken ifit5, two individual crispr rna (crrna) were designed with the highest-predicted score and lowest off-target affects; sgrna1 acaggagaagtctcgttacc and sgrna2 gcttggatctactaccacat. using dharmafect duo transfection reagents, df-1 cells were co-transfected with individual crrna and a common trans-activating crrna (tracrrna) as well as plasmid expressing cas9 nuclease and puromycin resistance gene (puro r ) separated by self-cleavage t2a sequence. cells were split after 48 hours transfection and selected for puromycin antibiotics (10 μg/ml) for one week or until complete eradication of non-transfected control cells. for fast and efficient enrichment of genetic modification, a population of cells with stable integration was enriched using facs. for this purpose, puromycin-selected cells were transfected with gfp-expression plasmid and individual cells were sorted by facs before being seeded in 96-well plates. at least 15 clones were expanded and the relative frequency of gene editing in the puromycin-resistant and facs-enriched cells was estimated from a dna mismatch detection assay using t7 endonuclease i (t7e1) (neb). the dna fragments flanking the target editing sites were amplified from genomic dna extracted by dneasy kits (qiagen) using primers mentioned in supplementary table 1. a total of 200 ng of the pcr products were denatured at 95 °c and allowed to anneal gradually at room temperature to form heteroduplex dna. the re-hybridized dna was digested with t7ei and resolved in a 1.8% agarose gel to determine the gene editing efficiency. additionally, the pcr products were sequenced using pcr-amplification primers and aligned with the corresponding wild-type genomic sequence to identify mutations, deletions and insertions. t7ei and sequence verified clones were used to monitor virus replication. rna was extracted from ifn-β (1000 u), lps (10 μg/ml), dsrna (5 μg/ml), or ndv-stimulated (moi of 1) df1 or cefs using trizol reagents (invitrogen, carlsbad, ca, usa). additionally, organs were collected from specific pathogen free (spf) chickens, which were infected or mock-treated (intranasally) for 2 days with 10 6 pfu of a/ chicken/pakistan/udl-01/2008 (h9n2). a total of 200 ng of rna was used in pcr reactions using superscript ® iii platinum ® sybr ® green one-step qrt-pcr kit (invitrogen, carlsbad, ca, usa). the abundance of specific mrna was compared to the 28s rrna (supplementary table 1 ) in the applied biosystems prism 7500 system. the reaction was carried out in abi 7500 light cycler using the following thermo profile; 50 °c for 5 minutes hold, 95 °c for 2 minutes hold, followed by 40 cycles of 95 °c for 3 seconds and 60 °c for 30 seconds. melting curve was determined at 95 °c for 15 seconds, 60 °c for 1 minute, 95 °c for 15 seconds and 60 °c for 15 seconds. primers for isgs including chifit5 are provided in supplementary table 1 . primers specific for a conserved region of the influenza a and ndv matrix genes were used as described previously 48,49 . short hairpin design and expression systems. to silence endogenous chifit5 gene in developing embryos, a total of three 22-nucleotides-long rna interference (rnai) short hairpin rnas (shrna) were designed using the genscript rnai target finder (https://www.genscript.com/ssl-bin/app/rnai). double stranded dna products for each of three chifit5 specific and a control scrambled target were generated by pcr using random and gene-specific oligonucleotides together with 1hp-l and 1hp-r (supplementary table 1 ) as described before 33 . the amplified pcr products were cloned between nhei and mlui into microrna (mirna) cloning sites of prfprnaic (kind gift of stuart wilson, the university of sheffield, uk). all shrna coding plasmids were sequenced to confirm the inserts and orientations. to evaluate the silencing potential of individual shrna, df-1 cells were transfected with 500 ng plasmid using lipofectamine 2000 according to the manufacturer's protocol and the knockdown effects on the chicken ifit5 was monitored and compared with the scrambled rna transfected control. next, the validated shrna cassette that included chicken u6 promoter, individual shrna, leader and trailer sequences was transferred to the rcasbp(a) retrovirus vector between noti and clai sites. rescue of rcasbp(a)-sh3ifit5 retroviruses and generation of mosaic transgenic chickens are detailed below. to determine responsiveness of chicken ifit5 promoter to chicken ifn-β, dsrna, and virus-stimulation, chicken fibroblasts were grown in 96-well plate format at 2 × 10 4 to 4 × 10 4 cells in addition pgl3-p-chmx-luc was used as a positive control whereas pgl3.1 basic vector was used as a negative control. correspondingly, df-1 cells were co-transfected with phrl-sv40 and pchifit5-101/+123-wt or pchifit5-101/+123-mut constructs. all transfections were performed using lipofectamine 7sk-as) was linearized with unique bamhi restriction digestion and the purified dna was used for in vitro transcription in the presence of bioin-16-utp using ribomax ™ large scale rna production system-sp6 (promega, cat# p1280) as recommended by the manufacturer and reported previously 17 with a few modifications. briefly, a reaction of 100 μl was established containing 20 μl 5 × sp6 buffer, 10 μl ntp-bioutp mixtures, 5 μg linearized plasmid, and 10 μl enzyme mix. the reaction mixture was first incubated at 37 °c for 3 hours followed by digestion of the dna remnant with 4u rnase-free dnase (thermo scientific) for another 30 minutes at 37 °c. the biotinylated uracil triphosphate (bioin-16-utp) was incorporated during in vitro transcription for purification of ribonucleoproteins and due to nascent nature of polymerase a 5′-ppprna was over-hanged as a signature for ifit5 protein interaction. after the completion of in vitro transcription, the quality of in vitro transcription rna was assessed by agarose gel electrophoresis and rna was purified with rneasy minelute cleanup kit (qiagen) according to the manufacturer's recommendations. a total of 30 μg of purified in vitro transcribed and biotinylated ppp-rna was either mock treated or rna samples were purified with rneasy minelute cleanup kit and eluted with 40 μl nuclease-free water for use in the rna-protein interactions to prepare chifit5 protein, chicken df-1 cells (1 × 10 6 ) were transfected with 5 μg v5-tagged chifit5 plasmid for 48 hours and lysed with tap buffer in the presence of protease and rnase inhibitors. the rna-coated beads were incubated with 2 mg chifit5 protein lysate on a rotary wheel for 4 hours at 4 °c, and washed three times to remove unbound proteins the gfp/gag expression-confirmed cell cultures were split into 25 cm 2 flasks and were passaged again into 75 cm 2 flasks after 3 days. finally cells were expanded into 150 cm 2 flasks until the desired number (10 6 cells/egg) was achieved. mosaic-transgenic chicken embryos were generated by inoculation of one million rcasbp(a)-chifn-β, rcasbp(a)-chifit5, rcasbp(a)-shrna3 and rcasbp(a)-wt infected df-1 cells at day 3 post-embryonation or were left un-infected. at day 9 post-embryonation (6 days post-infection), each egg was either left unchallenged or was challenged with 100 pfu ndv-gfp. embryo mortality was monitored daily and allantoic fluids were harvested at 14 days of embryonation and were subjected to the plaque assay for virus quantification. statistical analysis. pairwise comparisons of treated and control groups were performed using student's t-test interferons and 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ribonuclease p: a plurality of ribonucleoprotein enzymes when your cap matters: structural insights into self vs non-self recognition of 5′ rna by immunomodulatory host proteins sequestration by ifit1 impairs translation of 2′ o-unmethylated capped rna inhibition of translation by ifit family members is determined by their ability to interact selectively with the 5′-terminal regions of cap0-, cap1-and 5′ ppp-mrnas the broad-spectrum antiviral functions of ifit and ifitm proteins the rcas vector system the glucocorticoid attenuated response genes garg-16, garg-39, and garg-49/irg2 encode inducible proteins containing multiple tetratricopeptide repeat domains when two strands are better than one: the mediators and modulators of the cellular responses to double-stranded rna double-stranded rna is detected by immunofluorescence analysis in rna and dna virus infections, including those by negative-stranded rna viruses activation of the pkr/eif2α signaling cascade inhibits replication of newcastle disease virus the chicken mx-promoter contains an isre motif and confers interferon inducibility to a reporter gene in chick and monkey cells differential expression profile of chicken embryo fibroblast df-1 cells infected with cell-adapted infectious bursal disease virus triggering the innate antiviral response through irf-3 activation the rna polymerase ii core promoter genotyping with crispr-cas-derived rna-guided endonucleases distinct induction patterns and functions of two closely related interferoninducible human genes, isg54 and isg56 crystal structure and nucleotide selectivity of human ifit5/isg58 coordinated regulation and widespread cellular expression of interferon-stimulated genes (isg) isg-49, isg-54, and isg-56 in the central nervous system after infection with distinct viruses prolonged effect of baff on chicken b cell development revealed by rcas retroviral gene transfer in vivo antiviral activity of lambda interferon in chickens a robust system for rna interference in the chicken using a modified microrna operon association of rig-i with innate immunity of ducks to influenza rig-i in rna virus recognition a virological view of innate immune recognition identification and characterization of a functional, alternatively spliced toll-like receptor 7 (tlr7) and genomic disruption of tlr8 in chickens molecular evolutionary genetics analysis version 6.0 cell-culture methods a novel cytokine with antiviral activities tissue tropism in the chicken embryo of non-virulent and virulent newcastle diseases strains that express green fluorescence protein biological characterization and phylogenetic analysis of a novel genetic group of newcastle disease virus isolated from outbreaks in commercial poultry and from backyard poultry flocks in pakistan a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity development of a real-time reverse-transcription pcr for detection of newcastle disease virus rna in clinical samples development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h5 and h7 hemagglutinin subtypes construction and rescue of ifit5 expressing rcas system, and generation of transgenic embryos. the orf of chicken ifit5 and coding regions (signal and mature peptide sequence) of the chifn-β were amplified from rna extracted from the ndv-infected primary cefs. the amplified products were sub-cloned into an improved version of rcasbp(a)-δf1 (kindly provided by stephen h. hughes, national cancer institute, md, usa) via the clai and muli restriction sites which replace the src gene while maintaining the splice accepter signals. the resultant constructs were named as rcasbp(a)-chifit5 and rcasbp(a)-chifn-β. similarly, a gfp encoding rcasbp(a), referred as rcasbp(a)-egfp, was generated by introducing the coding sequence of the gfp in between the clai and muli sites. additionally, a codon optimized and shrna-silencing resistant chifit5 gene was chemically synthesized (geneart, invitrogen) and cloned in the corresponding sites and labelled as rcasbp(a)-shrna3. the inserted gene orientation and sequence validity were confirmed by dna sequencing.to rescue recombinant rcasbp(a) viruses, a total of 2.5 × 10 5 df-1 cells were seeded in 25 cm 2 flasks and maintained at 37 °c, 5% (vol/vol) co 2 for 24 hours (~80% confluent). cells were washed with pbs and transfected ethics statement. all animal studies and procedures were carried out in strict accordance with the guidance and regulations of european and united kingdom home office regulations under animal risk assessment numbers ar000789 and ar000791. as part of this process the work has undergone scrutiny and approval by the ethics committee at the pirbright institute. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-24905-y. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-342189-ya05m58o authors: banerjee, abhik k.; blanco, mario r.; bruce, emily a.; honson, drew d.; chen, linlin m.; chow, amy; bhat, prashant; ollikainen, noah; quinodoz, sofia a.; loney, colin; thai, jasmine; miller, zachary d.; lin, aaron e.; schmidt, madaline m.; stewart, douglas g.; goldfarb, daniel; de lorenzo, giuditta; rihn, suzannah j.; voorhees, rebecca; botten, jason w.; majumdar, devdoot; guttman, mitchell title: sars-cov-2 disrupts splicing, translation, and protein trafficking to suppress host defenses date: 2020-10-08 journal: cell doi: 10.1016/j.cell.2020.10.004 sha: doc_id: 342189 cord_uid: ya05m58o sars-cov-2 is a recently identified coronavirus that causes the respiratory disease known as covid-19. despite the urgent need, we still do not fully understand the molecular basis of sars-cov-2 pathogenesis. here, we comprehensively define the interactions between sars-cov-2 proteins and human rnas. nsp16 binds to the mrna recognition domains of the u1 and u2 splicing rnas and acts to suppress global mrna splicing upon sars-cov-2 infection. nsp1 binds to 18s ribosomal rna in the mrna entry channel of the ribosome and leads to global inhibition of mrna translation upon infection. finally, nsp8 and nsp9 bind to the 7sl rna in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. our results uncover a multipronged strategy utilized by sars-cov-2 to antagonize essential cellular processes to suppress host defenses. coronaviruses are a family of viruses with notably large single-stranded rna genomes and broad species tropism among mammals (graham and baric, 2010) . recently, a coronavirus, sars-cov-2, was discovered to cause the severe respiratory disease known as covid-19. it is highly transmissible within human populations and its spread has resulted in a global pandemic with more than a million deaths to date (andersen et al., 2020; zou et al., 2020) . we do not fully understand the molecular basis of infection and pathogenesis of this virus in human cells. accordingly, there is an urgent need to understand these mechanisms to guide the development of therapeutics. sars-cov-2 encodes 27 proteins with diverse functional roles in viral replication and packaging( bar-on et al., 2020; wang et al., 2020) . these include 4 structural proteins: the nucleocapsid (n, which binds the viral rna), and the envelope (e), membrane (m), and spike (s) proteins, which are integral membrane proteins. in addition, there are 16 non-structural proteins (nsp1-16) which encode the rna-directed rna polymerase, helicase, and other components required for viral replication (da silva et al., 2020) . finally, there are 7 accessory proteins (orf3a-8) whose function in viral replication or packaging remain largely uncharacterized (chen and zhong, 2020; finkel et al., 2020) . as obligate intracellular parasites, viruses require host cell components to translate and transport their proteins and to assemble and secrete viral particles (maier et al., 2016) . upon viral infection, the mammalian innate immune system acts to rapidly detect and block viral infection at all stages of the viral life cycle (chow et al., 2018; jensen and thomsen, 2012; wilkins and gale, 2010) . the primary form of intracellular viral surveillance engages the interferon pathway, which amplifies signals resulting from detection of intracellular viral components to induce a systemic type i interferon response upon infection (stetson and medzhitov, 2006) . specifically, cells contain various rna sensors (such as rig-i and mda5) that detect the presence of viral rnas, promote nuclear translocation of the transcription factor irf3 leading to transcription, translation, and secretion of interferon (e.g. ifn-α and ifn-β) . binding of interferon to cognate cell-surface receptors leads to transcription and translation of hundreds of antiviral genes. j o u r n a l p r e -p r o o f 4 in order to successfully replicate, viruses employ a range of strategies to counter host antiviral responses (beachboard and horner, 2016) . in addition to their essential roles in the viral life cycle, many viral proteins also antagonize core cellular functions in human cells to evade host immune responses. for example, human cytomegalovirus (hcmv) encodes proteins that inhibit class 1 major histocompatibility (mhc) display on the cell surface by retaining mhc proteins in the endoplasmic reticulum (miller et al., 1998) , polioviruses encode proteins that degrade translation initiation factors (eif4g) to prevent translation of 5'-capped host mrnas (kempf and barton, 2008; lloyd, 2006) , and influenza a encodes a protein that modulates mrna splicing to degrade the mrna that encodes rig-i (kochs et al., 2007; zhang et al., 2018) . suppression of the interferon response has recently emerged as a major clinical determinant of covid-19 severity , with almost complete loss of secreted ifn characterizing the most severe cases (hadjadj et al., 2020) . the extent to which sars-cov-2 suppresses the interferon response is a key characteristic that distinguishes covid-19 from sars and mers (lokugamage et al., 2020) . several strategies have been proposed for how the related sars-and mers-causing viruses may hijack host cell machinery and evade immune detection, including repression of host mrna transcription in the nucleus (canton et al., 2018) , degradation of host mrna in the nucleus and cytoplasm (kamitani et al., 2009; , and inhibition of host translation (nakagawa et al., 2018) . nonetheless, the extent to which sars-cov-2 uses these or other strategies, and how they may be executed at a molecular level remains unclear. understanding the interactions between viral proteins and components of human cells is essential for elucidating their pathogenic mechanisms and for development of effective therapeutics. because sars-cov-2 is an rna virus and many of its encoded proteins are known to bind rna (sola et al., 2011) , we reasoned that these viral proteins may interact with specific human mrnas (critical intermediates in protein production) or non-coding rnas (critical structural components of diverse cellular machines) to promote viral propagation. here, we comprehensively define the interactions between each sars-cov-2 protein and human rnas. we show that 10 viral proteins form highly specific interactions with mrnas or ncrnas, including those involved in progressive steps of host cell protein production. we show j o u r n a l p r e -p r o o f 5 that nsp16 binds to the mrna recognition domains of the u1 and u2 rna components of the spliceosome and acts to suppress global mrna splicing in sars-cov-2-infected human cells. we find that nsp1 binds to a precise region on the 18s ribosomal rna that resides in the mrna entry channel of the initiating 40s ribosome. this interaction leads to global inhibition of mrna translation upon sars-cov-2 infection of human cells. finally, we find that nsp8 and nsp9 bind to discrete regions on the 7sl rna component of the signal recognition particle (srp) and interfere with protein trafficking to the cell membrane upon infection. we show that disruption of each of these essential cellular functions acts to suppress the type i interferon response to viral infection. together, our results uncover a multipronged strategy utilized by sars-cov-2 to antagonize essential cellular processes and robustly suppress host immune defenses. we cloned all 27 of the known sars-cov-2 viral proteins into mammalian expression vectors containing an n-terminal halotag (los et al., 2008) (figure s1a , methods), expressed each in hek293t cells, and exposed them to uv light to covalently crosslink proteins to their bound rnas. we then lysed the cells and purified each viral protein using stringent, denaturing conditions to disrupt any non-covalent associations and capture those with a uv-mediated interaction ( figure 1a , methods). as positive and negative controls, we purified a known human rna binding protein (ptbp1) and a metabolic protein (gapdh) (figure s1a-e). we successfully purified 26 of the 27 viral proteins (figure s1a ; full-length spike was not soluble when expressed). we found that 10 viral proteins (nsp1, nsp4, nsp8, nsp9, nsp12, nsp15, nsp16, orf3b, n, and e protein) bind to specific host rnas (p-value < 0.001, figure 1b , table s1), including 6 structural ncrnas and 142 mrnas (table s1). these include mrnas involved in protein translation (e.g. cops5, eif1, and rps12,), protein transport (atp6v1g1, slc25a6, and tomm20), protein folding (hspa5, hspa6, and hspa1b), transcriptional regulation (yy1, id4, and ier5), and immune response (jun, aen, and j o u r n a l p r e -p r o o f 6 rack1) (fdr < 0.05, figure 1b , s1f). importantly, the observed interactions are highly specific for each viral protein, and each protein binds to a precise region within each rna ( figures 1c, s1f ). using these data, we identified several viral proteins that interact with structural ncrna components of the spliceosome (u1 and u2 snrna), the ribosome (18s and 28s rrna), and the signal recognition particle (7sl) (figure 1b) . because these molecular machines are essential for three essential steps of protein production -mrna splicing, translation, and protein trafficking -we focused on their interactions with viral proteins to understand their functions and mechanisms in sars-cov-2 pathogenesis. after transcription in the nucleus, nascent pre-mrnas are spliced to generate mature mrnas which are translated into protein. splicing is mediated by a complex of ncrnas and proteins known as the spliceosome. specifically, the u1 small nuclear rna (snrna) hybridizes to the 5' splice site at the exon-intron junction and the u2 snrna hybridizes to the branchpoint site within the intron to initiate splicing of virtually all human mrnas (séraphin et al., 1988) . we identified a highly specific interaction between the nsp16 viral protein and the u1 and u2 snrnas ( figure 1b) . because u1 and u2 are small rnas (164 and 188 nucleotides, respectively), we noticed strong enrichment of nsp16-associated reads across the entire length of each. to more precisely define the binding sites, we exploited the well-described tendency of reverse transcriptase to preferentially terminate when it encounters a uv-crosslinked protein on rna (konig et al., 2010) (figures 1a, s1d) . we determined that nsp16 binds to the 5' splice site recognition sequence of u1 (figures 2a-b, s2a based on the locations of the nsp16 binding sites relative to the mrna recognition domains of the u1/u2 spliceosomal components, we hypothesized that nsp16 might disrupt splicing of newly transcribed genes ( figure 2f ). to test this, we co-expressed nsp16 in human cells along with a splicing reporter derived from irf7 (an exon-intron-exon minigene) fused to gfp (majumdar et al., 2018) . in this system, if the reporter is spliced, then gfp is made; if not, translation is terminated (via a stop codon present within the first intron) and gfp is not produced ( figure 3a) . we observed a >3-fold reduction in gfp levels in the presence of nsp16 compared to a control human protein (figures 3b, s3a ). to explore whether nsp16 has a global impact on splicing of endogenous mrnas, we measured the splicing ratio of each gene using nascent rna sequencing. specifically, we metabolically labeled nascent rna by feeding cells for 20 minutes with 5-ethynyl uridine (5eu), purified and sequenced 5eu-labeled rna, and quantified the proportion of unspliced fragments spanning the 3' splice site of each gene (figure 3c, s3b) . we observed a global increase in the fraction of unspliced genes in the presence of nsp16 compared to controls ( figure 3d, s3c,d) . given that nsp16 is sufficient to suppress global mrna splicing, we expect that its expression in sars-cov-2-infected cells would result in a global mrna splicing deficit. to test this, we infected human lung epithelial cells (calu3) with sars-cov-2 and measured splicing levels of newly transcribed mrnas compared to a mock infected control. as expected, we observed a global increase in the fraction of unspliced transcripts upon sars-cov-2 infection, with ~90% of measured genes showing increased intron retention (figure 3e, s3e ). together these results indicate that nsp16 binds to the splice site and branch point sites of u1/u2 to suppress global mrna splicing in sars-cov-2 infected cells ( figure 3f) . although nsp16 is known to act as an enzyme that deposits 2'-o-methyl modifications on viral rnas (decroly et al., 2011) , our results demonstrate that it also acts as a host virulence factor. global disruption of mrna splicing may act to decrease host protein and mrna levels by triggering nonsense-mediated decay of improperly spliced mrnas (kurosaki et al., 2019) . consistent with this, we observed a strong global decrease in steady-state mrna levels (relative to ncrna levels) upon sars-cov-2 infection ( figure s3f ). j o u r n a l p r e -p r o o f 8 inhibition of mrna splicing suppresses host interferon response to viral infection because many of the key genes stimulated by interferon (ifn) are spliced, we reasoned that mrna splicing would be critical for a robust ifn response. to test this, we utilized a reporter line engineered to express alkaline phosphatase upon ifn signaling (mimicking an antiviral response gene). this ifn stimulated gene (isg) reporter line can be stimulated using ifn-β and assayed for reporter induction. we observed strong repression of this ifn responsive gene upon expression of nsp16 ( figure 3g ) and upon addition of a small molecule that interferes with spliceosomal assembly (figure s3g ). these results demonstrate that one outcome of nsp16mediated inhibition of mrna splicing is to reduce the host cells' innate immune response to viral recognition. consistent with such a role, we observed an increase in intron retention within multiple ifn-responsive genes (such as isg15 and rig-i) upon sars-cov-2 infection ( figure 3h , s3h-i). once exported to the cytoplasm, spliced mrna is translated into protein on the ribosome. initiation of translation begins with recognition of the 5' cap by the small 40s subunit (which scans the mrna to find the first start codon). we observed that nsp1 binds exclusively to the 18s ribosomal rna (figure 1b and s4a ) -the structural rna component of the 40s ribosomal subunit. several roles for nsp1 have been reported in sars-cov and mers-cov including roles in viral replication, translational inhibition, transcriptional inhibition, mrna degradation, and cell cycle arrest (brockway and denison, 2005; kamitani et al., 2009; lokugamage et al., 2015; narayanan et al., 2015) . one of the reported roles for nsp1 in sars-cov is that it can associate with the 40s ribosome to inhibit host mrna translation (kamitani et al., 2009; tanaka et al., 2012 ), yet it remains unknown whether this association is due to interaction with the ribosomal rna, protein components of the ribosome, or other auxiliary ribosomal factors. accordingly, the mechanisms by which nsp1 acts to suppress protein production remain elusive. j o u r n a l p r e -p r o o f 9 we mapped the location of nsp1 binding to a 37 nucleotide region corresponding to helix 18 ( figure 4a) , adjacent to the mrna entry channel (simonetti et al., 2020) (figure 4b ). the interaction would position nsp1 to disrupt 40s mrna scanning and prevent translation initiation (figure 4b) , and disrupt trna recruitment to the 80s ribosome and block protein production ( figure s4b) . interestingly, the nsp1 binding site includes the highly conserved g626 nucleotide which monitors the minor groove of the codon-anticodon helix for trna binding fidelity (ogle et al., 2001) . we noticed that the c-terminal region of nsp1 has similar structural regions to serbp1 (brown et al., 2018) and stm1 (ben-shem et al., 2011a) , two known ribosome inhibitors that bind within the mrna entry channel to preclude mrna access ( figure s4c ). consistent with this, a recent cryo-em structure confirms that nsp1 binds to these same nucleotides of 18s within the mrna entry channel (thoms et al., 2020) . given the location of nsp1 binding on the 40s ribosome, we hypothesized that it could suppress global initiation of mrna translation. to test this, we performed in vitro translation assays of a gfp reporter in hela cell lysates and found that addition of nsp1 led to potent inhibition of translation ( figure s4d ). we observed a similar nsp1-mediated translational repression when we co-expressed nsp1 and a gfp reporter gene in hek293t cells (figure 4c-d) . in contrast, we did not observe this inhibition when we expressed other sars-cov-2 proteins (nsp8, nsp9, m) or human proteins (gapdh) ( figure 4d ). to determine if nsp1 leads to translational inhibition of endogenous proteins in human cells, we used a technique called surface sensing of translation (sunset) to measure global protein production levels (schmidt et al., 2009) . in this assay, translational activity is measured by the level of puromycin incorporation into elongating polypeptides ( figure s4e ). we observed a strong reduction in the level of global puromycin integration in cells expressing nsp1 compared to cells expressing gfp (figure s4f-g) . because nsp1 expression is sufficient to suppress global mrna translation in human cells, we hypothesized that sars-cov-2 infection would also suppress global translation. to test this, we infected a human lung epithelial (calu3) or monkey kidney (vero) cell line with sars-cov-2 and measured nascent protein synthesis levels using sunset. we observed a strong reduction of to explore whether nsp1 binding to 18s rrna is critical for translational repression, we generated a mutant nsp1 in which two positively charged amino acids (k164 and h165) in the c-terminal domain were replaced with alanine residues (figure s4c ) (narayanan et al., 2008) . we observed a complete loss of in vivo contacts with 18s ( figure 4g) ; because this mutant disrupts ribosome contact, we refer to it as nsp1∆rc. we co-expressed gfp and nsp1∆rc in hek293t cells and found that the mutant fails to inhibit translation ( figure 4h and s4j) . in contrast, mutations to the positively charged amino acids at positions 124/125 do not impact 18s binding ( figure 4g ) or the ability to inhibit translation ( figure 4h ). together, these results demonstrate that nsp1 binds within the mrna entry channel of the ribosome and that this interaction is required for translational inhibition of host mrnas upon sars-cov-2 infection. we explored whether nsp1 binding to 18s rrna suppresses the ability of cells to respond to ifn-β stimulation upon viral infection. we transfected isg reporter cells with nsp1, stimulated with ifn-β, and observed robust repression of the ifn responsive gene (>6-fold, figure 4i ). to confirm that this nsp1-mediated repression occurs in human cells upon activation of double stranded rna (dsrna)-sensing pathways typically triggered by viral infection, we treated a human lung epithelial cell line (a549) with poly(i:c), a molecule that is structurally similar to dsrna and known to induce an antiviral innate immune response (alexopoulou et al., 2001; kato et al., 2006) (figure s4k ). we observed a marked downregulation of ifn-β protein and endogenous ifn-β responsive mrnas in the presence of nsp1, but not in the presence of nsp1∆rc ( figure s4l, m) . these results demonstrate that nsp1, through its interaction with 18s rrna, suppresses the innate immune response to viral recognition ( figure 4j ). j o u r n a l p r e -p r o o f 11 because nsp1 blocking the mrna entry channel would impact both host and viral mrna translation, we explored how translation of viral mrnas is protected from nsp1-mediated translational inhibition. many viruses contain 5' untranslated regions that regulate viral gene expression and translation (gaglia et al., 2012) ; all sars-cov-2 encoded subgenomic rnas contain a common 5' leader sequence that is added during negative strand synthesis (kim et al., 2020b) . we explored whether the leader sequence protects viral mrnas from translational inhibition by fusing the viral leader sequence to the 5' end of gfp or mcherry reporter genes ( figure s5a ). we found that nsp1 fails to suppress translation of these leader-containing mrnas ( figure 5a -b, s5b). we dissected the leader sequence and found that the first stem loop (sl1) is sufficient to prevent translational suppression upon nsp1 expression ( figure 5c) or sars-cov-2 infection ( figure 5d ). we considered three models for how the leader could protect viral mrnas: (i) it could compete with the ribosome for nsp1 binding, (ii) it could directly recruit free ribosomes or (iii) nsp1 could bind to the leader independently of its ribosome interaction to allosterically modulate the nsp1-ribosome interaction. we reasoned that if the leader competes for nsp1 binding or directly recruits free ribosomes, then the presence of sl1 should be sufficient for protection, regardless of its precise position in the 5' utr. in contrast, if the leader allosterically modulates ribosome binding then the spacing between the 5' cap (which is bound to nsp1-40s) and sl1 would be critical for protection. to distinguish between these models, we swapped the location of sl1 and sl2 in the 5' leader or inserted 5 nucleotides between the 5' cap and sl1 ( figure s5c ) and found that both mutants ablate protection ( figure 5e, s5d) . these results indicate that an mrna requires the 5' leader to be precisely positioned relative to the nsp1-bound 40s ribosome to enable translational initiation ( figure 5f ). while many aspects of this allosteric model remain to be explored, it would explain how leader-mediated protection can occur on an mrna only when present in cis. moreover, this model suggests that nsp1 might also act to further increase viral mrna translation by actively recruiting the ribosome to its own mrnas. consistent with this, we observe a consistent ~20% increase in 12 translation of leader-containing reporter levels upon viral infection ( figure 5d ) or expression of nsp1 ( figure s5e ). upon engaging the start codon in an mrna, the 60s subunit of the ribosome is recruited to form the 80s ribosome which translates mrna. the signal recognition particle (srp) is a universally conserved complex that binds to the 80s ribosome and acts to co-translationally scan the nascent peptide to identify hydrophobic signal peptides present in integral membrane proteins and proteins secreted from the plasma membrane (akopian et al., 2013) . when these are identified, srp triggers ribosome translocation to the endoplasmic reticulum (er) to ensure proper folding and trafficking of these proteins to the cell membrane (akopian et al., 2013) . we identified two viral proteins -nsp8 and nsp9 -that bind at distinct and highly specific regions within the s-domain of the 7sl rna scaffold of srp ( figure 6a , s6a). nsp8 interacts with 7sl in the region bound by srp54 (the protein responsible for signal peptide recognition, srp-receptor binding, and ribosome translocation) (akopian et al., 2013; holtkamp et al., 2012) ( figure 6b ). nsp9 binds to 7sl in the region that is bound by the srp19 protein ( figure 6b ), which is required for proper folding and assembly of srp (including proper loading of srp54) (akopian et al., 2013) . because srp scans nascent peptides co-translationally, we were intrigued to find that nsp8 also forms a highly specific interaction with 28s rrna (the structural component of the 60s subunit) ( figure 6c, s6b) . the binding site on 28s rrna corresponds to the largest human-specific expansion segment within the ribosome, referred to as es27 (parker et al., 2018) . es27 is highly dynamic, and thus has not been resolved in most ribosome structures (zhang et al., 2014) . however, when engaged by specific factors, es27 can become ordered, and was recently shown to be capable of interacting with the ribosome exit tunnel, adjacent to the 60s binding site of srp ( figure 6d , s6c) (wild et al., 2020) . together, these observations suggest that nsp8 and nsp9 bind to the co-translational srp complex. consistent with this, we find that nsp8 and nsp9 localize broadly throughout the j o u r n a l p r e -p r o o f 13 cytoplasm when expressed in human cells ( figure s6d ) or upon sars-cov-2 infection ( figure s6e -f). because nsp8 and nsp9 binding on 7sl are positioned to disrupt srp function, we hypothesized that they may alter translocation of secreted and integral membrane proteins ( figure s7a ). to test this, we expressed an srp-dependent membrane protein (nerve growth factor receptor, ngfr (izon et al., 2001a) ) fused via an internal ribosome entry site (ires) to a non-membrane gfp ( figure s7f ). in this system, if a perturbation specifically affects membrane protein levels we expect to see a decrease in the ratio of membrane to non-membrane protein levels. to ensure that the ngfr reporter accurately reports on srp function, we treated hek293t cells with sirnas against srp54 or srp19 and found that both lead to a dramatic reduction of the ngfrmembrane protein relative to the non-membrane gfp protein ( figure s7b) . similarly, we found that expression of nsp8 and nsp9 (alone or together) lead to a striking reduction in expression of ngfr relative to gfp ( figure 7a ). expression of control proteins did not specifically impact ngfr levels ( figure 7a, s7b) . to determine if there is a global effect on membrane protein levels, we utilized the sunset method to measure puromycin levels in membrane proteins using flow cytometry (see methods). we confirmed that disruption of srp leads to a global reduction in puromycin levels in the cell membrane ( figure s7c ). we observed a comparable global reduction of puromycin-labeled membrane proteins upon expression of nsp8 or nsp9 individually or together, but not with control proteins (figure 7b, s7c) . because nsp8 and nsp9 are each sufficient to suppress protein integration into the cell membrane, we anticipate that sars-cov-2 infection would lead to similar suppression. j o u r n a l p r e -p r o o f 14 however, determining whether sars-cov-2 infection specifically impacts membrane protein expression is confounded by the fact that nsp1 inhibits translation of membrane and nonmembrane proteins upon infection. to address this, we co-expressed a membrane protein reporter (ngfr) containing the 5' viral leader along with a non-membrane gfp reporter containing the viral leader. upon viral infection, we observed a strong reduction of membrane protein levels ( figure 7c ), but no reduction in non-membrane gfp levels ( figure 5d ). to ensure that these effects are specific to sars-cov-2 infected cells, we separated individual cells within the infected population into those expressing the viral spike protein (s+) and those not expressing the protein (s-). we found that the shift in membrane protein levels only occurs in s+ cells ( figure 7d ), while the spopulation resembled the mock infected samples ( figure 7c ). we observed a strong relationship between the level of spike protein -likely reflecting the amount of viral replication within each cell -and the level of membrane protein suppression ( figure 7c ). we observed this membrane protein-specific decrease upon infection of human lung epithelial (calu3, figure s7d ) and monkey kidney (vero, figure 7c -d) cell lines. together, these results demonstrate that nsp8 and nsp9 bind to 7sl to disrupt srp function and suppress membrane protein trafficking in sars-cov-2 infected cells. although nsp8 and nsp9 are thought to be components of the viral replication machinery (sutton et al., 2004) , our results indicate that they play an additional role as host virulence factors. because viral membrane proteins also require trafficking to the er, viral disruption of srp might negatively impact viral propagation, unless viral proteins are trafficked in an srp-independent manner ( figure s7e ) or if nsp8/9 selectively impacts host (but not viral) proteins. next we explored how disruption of srp might be advantageous for viral propagation. because secretion of ifn and other cytokines is dependent on the srp complex for secretion ( figure s7f ), a central component of the ifn response is dependent on srp. accordingly, we hypothesized that nsp8/9-mediated viral suppression of srp would act to suppress the ifn j o u r n a l p r e -p r o o f 15 response upon infection. to test this, we co-expressed nsp8 and nsp9 and observed a significant reduction in the ifn response relative to a control protein ( figure s7g ). together, these results suggest that sars-cov-2 mediated suppression of srp-dependent protein secretion enables suppression of host immune defenses ( figure 7e) . interestingly, many proteins involved in anti-viral immunity -including most cytokines and class i major histocompatibility complex -are membrane-anchored or secreted, and are known to use the srp pathway for transport (vermeire et al., 2014) (figure s7f ), suggesting that there may be other effects of srp pathway inhibition on sars-cov-2 pathogenesis. we identified several pathogenic functions of sars-cov-2 in human cells -including global inhibition of host mrna splicing, protein translation, and membrane protein trafficking -and described the molecular mechanisms by which the virus acts to disrupt these essential cell processes. interestingly, all of the viral proteins involved (nsp1, nsp8, nsp9, and nsp16) are produced in the first stage of the viral life cycle, prior to generation of double stranded rna (dsrna) products during viral genome replication. because dsrna is detected by host immune sensors and triggers the type i interferon response, disruption of these cellular processes would allow the virus to replicate its genome while minimizing the host innate immune response. disruption of these three non-overlapping steps of protein production may represent a multipronged mechanism that synergistically acts to suppress the host antiviral response ( figure 7f) . specifically, the ifn response is usually boosted >1,000-fold upon viral detection (through amplification and feedback, figure s4k ), yet each individual mechanism impacts ifn levels on the order of ~5-10-fold. accordingly, if each independent mechanism impacts ifn levels moderately, the three together may be able to achieve dramatic suppression of ifn (10 3 =1,000fold). this multi-pronged mechanism may explain the molecular basis for the potent suppression of ifn observed in severe covid-19 patients. interferon is emerging not only as a determinant of disease severity, but also a potential treatment option (zhou et al., 2020) . as such, our work identifies several therapeutic opportunities for boosting ifn levels upon sars-cov-2 infection. for example, disrupting the interaction between nsp1 and 18s rrna could allow cells to detect and respond to viral infection. because many small-molecule drugs target ribosomal rnas (liaud et al., 2019) , it may be possible to develop drugs to block the nsp1-18s and other interactions. additionally, disrupting the 5' viral leader may be a potent antiviral strategy since it is critical for translation of all viral proteins. because sl1 is a structured rna, it may be possible to design small molecules that specifically bind this structure to suppress viral protein production (hermann, 2016) . viral suppression of these cellular functions is not exclusive to the ifn response and will also impact other spliced, translated, secreted, and membrane proteins. many proteins involved in anti-viral immunity are spliced and/or membrane-anchored or secreted. for example, class i major histocompatibility complex (mhc), which is critical for antigen presentation to cd8 t cells at the cell surface of infected cells (hansen and bouvier, 2009 ). by antagonizing membrane trafficking, sars-cov-2 may prevent viral antigens from being presented on mhc and allow infected cells to escape t-cell recognition and clearance. in this way, interference with these essential cellular processes might further aid sars-cov-2 in evading the host immune response. more generally, we expect that insights gained from the sars-cov-2 protein-rna binding maps will be critical for exploring additional viral mechanisms. specifically, we identified many other interactions, including highly specific interactions with mrnas. for example, nsp12 binds to the jun mrna ( figure s1e ) which encodes the critical immune transcription factor c-jun which is activated in response to multiple cytokines and immune signaling pathways (weston and davis, 2007) . we also identified an interaction between nsp9 and the start codon of the mrna that encodes cops5 (figure 1c) , the enzymatic subunit of the cop9 signalosome complex which regulates protein homeostasis (cope and deshaies, 2003) , suggesting that it might disrupt its translation. interestingly, cops5 (also known as jab1) is known to bind and stabilize c-jun protein levels (claret et al., 1996) and several viruses are known to disrupt this protein (lungu et al., 2008; oh et al., 2006; tanaka et al., 2006) . while it remains unknown what, if any, role these interactions play in virally infected cells, the specificity suggests that they may provide a selective advantage for viral propagation. together, our results demonstrate that global mapping of rna binding by viral proteins could enable rapid characterization of mechanisms for emerging pathogenic rna viruses. we note several limitations of our current study that will need to be explored in future work. (i) our mapping experiments were performed in uninfected human cells expressing tagged viral proteins. accordingly, it remains possible that our maps may not fully capture all of the interactions that occur when human cells are infected, such as interactions that occur with viralinduced rnas, in specific viral compartments, or that require multiple viral proteins. (ii) while we characterized the functional and mechanistic roles of several viral proteins and structural ncrnas, we did not explore what roles viral protein interactions with mrnas might play. (iii) how the virus disrupts fundamental cellular processes while still maintaining its own production is still largely undefined. while we showed that the 5' leader is sufficient to relieve translational inhibition by nsp1, we still do not fully understand how this protection occurs and specifically how nsp1 might interact with the viral leader or allosterically modulate ribosome binding. similarly, viral membrane proteins are dependent on trafficking to the er and how nsp8/9 might selectively impact er translocation of host -but not viral -proteins remains to be explored. (iv) while we showed that viral disruption of these essential cellular functions can suppress ifn, what other roles host cell shutdown might play in viral pathogenesis and in suppressing other aspects of anti-viral immunity, including possible roles in adaptive immune responses, have not been explored. the authors declare no competing interests. further information and requests for reagents and resources should be directed to and will be fulfilled by the lead contact, mitchell guttman (mguttman@caltech.edu). all constructs and plasmids generated in this study will be made available on request sent to the lead contact with a completed materials transfer agreement. all datasets generated during this study are available at ncbi short read archive: bioproject prjna665692 (viral protein purifications) and prjna665581 (nascent and total rna-seq) essential medium (atcc) containing 10% fbs and 1% penicillin-streptomycin purchased from thermo fisher scientific. all cell lines were maintained at 37°c under 5% co 2 . cells were grown in a humidified incubator at 37ºc with 5% co 2 . all experiments using infectious sars-cov-2 conducted at the uvm bsl-3 facility were cloning of expression constructs. sars-cov-2 protein constructs (with the exception of nsp11) were a gift from fritz roth (see table s3 for addgene information) (kim et al., 2020a) and were lr-cloned (invitrogen gateway cloning, thermo fisher scientific) into mammalian expression destination vector pcag-halo-tev-dest-v5-ires-puror. note that following lr cloning, proteins were not v5-tagged because all entry clones contained stop codons. for nsp11, an entry clone was generated by bp cloning (invitrogen gateway cloning, thermo manganese/calcium mix (0.5mm cacl 2 , 2.5 mm mncl 2 ). samples were incubated on ice for 10 minutes to allow lysis to proceed. the lysates were then incubated at 37°c for 10 minutes at 700 rpm shaking on a thermomixer (eppendorf). lysates were cleared by centrifugation at 15,000× g for 2 minutes. the supernatant was collected and kept on ice until bound to the halolink resin (promega). of the 1ml lysis volume, 50ul was set aside for input, 20ul used for protein expression confirmation, and the rest for capture on halolink resin as described below. for qpcr analysis, cdna was generated from purified rna using maxima h-reverse transcriptase (thermo fisher scientific) following manufacturer's recommendations. amplification reactions were assembled with primer sets indicated in table s2 and lightcycler® 480 sybr green i master (roche) following manufacturer's protocols and read out in a roche lightcycler 480. library construction. rna-seq libraries were constructed from purified rna as previously described (van nostrand et al., 2016) . briefly, after proteinase k elution, the rna was dephosphorylated (fast ap) and cyclic phosphates removed (t4 pnk) and then cleaned using silane beads as previously described (van nostrand et al., 2016 ). an rna adapter containing a rt primer binding site was ligated to the 3' end of the cleaned and end-repaired rna. the ligated rna was reverse transcribed (rt) into cdna, the rna was degraded using naoh, and a second adapter was ligated to the single stranded cdna. library preparation was the same for input samples except that an initial chemical fragmentation step (90°c for 2 min 30 s in 1x fastap buffer) was included prior to fastap treatment. this chemical fragmentation step was designed to be similar to the fragmentation conditions used for purified halo bound samples. the for staining of infected cells, cells were fixed and permeabilized in 8% formaldehyde 1% triton, and subsequently labelled with primary antibodies raised in sheep to sars-cov-2 at 1/500 dilution, followed by incubation with a rabbit anti-sheep alexa 555 secondary antibody (abcam, ab150182) at 1/1000 dilution and mounted with dapi in the medium (thermo fisher scientific, cat# p36395). cells were imaged with a zeiss lsm 880 confocal microscope, with 1 airy unit pinhole for all primary antibody channel acquisitions and pixel size 0.07 µm x 0.07 µm. the objective lens used was a zeiss plan-apochromatic 63x/1.4na m27. structure modeling nsp1 homology model. the predicted model of sars-cov-2 nsp1 was generated using the transform-restrained rosetta (trrosetta) algorithm, a deep learning-based modeling method based on the rosetta energy minimization pipeline with additional distance and interaction restraints generated from co-evolution (yang et al., 2020) . all figures were generated using pymol (www.pymol.org). nsp1-ribosome model. the model of nsp1 bound to the ribosome was generated using modeller version 9.24 (webb and sali, 2016) . the c-terminal sequence of nsp1 (khssgvtrelmrelngg) was modeled using the structure of serbp1 bound to the ribosome (pdb id: 6mte, chain w) as a template. the default modeller parameters were used to create an alignment of nsp1 and serbp1 and to generate the model, and all atoms within 6å of serbp1 were included in the model to define the neighboring environment. twenty models were generated and the model with the lowest dope score was selected to visualize with pymol (delano, 2002) . x-ray crystal structures and cryo-electron microscopy structures were obtained from the protein data bank (www.rcsb.org) (berman et al., 2000) and visualized with pymol (delano, 2002) . for u1 and u2 structural analysis, we used a cryo-em structure of the pre-catalytic human spliceosome (pdb id: 6qx9). for 7sl structural analysis, we used an x-ray crystal structure of the human signal recognition particle (pdb id: 1mfq). to examine human srp in the context of the ribosome, we used a cryo-em structure of the mammalian srp-ribosome complex (pdb id: 3jaj). to analyze the ribosomal es27 expansion segment, we superimposed a cryo-em structure of the expansion segment (pdb id: 6sxo) onto the complete ribosome structure (pdb id: 3jaj) using the pymol command "super." finally, for nsp1-18s rrna structural analysis, we used multiple structures of the ribosome, including structures of the pre-40s subunit (pdb id: 6g5h), 48s late-stage initiation complex (pdb id: 6yal), 80s in complex with serbp1 (pdb id: 6mte), and 80s in complex with stm1 (pdb id: 4v88). j o u r n a l p r e -p r o o f 37 nsp1 was cloned into a bacterial expression vector resulting in n-terminally tagged halo-6xhistagged nsp1. the nsp1 sequence was pcr amplified from addgene nsp1 entry vector to add a n-terminal 6x his tag and restriction enzyme sites for digestion and ligation into n-terminal halo bacterial expression vector. this construct was transformed into bl21 de3 e. coli (agilent), expanded to a 500ml liquid culture, and grown until od 600 reached 1.0. iptg was added to a final concentration of 1mm. after 3 hours of iptg induction, bacteria was centrifuged for 15 min at 5000× g. pellet was lysed with binding buffer (50mm hepes, ph 7.5, 20mm mgcl 2 , 600mm nacl, 2mm tcep, 10mm imidazole, 2mm atp, 1% triton x-100) supplemented with atp (2mm), protease inhibitor cocktail (promega), benzonase (sigma) and triton-x 100 (sigma) using 5ml of lysis mix per gram of wet cell paste. cell suspension was rocked for 20 min at room temperature and then centrifuged at 16,000× g for 20 min at 4°c. supernatant was incubated with washed imac resin (bio-rad) and rocked for 20 min at room temperature. we loaded the resin-lysate mixture into an appropriately-sized column and washed with 5 column volumes of binding buffer (50mm hepes, ph 7.5, 20mm mgcl 2 , 600mm nacl, 2mm tcep, 10mm imidazole, 2mm atp, 1% triton x-100) followed by 10 column volumes of wash buffer (50mm hepes, ph 7.5, 600mm nacl, 2mm tcep, 20mm imidazole, ph 8). recombinant nsp1 (rnsp1) was eluted with 5 column volumes of elution buffer by adding 1 column volume at a time with column flow stopped, collecting eluate after each addition, and waiting 15 min between each elution buffer addition. we dialyzed these eluates with a 10ml spectra-por® float-a-lyzer® g2 (spectrum laboratories) into storage buffer (50mm hepes, ph 7.5, 150mm nacl, 10% glycerol) at 4°c using 2 exchanges, one after 2 hours and then overnight. pierce 1-step human coupled ivt-dna (thermo fisher scientific) in vitro translation kit was used to measure rnsp1-dependent translation inhibition. bovine serum albumin (bsa), and buffer only controls were used to control for the addition of excess protein or changes in buffer composition. to measure translation inhibition, 5µl in vitro translation reactions were assembled, scaled according to manufacturer's recommendations. the included control plasmid pcfe-gfp was used to measure translational output of the reactions. gfp fluorescence was measured on a biotek cytation3 plate reader using emission filters for gfp fluorescence. 1.5µm j o u r n a l p r e -p r o o f 38 stock dilutions of rnsp1 and bsa were made in storage buffer (50mm hepes, ph 7.5.,150mm nacl,10% glycerol). subsequent 10 fold dilutions were made in storage buffer to span a concentration range of 1000 nm to 1 nm for each protein in the final reaction. 10 µl of the diluted protein solution was added to the 5µl translation reactions, and incubated for 5 minutes at room temperature prior to the addition of the gfp reporter plasmid. duplicate reactions were made to measure variability for each condition. in addition, a buffer only control was included to measure the effect of dilution of the translation reaction by the storage buffer. after the 5 minute incubation, 50 ng of gfp reporter plasmid was added to each reaction and incubated at 30°c for 4 hours prior to fluorescence detection. two microliters from each reaction was measured in duplicate on a biotek cytation3 microplate reader using excitation and emission filters for gfp. sample readings were blanked by subtracting values obtained from the buffer only control. promega's rabbit reticulocyte lysate system was also used to assay translation inhibition. to measure translation inhibition, 10µl in vitro translation reactions were assembled, scaled according to manufacturer's recommendations. for each translation reaction, either 10µl of recombinant protein storage buffer or rnsp1 was added, followed by 500ng of mrna. after 4 hours of incubation at 30°c, luciferase was read out using the bright-glo luciferase assay (promega) or gfp fluorescence was measured, both on a biotek cytation3 plate reader. we assayed translation in hek293t cells transfected with mammalian expression vectors, mrnas, or combinations of these. for mrna transfections of fluorescence protein translation reporters (including unmodified, +sars-cov2 leader sequence, +sl1, +sl2-sl1, and +5nts), dna templates for in vitro transcription were generated with sequences appended to the 5' end of gfp and mcherry (see tables s4 and s5 for primers and templates, respectively) and transcribed using hiscribe™ t7 arca mrna kit with tailing (new england biolabs). for nsp1 mrna transfection, indicated primers from table s4 were used to add restriction enzyme sites for cloning into pt7cfe1-chis backbone provided in the pierce human 1-step coupled acquistion files were analyzed with flowjo analysis software. to assay global protein translation, a sunset assay was performed as previously described (schmidt et al., 2009) we transfected these mammalian expression vectors for nsp1 and gfp into hek293t using biot transfection reagent. after 3 hours, doxycycline (sigma) was added to a final concentration of 2µg/ml. after 24 hours, cells were incubated with puromycin (10µg/ml) for 10 min, then washed with fresh media, and harvested with cold pbs. pelleted cells were lysed for 10 min on j o u r n a l p r e -p r o o f 40 ice (mixing after 5 min) with 100ul ripa buffer supplemented with protease inhibitor cocktail (promega). insoluble debris was pelleted by centrifuging at 12,500 × g for 2.5 minutes and supernatant was run on a bolt™ 4-12% bis-tris plus gel (thermo fisher scientific). proteins were then transferred to nitrocellulose using the iblot transfer system (thermo fisher scientific) and western blotting carried out using an anti-puro antibody (clone 12d10, emd millipore). sunset in sars-cov-2 infection was performed as above with the following modifications. cells were infected or not (mock) with sars-cov-2, and 48 hpi cells were incubated with puromycin (10µg/ml) for 20 min. media was aspirated and cells lysed directly in 2x laemmli's buffer (biorad), heated at 95ºc for ten minutes and run on a 4-12% nupage gel (thermo fisher scientific). proteins were transferred to nitrocellulose using the iblot transfer system and probed as above. to assay srp-dependent membrane protein transport to the cell surface, we monitored surface arrival of exogenously expressed neuronal growth factor receptor (ngfr) by flow cytometry in the presence of nsps. mammalian expression vectors were exchanged for versions that contained an ires-ngfr to co-express a membrane reporter and thus, for these experiments, lr reactions were carried out with destination vector pb-6xhis-gfp-dest-ires-ngfr. resulting expression vectors drive protein expression by a dox-inducible promoter, contain the rtta needed for dox induction, and produce an n-terminally-tagged his-gfp fusion protein and a co-expressed ngfr. the gfp here is an enhanced gfp containing an amino acid substitution (a205k) to generate a monomeric variant based on previous literature (alberti et al., 2018) . we transfected these mammalian expression vectors for nsp8, nsp9, nsp1∆rc mutant and to knockdown srp19 and srp54, sirnas targeting each (dharmacon cat# l-019729-01-0005 and l-005122-01-0005, respectively) were transfected into hek293t cells using lipofectamine rnaimax (invitrogen) according to manufacturer's protocols. to validate knockdown, transfected cells were assayed by qpcr using primer sets (table s2 ) to amplify each target as well as normalizer calm3. transfections were carried out 48 hours prior to assaying cells, either by qpcr, membrane reporter, or membrane sunset (see below) experiments. calu3 and vero cells were transfected with mrnas encoding leader-ngfr and leader-gfp using transit-mrna transfection kit (mirus) and subsequently infected with sars-cov-2 at an moi of 0.1. after 24 hours, cells were washed with pbs, trypsinized and fixed in 4% pfa for 20 minutes before staining with biotinylated anti-ngfr (biolegend) and anti-sars-cov-2 spike antibody (sino) and subsequently stained with pe-labeled anti-rabbit (thermo, p-2771mp) and pacblue-labeled streptavidin (thermo, s1222). facs was performed on a macsquant flow cytometer and analyzed using flojo analysis software; facs distributions were compared using a 2-tailed kolmogorov-smirnov test. for these experiments, rna was transcribed from a pcr template (see table s4 ) using the hiscribe t7 arca mrna kit (with tailing). to assay transport to the cell surface of all plasma membrane proteins, the sunset assay was adapted to puro-label surface proteins as previously described (schmidt et al., 2009) , and read out by flow cytometry. briefly, cells were incubated with puromycin as described above, followed by two quick washes and a chase with fresh complete media for 50 min. cells were lifted with 1mm edta as described above and stained with an anti-puro antibody (clone 12d10, emd millipore) conjugated to alexa-647. for these experiments, nsp was expressed from the same vector described above for membrane reporter assays. fluorescence intensity j o u r n a l p r e -p r o o f 42 measurements were taken for gfp and alexa-647 on a macsquant flow cytometer and analyzed using flojo analysis software; distributions were compared using a 2-tailed kolmogov-smirnov. to assess splicing efficiency, exons 5-6 of mouse irf7 (enmust00000026571.10) containing its endogenous intron were fused upstream of 2a self-cleaving peptide and egfp and cloned into an mscv vector (pig, addgene) (mayr and bartel, 2009 ). this construct was co-transfected into hek293ts with nsp16 or gfp and measured 24 hours after transfection by flow cytometry (macsquant) and analyzed using flojo analysis software. sars-cov2 or mock infected calu3 cells and nsp16-or gapdh-expressing hek293ts were labeled with 5-ethynyl-uridine (5eu; jena bioscience) by adding 5eu containing media to cells for 20 min at a final concentration of 1mm, as previously described (jao and salic, 2008) . after the pulse label, cells were washed with warm pbs and lysed in rlt buffer (qiagen). total rna was isolated from cells using manufacturer's protocols for qiashredder and rneasy rna isolation (both qiagen), followed by turbo dnase treatment (ambion, thermo scientific), and zymo rna clean and concentrate. for each sample, 2µg of rna was used for ligation of a unique barcoded rna adaptor, following the relevant steps in the protocol described above in library construction of rna-seq libraries. samples were then pooled before proceeding to biotinylation steps. to biotinylate 5eu-labeled rna, samples were first mixed, in order, with water, hepes (100 mm), biotin picolyl azide (1 mm; click chemistry tools) and ribolock rnase inhibitor, then added to premixed cuso 4 (2 mm) and thpta (10mm), and finally added to freshly prepared j o u r n a l p r e -p r o o f 43 sodium ascorbate (12mm), as previously described (hong et al., 2009) . the click reaction was incubated for 1 hour at 25ºc with 1000rpm shaking on an eppendorf thermomixer followed by rna purification using >17nt protocol for zymo clean and concentrate. we completed three rounds of sequential capture on streptavidin beads to isolate nascent transcripts (see figure s3b ). to capture biotinylated rna, myone streptavidin c1 dynabeads (thermofisher scientific) were first washed three times in urea buffer (10mm hepes, ph 7.5, 10mm edta, 0.5m licl, 0.5% triton x-100, 0.2% sds, 0.1% sodium deoxycholate, 2.5mm tcep, 4m urea) followed by three additional washes in m2 buffer (20mm tris, ph 7.5, 50mm nacl, 0.2% triton x-100, 0.2% sodium deoxycholate, 0.2% np-40). washed beads were mixed with 3 parts 4m urea buffer and 1 part biotinylated rna and incubated for 60 min with 900rpm thermomixer shaking at room temperature. after magnetic separation, beads were washed 3 times with m2 buffer followed by 3 washes with urea buffer at 37 ºc at 750rpm for 5 min. rna was eluted from beads in 2 sequential elutions by incubating with elution buffer (5.7m guanidine thiocyanate , 1% n-lauroylsarcosine; both sigma) at 65 ºc for 2 minutes, repeating with more elution buffer for a second elution. the elutions were pooled, diluted with urea buffer, incubated with pre-washed streptavidin beads, washed, and eluted for 2 additional rounds exactly as described above for a total of 3 sequential captures. final elutions were pooled, cleaned with zymo rna clean and concentrate following manufacturer's protocols, and carried through rna-seq library preparation as described above starting with the reverse transcription step. hek-blue™ isg cells were seeded in 96 well plates, transfected with nsp1 mammalian expression vectors using biot and stimulated with 50 ng/ml human ifn-b (r&d systems). supernatants were assayed for alkaline phosphatase as per manufacturer instructions using j o u r n a l p r e -p r o o f 44 quanti-blue reagent (invivogen). hek-293t cells were seeded in 6 well plates, transfected with either halo-tagged gapdh, nsp1, nsp8 and nsp9 in combination, or nsp16 mammalian expression vectors using biot. 24 hours later, the media was replaced with media containing 50 ng/ml human ifn-β (r&d systems). expression was assayed using live cell halo-imaging. halo-tmr ligand was diluted 1:200 in media and added to the culture for a 1:1000 final dilution. samples were incubated 30 minutes at 37°c, 5% co 2 and then the media was aspirated. wells were rinsed twice with pbs, then media was added back to the wells. samples were incubated 30 minutes at 37°c, 5% co 2 to allow uncoupled ligand to diffuse out of the cells. media was then aspirated and replaced, and cells were imaged by widefield fluorescence microscopy. cultures were ultimately harvested for rna 24 hours later, or 48 hours post transfection. a549s were seeded in 6 well plates, transfected with nsp1 mammalian expression vectors using lipofectamine 2000 and stimulated with 1 µg/ml hmw poly(i:c) (invivogen) 24h after transfection. supernatant was assayed for secreted ifn-β by elisa (human ifn beta elisa, high sensitivity, pbl) 24 hours after stimulation, and rna from cells was purified and assessed for isg gene expression as normalized to gapdh expression (sybr green master mix, bio-rad). primers used for qpcr are listed in table s2 . sars-cov-2 leader sequence was appended to the 5' end of gfp and mcherry reporter templates via pcr. pcr templates were then transcribed using hiscribe t7 arca mrna kit (with tailing). leader mutants, including sl1 only, sl1/sl2 swap, and +5nts mutants were likewise appended to the 5' end of fluorescent reporter templates via pcr and transcribed using hiscribe t7 arca kit. mrna reporters were transfected in hek-293t cells with lipofectamine messengermax. to measure fluorescence of mcherry and gfp reporters, 24 hours post transfection cells were either lifted with pbs and transferred into black 96 well plates for fluorescence readout on a biotek cytation 3 or trypsinized and processed for flow cytometry. sequence alignment and analysis. for halo purifications and rna binding mapping sequencing reads were aligned to a combined genome reference containing the sequences of structural rnas (ribosomal rnas, snrnas, snornas, 45s pre-rrna) and annotated mrnas (refseq hg38) using bowtie2. to distinguish between the nascent pre-ribosomal rna and mature 18s, 28s, and 5.8s rrna, we separated each of the components of the 45s into separate sequence units for alignment (e.g. its, ets). we excluded all low quality alignments (mapq < 2) from the analysis. for mrna analysis, we removed pcr duplicates using the picard markduplicates function (https://broadinstitute.github.io/picard/). for each rna, we enumerated 100 nucleotide windows across the entire rna. for each window, we calculated the enrichment by computing the number of reads overlapping the window in the protein elution sample divided by the total number of reads within the protein elution sample. we normalized this ratio by the number of reads in the input sample divided by the total number of reads in the input sample. because all windows overlapping a gene should have the same expression level in the input sample (which represents rna expression), we estimated the number of reads in the input as the maximum of either (i) the number of reads over the window or (ii) the median read count over all windows within the gene. this approach provides a conservative estimation of enrichment because it prevents windows from being scored as enriched if the input values over a given window are artificially low, while at the same time accounting for any non-random issues that lead to increases in read counts over a given window (e.g. fragmentation biases or alignment artifacts leading to non-random assignment or pileups). j o u r n a l p r e -p r o o f 46 we calculated a multiple testing corrected p-value using a scan statistic, as previously described (guttman et al., 2009 (guttman et al., , 2010 . briefly, n was defined as the number of reads in the protein elution plus the number of reads in the control sample. p was defined as the total number of reads in the protein elution sample divided by the sum of the protein elution sample total reads and total reads in the control sample. w was the size of the window used for the analysis (100 nucleotides). the scan statistic p-value was defined using the poisson estimations based on standard distributions previously described (naus, 1982) . because rna within input samples are fragmented differently than the protein elution samples, we noticed that the overall positional distribution of protein elution samples was distinct from input distributions. accordingly, we used the remaining protein elution samples (rather than input) as controls for each protein. specifically, this enabled us to test whether a given protein is enriched within a given window relative to all other viral and control proteins. enrichments were computed as described above. these values are plotted in figure 1 and table 1 . igv (robinson et al., 2011) and were generated by either: (i) computing the enrichment for each nucleotide as described above. in this case, the read count for each nucleotide was computed as the total number of reads that overlapped the nucleotide. (ii) counting the number of rt stop sites at a given nucleotide. in this case, we compute the alignment start position of the second in pair read and computed a count of each nucleotide. we normalized this count by the total number of reads in the sample to account for sequencing depth generated. we then normalized this ratio by the same ratio computed for the control sample (merge of all other protein samples) for each nucleotide. heatmaps were generated using morpheus (https://software.broadinstitute.org/morpheus/). all values were included if they contained a significant 100nt window with a p-value<0.001 (see above) and minimum enrichment of 3-fold above the control sample. gene ontology analysis. the 66 non-n enriched mrnas were analyzed against the gene ontology biological processes and reactome gene sets using the molecular signatures database (msigdb) (liberzon et al., 2015) . significantly enriched gene sets with an fdr<0.05 were used. j o u r n a l p r e -p r o o f 47 to ensure that significant gene sets were not being driven by the multiple ribosomal proteins or histone proteins, these analyses were also carried out excluding these proteins. sequenced reads were demultiplexed according to barcoded rna adaptor sequences ligated to each respective sample. trimmomatic (https://github.com/timflutre/trimmomatic ) was used to remove any contaminating illumina primer sequences in the reads and low quality reads. demultiplexed and trimmed files were then aligned to a hg19 reference genome using the spliceaware star aligner (https://github.com/alexdobin/star). alignments were then deduplicated for pcr duplicates using picard markduplicates (https://broadinstitute.github.io/picard/). aligned read-fragments were defined as read1 and read2 contained within a paired-end read fragment along with the insert between these two reads. we defined a set of high-quality represented isoforms per gene using the appris database (rodriguez et al., 2013) . all readfragments that spanned any 3' splice site within an isoform of one of these genes was retained. for each 3' splice site spanning fragment, we classified the read-fragment as a spliced fragment if it spanned an exon-exon junction (e.g. aligned entirely within 2 distinct exons) or an unspliced fragment if it spanned an intron-exon junction (e.g. one of the reads was contained -or partially contained -within the intron). for each isoform, we computed an unspliced ratio by counting the total number of reads that were classified as unspliced divided by the total number of readfragments spanning 3' splice sites within that gene. to ensure that the splicing ratio that we measured is a reliable metric and not inflated/deflated due to low read counts, we only included genes that contained at least 10 read-fragments in each sample and where the total number of reads in the control and sample conditions (when merged together) contained a significant number of reads to reliably measure a difference between the two groups as measured by a hypergeometric test (p<0.01). because different genes contain different baseline splicing ratios due to gene length and coverage, we computed a change in the splicing ratio for each gene independently. to do this, we subtracted the unspliced ratio for each sample from the average unspliced ratio for that gene j o u r n a l p r e -p r o o f 48 in all of the control samples. we plotted the overall distribution of these differences in splicing ratios as violin plots for each sample. if there is no change in splicing ratio, we would expect that some genes would have higher splicing ratios and others lower splicing ratios but that the overall distribution would be centered around 0. total rna-seq libraries were generated from the same mock infected and sars-cov-2 virally infected calu3 samples treated with 5eu. prior to 5eu purification, total rna was taken and an rna-seq library constructed as described above using barcoded rna adapters. cytoplasmic ribosomal rnas (18s and 28s) were depleted using nebnext ribosomal rna depletion kit (neb e6310l) per manufacturers recommendations. demultiplexed reads were aligned using bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) to custom genomes encoding classical noncoding rnas (ncrnas) or human messenger rnas (mrnas). expression levels were computed for each mrna by counting the total number of sequencing reads aligned to the mature mrna. to normalize across the different libraries, we computed the read counts for each sample that align to non-spliced structural non-coding rnas -excluding rrna but including snrnas, 7sl, 7sk, etc. we then divided each mrna count by the sum of all ncrna counts. this normalized value for each gene per sample was then converted into a fold-change by dividing this normalized value to the mean value for both mock infected samples. the fold change of each gene relative to mock was plotted across all mrnas as a violin plot. and enrichment >3-fold for any of the viral proteins is reported. • nsp16 binds mrna recognition domains of u1/u2 snrnas and disrupts mrna splicing • nsp1 binds in the mrna entry channel of the ribosome to disrupt protein translation • nsp8 and nsp9 bind the signal recognition particle and disrupt protein trafficking • these disruptions to protein production suppress the interferon response to infection in brief -sars-cov-2 proteins directly engage host rnas and dysregulate rna-based processes to suppress the interferon response j o u r n a l p r e -p r o o f ); office of the vice president for research at uvm m.g. is a nyscf-robertson investigator signal recognition particle: an essential protein-targeting machine a user's guide for phase separation assays with purified proteins recognition of doublestranded rna and activation of nf-κb by toll-like receptor 3 visualizing late states of human 40s ribosomal subunit maturation the proximal origin of sars-cov-2 sars-cov-2 (covid-19) by the numbers innate immune 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title: applications of replicating-competent reporter-expressing viruses in diagnostic and molecular virology date: 2016-05-06 journal: viruses doi: 10.3390/v8050127 sha: doc_id: 2076 cord_uid: 7t4d4vvo commonly used tests based on wild-type viruses, such as immunostaining, cannot meet the demands for rapid detection of viral replication, high-throughput screening for antivirals, as well as for tracking viral proteins or virus transport in real time. notably, the development of replicating-competent reporter-expressing viruses (rcrevs) has provided an excellent option to detect directly viral replication without the use of secondary labeling, which represents a significant advance in virology. this article reviews the applications of rcrevs in diagnostic and molecular virology, including rapid neutralization tests, high-throughput screening systems, identification of viral receptors and virus-host interactions, dynamics of viral infections in vitro and in vivo, vaccination approaches and others. however, there remain various challenges associated with rcrevs, including pathogenicity alterations due to the insertion of a reporter gene, instability or loss of the reporter gene expression, or attenuation of reporter signals in vivo. despite all these limitations, rcrevs have become powerful tools for both basic and applied virology with the development of new technologies for generating rcrevs, the inventions of novel reporters and the better understanding of regulation of viral replication. the commonly used tests based on wild-type viruses, such as immunostaining, are often time-consuming and labor-intensive. furthermore, these methods cannot meet the demands for high-throughput screening (hts) of antivirals, rapid, sensitive and quantitative detection of neutralizing antibodies (nabs), visual tracking of viral proteins or viruses in vitro and in vivo and other fields of virology. replicating-competent reporter-expressing viruses (rcrevs) are one type of artificially modified viruses that not only retain the viral genetic characteristics but also possess the new properties of the reporter genes, which represent a useful tool for quantitative analysis of viral replication and tracking viral protein transport in both living cells and animals. to date, advances in technologies enable the generation of rcrevs, which have been successfully applied in diagnostic and molecular virology. currently, reverse genetics systems for many viruses have been well-established [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] , providing powerful tools for generating rcrevs. since some viruses possess a large genome, they usually permit a large extrinsic genetic insertion without impairing viral replication. for example, vaccinia virus (vacv) contains a 192 kb genome, capable of accepting up to 25 kb insertion [14] . however, for most rna and some dna viruses containing a small-sized genome, a recurring difficulty in generating rcrevs is the genetic instability, especially for a larger reporter gene. for some viruses with a segmented rna genome, the insertion of a large reporter gene into the genome is difficult or even impossible to achieve. commonly used reporters in rcrevs include fluorescent proteins, such as enhanced green fluorescent protein (egfp) (green), gfp mutants (enhanced cyan fluorescent protein (ecfp) (blue), mcherry (red) and venus (yellow)), far-red fluorescent reporters (red fluorescent protein (rfp), katushka 2, dtomato and dsred)), near-infrared fluorescent proteins (irfps) and tetracysteine (tc); bioluminescent reporters, such as firefly luciferase (fluc), renilla luciferase (rluc) and gaussia luciferase (gluc); in addition to other reporters, such as neomycin-resistance gene (neo r ) and cre recombinase. these reporters are mainly used to rapidly quantify viral replication and track viral proteins or viruses by living imaging in vitro and in vivo. however, different reporters may have different influences on the biological properties of various viruses, and the loss of the reporter gene expression is a significant concern for some rcrevs. therefore, choosing a suitable reporter is a critical decision on designing rcrevs. for the planned applications, the reporters with a smaller size may be a promising option due to their minimum effects on the viral biology. for example, the rluc gene (933 bp) is better than the fluc gene (1653 bp) and has a minimal influence on the growth of the engineered classical swine fever virus (csfv) expressing the reporters [15, 16] . various strategies associated with the reporter gene expression have been developed. an extensively used expression strategy is to fuse the reporters to one of the viral proteins. for instance, the rluc activities from engineered csfv carrying the rluc fused to the viral n pro protein were detected [15] . a nonessential viral gene can be replaced with a reporter gene to generate a reporter virus. in addition, the cre-loxp recombination is widely used to control reporter gene expression in cell cultures or animal models. notably, reporters can be expressed from an additional transcriptional unit (atu), in which a reporter gene is generally flanked by highly conserved gene start-and-stop signals. for instance, gfp was expressed as a separate protein from the atu in the recombinant peste des petits ruminants virus (pprv) [17] . furthermore, the reporters can be expressed separately by introduction of an internal ribosome entry site (ires) or foot-and-mouth disease virus (fmdv) 2a self-cleaving peptide (2a) (llnfdllklagdvesnpgóp), which is able to undergo self-cleavage allowing simultaneous expression. for example, recombinant alphaviruses expressing a separate fluc by 2a-mediated cleavage were successfully used to screen viral receptors [18] . since the properties of rcrevs and the stability of reporter genes may vary among different strategies, the selection of expression strategy is another principal consideration on designing rcrevs for specific applications. notably, the same strategy might lead to different effects on the growth of the same virus due to the distinct insertion site. for example, a recombinant respiratory syncytial virus (rsv) expressing a reporter protein from an atu upstream of ns1 displayed negligible attenuation in cell cultures [19] , whereas the rsv expressing a reporter from an atu inserted between f and g genes was significantly attenuated in cell cultures [20] . additionally, the use of 2a peptide to achieve expression of a separate reporter might constitute a promising approach as 2a peptide is small and can readily be self-cleaving while minimizing the possibility of the loss of functions of the viral proteins. the neutralization immunofluorescence test (nift) is currently a gold standard for the detection of nabs against many noncytopathogenic viruses. however, nift is labor-intensive and time-consuming due to the necessary incubation and staining procedures. it would be convenient to use rcrevs to detect nabs directly without immunostaining. there are many successful applications of rcrevs harboring egfp, rluc or fluc in the rapid neutralization tests [17, [21] [22] [23] [24] [25] . for viruses causing slight or no cytopathic effects (cpes) in cultured cells, the egfp reporter can be chosen to generate rcrevs for quantifying nabs with higher specificity through direct observation of egfp fluorescence. due to the structural characteristics of egfp, the fluorescence of egfp fused to a viral protein may be attenuated or quenched. therefore, egfp should be separated from the viral protein by introduction of an atu, ires or 2a sequence when constructing the rcrevs. owing to the simple assaying for gluc activity compared with the fluc, rluc and other bioluminescent reporters, it is advantageous to determine the neutralizing antibody titers based on gluc-expressing viruses. notably, attenuated rcrevs can also be applied for rapid neutralization tests due to high sensitivity and operational simplicity for detection of the reporters. antiviral compounds, interferon-stimulated genes (isgs) or small interfering rnas (sirnas) have potential applications in the treatment of many diseases. the traditional screening methods of them are developed by a cell-based hts, in which the treated cells were observed under a microscope for the inhibitory activity of the compounds for cpes [26], enzyme-linked immunosorbent assay (elisa) [27] or fluoresces-linked immunosorbent assay [28] . using these approaches, the scientists have screened and identified a series of small antiviral molecules or inhibitors [29, 30] . however, the traditional methods based on wild-type viruses are inefficient for antiviral screening. to overcome this problem, rcrevs have been applied for the purpose of antiviral screening, because rcrevs can target the complete virus life cycle and offer a higher throughput of antiviral screening than traditional assays. rcrevs represent powerful screening tools for identifying antiviral compounds against various highly pathogenic viruses [31-34]. for example, a high-throughput assay for zaire ebov has been established using the recombinant ebov expressing the egfp reporter gene [31] . interestingly, reporter viruses in combination with other approaches, such as rnai, have been applied to screen anti-csfv isgs [15] , which is time-and cost-effective. importantly, rcrevs with slightly reduced growth ability compared with the wild-type viruses can also be applied for screening antiviral isgs. in addition, rcrevs can be used for sirnas hts with high efficiency. for instance, a reporter csfv expressing the fluc gene has been used to screen antiviral sirnas efficiently [16] . recently, a recombinant ebov carrying a luciferase reporter was used to screen sirnas with higher screening efficiency than the wild-type virus [25] . however, there are some problems associated with rcrevs in hts applications. first, the interference of compound fluorescence may occur when screening antivirals using fluorescent reporter-expressing viruses. second, the antiviral effects of screened out antivirals by rcrevs need to be verified with the parental viruses. furthermore, the antiviral effects may be different between rcrevs and the wild-type viruses due to the occasionally inclusive fluorescence signals for the wild-type viruses in indirect immunofluorescent assay (ifa) and higher sensitivity for rcrevs in fluc/rluc activity assay. third, rcrevs are not ideal tools for screening of antivirals targeting specific step(s) of viral infection, since rcrevs can undergo a complete virus life cycle. for example, currently, a set of isgs against hepatitis c virus (hcv), yellow fever virus (yfv), west nile virus (wnv), chikungunya virus (chikv), venezuelan equine encephalitis virus (veev) and human immunodeficiency virus (hiv-1) have been documented, but their exact antiviral step(s) remain(s) unknown [35] [36] [37] [38] . a practical challenge lies in the explanations of their antiviral mechanisms for antiviral isgs screened by rcrevs. despite these limitations, the following strategies may address some of the above issues. fluc, rluc and other bioluminescent reporters provide a viable alternative to fluorescent reporters in hts assays for drug discovery [39] . this facilitates the development of highly sensitive, cell-based reporter assays [40] , eliminates the problem of compound fluorescence [41] , and possesses several advantages such as high reliability, convenience and adaptability to hts assays. remarkably, primary hts followed by validation using traditional assays based on the parental viruses will greatly aid the discovery of novel antivirals against infectious diseases. finally, the use of replicons or pseudoparticles would help to identify the step(s) of the viral life cycle as the potential targets of antivirals. identification of cellular receptors facilitates understanding of the mechanisms of virus entry into host cells [42, 43] . moreover, the receptors are regarded as promising targets for development of novel antivirals [44] [45] [46] [47] . while reporter-expressing pseudoparticles are widely used to screen viral receptors [48, 49] , rcrevs carrying fluc [18, 50] , gfp [51] or neo r [52] as new useful tools have been applied for screening of viral receptors (table 1 ). since rcrevs can infect the cells with multiple life cycles in contrast to pseudoparticles, more false-positive receptors may be screened. in spite of these few limitations, rcrevs are still powerful tools to screen viral receptors in combination with unsusceptible cells and cdna library derived from susceptible cells [51, 52] or a set of sirnas against a number of genes encoding cell membrane proteins [18, 50] (table 1) . with the development of reverse genetics systems, rcrevs provide an ideal tool for monitoring the dynamics of viral infection progression in vitro and in vivo due to eliminating the need for secondary labeling, which represents a significant advance in the study of the biology of viruses (table 2) . rcrevs carrying a gfp reporter gene have been successfully used for tracking viral protein(s) or viral infection in vitro and in vivo [53] [54] [55] [56] , which indicates that the gfp reporter gene is suitable for generating rcrevs to track viral proteins either in cell cultures or animal models. furthermore, gfps in rcrevs can be expressed efficiently in rodent brain for a long time [57] and show lower autofluorescence in the tissue [56] . therefore, gfp may be a promising option when rcrevs are used to study the infection of viruses replicating in the brain. additionally, an engineered virus expressing the split-green fluorescent protein (split-gfp) in the presence of cell lines expressing the complementing gfp can facilitate the tracking of viral infection in living cells [58] . compared with the most commonly used egfp tag, the tc tag enables the fusion protein to fluoresce more quickly, with a minimum risk of disrupting the overall structure and function of the targeted protein [59] . the tc-labeling technology has led to successful tracking of the nonstructural or structural proteins of diverse viruses [60] [61] [62] [63] [64] [65] . however, since the tc-tag technology contains a biarsenical labeling process [66, 67] , the engineered replication-competent tc-tagged viruses are not suitable for tracking viral protein in vivo. in addition, recombinant canine distemper virus (cdv) expressing dtomato was used to investigate the routes of virus spread in vivo [56] . a fully functional recombinant pneumonia virus of mice (pvm) with katushka 2 has been developed to track infection of target cells in vivo [68] . compared with far-red gfp-like proteins, irfp has a substantially higher signal-to-background ratio in a mouse model due to its infrared-shifted spectra [69, 70] . interestingly, the cre recombinase as a reporter is used to generate rcrevs for visualizing virus infection in engineered cell lines or transgenic animals harboring a loxp-flanked fluorescent marker upstream of another otherwise silenced fluorescent reporter [71] . table 2 . applications of representative rcrevs in virus tracking and live imaging in vitro and in vivo. green fluorescent protein (gfp) herpes simplex virus (hsv) compartmentalization of protein by autofluorescent particles [54] borna disease virus (bdv) in rodent brains [57] canine distemper virus (cdv) routes of virus spread in vivo [56] vesicular stomatitis virus (vsv) intracellular transport [55] tetracysteine (tc) influenza a virus visualization of ns1 protein nuclear import in virus-infected cells in real time [61] classical swine fever virus (csfv) nucleus import and export [62] hepatitis c virus (hcv) virus particle assembly [63] human immunodeficiency virus (hiv) viral component complexes [64] de novo hiv production [ recently, several influenza viruses expressing fluorescent proteins of different colors ("color-flu" viruses expressing ecfp, egfp, venus or mcherry) or a toolbox of influenza a and b reporter viruses were generated to facilitate the study of viral infection in in vivo models. whole-mount images of transparent lung tissues were obtained using a fluorescent stereomicroscope [72] [73] [74] [75] [76] . in addition, bioluminescent and fluorescent dual-reporter marek's disease viruses are engineered to track viral replication in cell cultures or animal models [77] . in the future, "color" or dual-reporter viruses will be powerful tools to analyze viral infection at the cellular level in vivo to better understand the pathogenesis of various viruses. notably, reporters fused with viral proteins are very suitable for investigating the localization and distribution of the proteins in infected living cells. rcrevs will help advance virus-related live-imaging studies in vitro and in vivo, which allow localization of the infection and tracking of changes in the distribution of viruses in animals in real time. elucidating various aspects of pathogen-host interactions is essential for the comprehensive understanding of pathogenesis. compared with the most frequently used techniques for mapping of virus-host interactions, the approaches based on rcrevs can recapitulate the virus life cycle [78] . split-gluc (gluc1 and gluc2) has been applied for identification of virus-host interactions. for example, a recombinant influenza virus carrying a gluc1-tagged polymerase subunit is used to infect the cultured cells expressing a pool of gluc2-tagged cellular proteins involved in nucleocytoplasmic-transporting pathways for confirming virus-host interactions [79] . in addition, split-gfp reporter has huge potential in this application. however, the reporter activity based on the interactions of rcrevs with the cellular proteins may not be detected due to the interference of the space structure. the rcrevs are also useful in modified live vaccines containing genetic markers, which have been developed for many viruses by inserting egfp as a positive marker [80] [81] [82] . for example, a genetically marked recombinant rinderpest vaccine expressing gfp has been developed [81] . in addition, a recombinant gfp-tagged prrsv containing a deletion of an immunogenic epitope, in accompany with the diagnostic tests (gfp-and epitope-based elisas), enables serological differentiation between the marker virus-infected animals and those infected with the wild-type virus [82] . a recombinant viral hemorrhagic septicemia virus (vhsv) harboring rfp gene was utilized to evaluate vhsv-based viral-vectored vaccines [83] . more recently, the marker vaccine vsmegfp-hclv3'utr in the context of the csfv shimen strain was generated by inserting egfp to create a positive marker [84] . for those viruses causing cpes, rcrevs can be used as an intermediate to generate and purify expected variants. for example, a novel ge-deleted pseudorabies virus (prv) was obtained by ge/gi-deleted virus expressing egfp [85] . in addition, katushka 2 as a reporter was used to evaluate a novel reverse genetic system of rsv [19] . interestingly, oncolytic recombinant viruses harboring reporter genes have been developed and applied for the disease progression tracking and accurate visualization of tumor burden [14] . since oncolytic viruses selectively infect as well as replicate within cancer cells, the recombinant oncolytic viruses expressing reporter genes, particularly for far-red fluorescent proteins, will be a promising option for real-time monitoring of viral infection in cancer tissues [14] . while rcrevs harboring a reporter fused to a viral protein are the most suitable for studying the localization of the protein in infected cells, rcrevs carrying separate reporters are useful for other basic research purposes. for example, the preferential translation of viral rnas over host rnas during vsv infection has been demonstrated by the egfp reporter expressed from the recombinant vsv [86] . recently, the contribution of ebov proteins in modulating dendritic cells (dc) maturation was investigated using the recombinant virus carrying egfp [87] . furthermore, unique profiles of rfp expression acquired from thousands of co-infected cells with viable and defective viruses showed how the interference of defective viruses acts at multiple steps of infection [88] . firstly, a practical challenge for some viruses lies in not allowing the insertion of reporter genes. as we stated above, it is difficult to insert a reporter gene into the genome of influenza viruses. despite the challenge, reporter-expressing influenza viruses have been developed and applied in basic science [58, 61, [71] [72] [73] [74] [75] [76] . to address the question, there are three necessary considerations, including the reporter protein itself, expression strategy, and structure of the viral protein. for example, the loop/linker regions are usually chosen to insert the tc tag based on the structure of ns1 of influenza viruses [61] . although rcrevs have been developed and applied in vitro and in vivo, one question arises regarding the expression stability of the reporter gene in rcrevs during the viral replication in vitro and in vivo [53, 89] . one potential consequence of rcrevs' attenuation is the purging of the inserted reporter from the viral genome. in this regard, we need to better understand the mechanism of regulation of viral genome replication and gene expression [90, 91] , the association between structure and function of viral proteins, as well as the application of novel reporters such as nanoluc due to its small size [92] . one of the biggest challenges is that rcrevs are possibly attenuated and may not accurately reflect natural infections [93, 94] , which partially limits the applications of the rcrevs, especially in vivo. replacement of currently used expression strategies may be a promising approach to overcoming this problem. as an example, ires or 2a peptide-encoding sequence has been used to express separately the reporter from viral protein [71, 95] . importantly, the use of split-gfp or split-luciferase may not compromise viral replication competency due to their smaller sizes [58, 79] . however, whether these reporter viruses will be attenuated in vivo needs further investigation in the future. more recently, it has been reported that after mouse adaptation, influenza virus h5n1 expressing the venus reporter gene became more pathogenic to mice and the venus gene was more highly and stably expressed [96] , which may be another promising avenue that maintains the pathogenicity of the reporter viruses. luciferase imaging uses the luciferases to catalyze reactions that produce visible light in vivo at body temperature, which is used to determine the sites of virus replication, monitor viral dissemination in real time [97] . however, there are many caveats in the process of obtaining accurate luciferase imaging [98] . for example, the reporter signal from rcrevs is attenuated when in vivo imaging in tissues. despite these limitations, luciferases will still become major reporters for in vivo imaging in real time in the future as they have a number of advantages compared with the fluorescent reporters, such as no intrinsic autoluminescence. in addition, irfps are in high demand for in vivo imaging, which exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies with multicolor imaging. finally, in view of the advantages and disadvantages of different reporters, there seems no universal reporter for various applications. fortunately, ever-increasing novel reporters, including gfp mutants, "red-shifted" analogs of luciferase, variants of luciferase and novel luciferase nanoluc, can be chosen to design rcrevs for specific purposes. moreover, the dual-reporter rcrevs may be widely used to address the scientific questions. although reporter-based assays require costly automated imaging equipment, the detection of the reporter gene expression could be also performed with inexpensive, small and simple-to-use equipment, such as a pcr device based on the development of the technologies discussed in this article. rcrevs have proved themselves to be powerful tools for applied and basic sciences. despite their limitations, rcrevs have many more far-reaching benefits in virus research: a genome-wide rnai screening for host factors required for virus replication, identifying antivirals against viral infections using hts settings, monitoring viral infections in vitro and in vivo in real time, or evaluating vaccination approaches, as well as detecting antiviral nabs. classical swine fever virus: recovery of infectious viruses from cdna constructs and generation of recombinant cytopathogenic defective interfering particles infectious rna transcribed from an engineered full-length cdna template of the genome of a pestivirus a dna transfection system for generation of influenza a virus from eight plasmids rna polymerase ii-controlled expression of antigenomic rna enhances the rescue efficacies of two different members of the mononegavirales independently of the site of viral genome replication recovery of infectious murine norovirus using pol ii-driven expression of full-length cdna efficient rescue of infectious bursal disease virus using a simplified rna polymerase ii-based reverse genetics strategy rescue of newcastle disease virus from cloned cdna using an rna polymerase ii promoter minigenomes, transcription and replication competent virus-like particles and beyond: reverse genetics systems for filoviruses and other negative stranded hemorrhagic fever viruses generation of recombinant lentogenic newcastle disease virus from cdna generation of bovine respiratory syncytial virus (brsv) from cdna: brsv ns2 is not essential for virus replication in tissue culture, and the human rsv leader region acts as a functional brsv genome promoter enhanced genetic rescue of negative-strand rna viruses: use of an mva-t7 rna polymerase vector and dna replication inhibitors recovery of infectious rabbit hemorrhagic disease virus from rabbits after direct inoculation with in vitro-transcribed rna recovery of genetically defined murine norovirus in tissue culture by using a fowlpox virus expressing t7 rna polymerase oncolytic gene therapy with recombinant vaccinia strain glv-2b372 efficiently kills hepatocellular carcinoma rna interference screening of interferon-stimulated genes with antiviral activities against classical swine fever virus using a reporter virus generation of a recombinant classical swine fever virus stably expressing the firefly luciferase gene for quantitative antiviral assay a functional genomic screen reveals novel host genes that mediate interferon-alpha's effects against hepatitis c virus interferon-stimulated genes and their role in controlling hepatitis c 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fluorescent protein-tagged pb2 polymerase subunit allows live-cell imaging of the virus life cycle the green fluorescent protein biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged m protein allows dynamic imaging of m protein and virus uncoating in infected cells genetically engineered, biarsenically labeled influenza virus allows visualization of viral ns1 protein in living cells visualization of the n pro protein in living cells using biarsenically labeling tetracysteine-tagged classical swine fever virus trafficking of hepatitis c virus core protein during virus particle assembly quantitative four-dimensional tracking of cytoplasmic and nuclear hiv-1 complexes resolution of de novo hiv production and trafficking in immature dendritic cells specific covalent labeling of recombinant protein molecules inside live cells mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity priming of the respiratory tract with immunobiotic lactobacillus plantarum limits infection of alveolar macrophages with recombinant pneumonia virus of mice (rk2-pvm) bright and stable near-infrared fluorescent protein for in vivo imaging near-infrared fluorescent proteins for multicolor in vivo imaging generation of a variety of stable influenza a reporter viruses by genetic engineering of the ns gene segment multi-spectral fluorescent reporter influenza viruses (color-flu) as powerful tools for in vivo studies influenza a virus encoding secreted gaussia luciferase as useful tool to analyze viral replication and its inhibition by antiviral compounds and cellular proteins replication-competent fluorescent-expressing influenza b virus replication-competent influenza a viruses expressing a red fluorescent protein multi-modal imaging with a toolbox of influenza a reporter viruses a virulent bioluminescent and fluorescent dual-reporter marek's disease virus unveils an alternative spreading pathway in addition to cell-to-cell contact proteomic analysis of virus-host interactions in an infectious context using recombinant viruses exploration of binary virus-host interactions using an infectious protein complementation assay marker vaccine strategies and candidate csfv marker vaccines development of a genetically marked recombinant rinderpest vaccine expressing green fluorescent protein development of genetic markers in the nonstructural protein 2 region of a us type 1 porcine reproductive and respiratory syndrome virus: implications for future recombinant marker vaccine development optimal place of a foreign gene in the genome of viral haemorrhagic septicaemia virus (vhsv) for development of vhsv-based viral-vectored vaccines generation and evaluation of a chimeric classical swine fever virus expressing a visible marker gene a novel ge-deleted pseudorabies virus (prv) provides rapid and complete protection from lethal challenge with the prv variant emerging in bartha-k61-vaccinated swine population in china preferential translation of vesicular stomatitis virus mrnas is conferred by transcription from the viral genome the lack of maturation of ebola virus-infected dendritic cells results from the cooperative effect of at least two viral domains high-throughput single-cell kinetics of virus infections in the presence of defective interfering particles a single amino acid change resulting in loss of fluorescence of egfp in a viral fusion protein confers fitness and growth advantage to the recombinant vesicular stomatitis virus the use of transcription terminators to generate transgenic lines of chinese hamster ovary cells (cho) with stable and high level of reporter gene expression importance of codon usage for the temporal regulation of viral gene expression engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate rescue of influenza virus expressing gfp from the ns1 reading frame characterization of a neuraminidase-deficient influenza a virus as a potential gene delivery vector and a live vaccine multiple proteins from a self-processing polyprotein molecular determinants of virulence and stability of a reporter-expressing h5n1 influenza a virus applications of bioluminescence imaging to antiviral research and therapy: multiple luciferase enzymes and quantitation imaging luciferase-expressing viruses acknowledgments: this work was supported by natural science foundation of china (no. 31572540 and no. 31400146) and the natural science foundation of heilongjiang province of china (no. qc2015039). we thank lintao liu at lerner research institute, united states of america for improving the language of the manuscript. key: cord-348799-qu4zin3o authors: wu, nannan; nguyen, xuan-nhi; wang, li; appourchaux, romain; zhang, chengfei; panthu, baptiste; gruffat, henri; journo, chloé; alais, sandrine; qin, juliang; zhang, na; tartour, kevin; catez, frédéric; mahieux, renaud; ohlmann, theophile; liu, mingyao; du, bing; cimarelli, andrea title: the interferon stimulated gene 20 protein (isg20) is an innate defense antiviral factor that discriminates self versus non-self translation date: 2019-10-10 journal: plos pathog doi: 10.1371/journal.ppat.1008093 sha: doc_id: 348799 cord_uid: qu4zin3o isg20 is a broad spectrum antiviral protein thought to directly degrade viral rna. however, this mechanism of inhibition remains controversial. using the vesicular stomatitis virus (vsv) as a model rna virus, we show here that isg20 interferes with viral replication by decreasing protein synthesis in the absence of rna degradation. importantly, we demonstrate that isg20 exerts a translational control over a large panel of non-self rna substrates including those originating from transfected dna, while sparing endogenous transcripts. this activity correlates with the protein’s ability to localize in cytoplasmic processing bodies. finally, these functions are conserved in the isg20 murine ortholog, whose genetic ablation results in mice with increased susceptibility to viral infection. overall, our results posit isg20 as an important defense factor able to discriminate the self/non-self origins of the rna through translation modulation. isg20 was identified in 1997 by the mechti laboratory as a type-i interferon-induced protein associated to promyelocytic leukemia (pml) nuclear bodies and then to nucleoli and cajal bodies [1] [2] [3] . the protein belongs to the dnaq-like (or dedd, for asp-glu-asp-asp) 3'-5' exonuclease superfamily that includes several enzymes with dna or rna specificities. members of this superfamily share three conserved exonuclease motifs (exo i, ii and iii) that surround the protein's active site and contain the four dedd residues important for metal ions chelation [4, 5] . isg20 exhibits rna, but no apparent dna exonuclease activities [3, 6] and this property has been associated with inhibition of a broad range of rna viruses such as flaviviridae (yellow fever, west nile, dengue, hepatitis c and bovine viral diarrhea viruses), picornaviridae (hepatitis a virus), togaviridae (sindbis, chikungunya and venezuelan equine encephalitis viruses), rhabdoviridae (vesicular stomatitis virus, vsv), orthomyxoviridae (influenza virus), retroviridae (human immunodeficiency type 1 virus), hepadnaviridae (hepatitis b virus) and more recently several bunyaviridae [3, [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] , although certain rna viruses do resist isg20 as human coronavirus and phleboviruses [10, 17] . direct viral rna degradation by isg20 has been proposed as the main mechanism of viral inhibition based on two main evidences: its potent exonuclease functions in vitro and the loss of antiviral effects observed upon the mutation of dedd residues in the protein's catalytic site, which constitute at present the only isg20 mutants described in the literature. however, a number of studies have observed viral inhibition by isg20 in the absence of viral rna degradation [10, 11] , raising the possibility that alternative mechanisms may be at play. in this study, we determine that isg20 acts by modulating mrna translation and we provide evidence that this mechanism broadly targets mrna transcripts originated from exogenous genetic material independently of its viral origins (that we globally refer to here as of non-self origins), while sparing mrnas of endogenous chromosomal origins (self), or selfmimicking ones. using vsv as a model for highly replicative rna viruses, we show here that isg20 decreases translation from a broad spectrum of viral as well as non-viral mrnas, as long as they are ectopically expressed. while this mechanism of discrimination relies on an intact catalytic core, it takes place in the absence of target mrna degradation and correlates with the ability of isg20 to localize in cytoplasmic processing bodies (p bodies). the properties of human isg20 are also conserved in its murine orthologue and through the crispr/cas9-mediated generation of isg20 knockout mice, we provide clear evidence that isg20 plays an important role in the modulation of virus replication in vivo. overall, our study reveals a novel role for isg20 as a translational modulator capable of discriminating self from non-self mrnas, offering novel perspectives on strategies of viral control and an appropriate animal model in which to test them. to apprehend the role/s of isg20 during viral infection, dox-inducible jurkat, thp-1 and hela cell lines stably expressing isg20 were generated by retroviral-mediated gene transduction followed by the selection of the pool of transduced cells. isg20 was then expressed at levels comparable to those observed in primary monocyte-derived macrophages and dendritic cells stimulated with ifnα, not only to keep within physiologically relevant boundaries, but also because we noted that higher expression levels of isg20 were cytotoxic in established cell lines (s1a and s1b fig). as such, all subsequent experiments were carried out at non-cytotoxic levels of isg20 expression. under these conditions, wt isg20 inhibited vsv spread in the different cell lines tested, in contrast to a catalytically inactive isg20 mutant (fig 1a, m1 , that corresponds to the already described . however, when hela cells were examined closely, we noted only a moderate reduction in the levels of viral-coded gfp rna in isg20-expressing cells in contrast to the drastic decrease in the amount of accumulated viral proteins by wb (fig 1b, in this recombinant virus, gfp behaves as an additional transcriptional viral unit inserted between the m and the g genes, [18] ). this was not due to loss of the rna exonuclease abilities of isg20, as these were readily measurable when isg20 was immunoprecipitated from cell lysates and incubated with different exogenously-provided substrates (single or double stranded rnas, as well as dna:rna hybrids, although not dnas as expected [6] , s4a and s4b fig) . this lack of specificity was not present in cells however, given that the ectopic expression of isg20 did not lead to the indiscriminate degradation of cellular rnas (ribosomal as well as small nucleolar rnas previously shown to be associated to isg20 [2] ), nor of an hepatitis c virus (hcv)-derived luciferase reporter mrna produced in vitro and then transfected into cells (s4c and s4d fig) , suggesting that in cells the rnase activity of isg20 is tightly controlled. given that the protein accumulation defect was also observed after mg132-mediated inhibition of proteasome (s5 fig), we decided to determine whether the effects of isg20 related to translation inhibition. to this end, hela cells were metabolically labelled 16 hours post vsv infection for one hour with 35 s methionine and cysteine, prior to cell lysis and phosphor imager analysis (fig 1c and 1d ). under these experimental conditions, wt isg20 imposed a strong translational inhibition to the expression of viral-coded proteins (n and gfp) in contrast to the catalytically inactive m1-isg20 mutant. cellular translation rates remained overall unperturbed, indicating that under these experimental conditions isg20 was capable of discriminating viral from cellular mrnas. overall, these findings indicate that isg20 may exert a novel antiviral inhibitory mechanism through the selective translational control of viral mrnas. the antiviral activity of isg20 strictly correlates with its ability to act as a general translational modulator of non-self genetic material to determine whether this mechanism of control could target more broadly non-self genetic material, we used transient dna transfection which indeed represents a massive introduction of exogenous genetic material into the intracellular milieu. transfection of isg20-expressing hela cells with dnas coding for a gfp reporter yielded a mutually exclusive pattern of expression upon confocal microscopy analysis, so that cells expressing isg20 were rarely gfppositive and vice versa, mirroring the results obtained during ongoing vsv infection (fig 2a, for qualitative results). this pattern could be observed with wt isg20, but not with the catalytically inactive m1 mutant and interestingly, loss of gfp protein accumulation was observed in the absence of effects on several endogenous proteins tested, as assessed by wb and in the absence of detectable changes in gfp dna and mrna levels (fig 2b) . similar results were obtained with pcr amplicons placed at different location on the gfp mrna and with reverse transcription reactions started with random hexamers or at the polya tail with oligo-dt primers, excluding the possibility of partial end degradation of the gfp mrna (s6a fig) . at the protein level, similar defects in gfp accumulation were observed in the presence of isg20 both by wb using limiting dilutions of samples, as well as by facs by adding defects in both the accumulation of gfp-positive cells and in their mean fluorescent intensity (s6b and s6c fig). lastly, the inhibitory effect of isg20 was dose-dependent (s6d fig) and was . b) sixteen hours post infection, a fraction of hela cells expressing or not isg20 were lysed and analyzed by rt-qpcr for the viral coded gfp rna and by wb. c) as in b, but cells were incubated for one hour with 35 s met/cys prior to cell lysis. samples were then directly loaded onto an sds-page gel and analyzed by phosphor imager. d) viral translation was quantified on the viral-coded proteins n and gfp, while cellular translation was quantified on the indicated portion of the gel (normalized to each no dox. condition). nd, not determined. the graphs present means and sem of two to four individual experiments, while the panels present typical results obtained. � ; p�0.05 between the indicated condition and control, according to a student t test. observed for a large spectrum of ectopically expressed proteins independently from their dna backbone, transfection method used or origins (viral or cellular genes, s6e fig) . given the lack of described mutants in isg20 outside its dedd residues, a series of mutations were engineered in isg20 outside the exo domains, in sequences of potential interest (phosphorylation, sorting and nuclear import, through the expasy and the cbs protein sequence analysis prediction tools) to better apprehend the relationship between antiviral functions and translation control abilities. not all generated mutants could be detected upon transient transfection and wb analysis, likely due to a compromised protein stability. however, five novel isg20 mutants could be analyzed functionally (fig 2c) . although not pursued in depth in this study, these mutations could potentially affect phosphorylation on serine (m2 and m2-1), or trafficking via mutations in dileucine domains (m4 and m3), or in nuclear localization signals (m5). three isg20 mutants lost their ability to inhibit vsv spread in the antiviral properties of isg20 strictly correlate with its ability to act as a general modulator of translation of non self genetic elements. a) hela cell lines stably expressing isg20 were analyzed by confocal microscopy twenty-four hours after vsv-gfp challenge at an moi of 0.01, or after transfection with dna coding for gfp, as indicated. b) the same cells used in the bottom panels in a were also analyzed by wb, rt-qpcr (rna), qpcr (dna), as indicated. the pictures and the panels present typical patterns of expression, while the graph presents data obtained from four experiments. c) schematic representation of the isg20 mutants used here. the gray boxes indicate schematically the position of the three exo domains of isg20. the color code is used throughout the figure and refers to the antiviral activities of the indicated mutant: purple = lost; yellow = preserved. d) dox-inducible isg20 stable jurkat t cells expressing the different mutants were obtained and infected with vsv, as specified above at an moi of 0.001. the graph presents typical replication curves obtained out of three independent experiments. e) hek293t cells were ectopically transfected with dnas coding for isg20 mutants and trex1, used here as an additional reporter of isg20 activity on ectopic gene expression. cells were then metabolically labelled for one hour with 35 s met/cys prior to cell lysis. a fraction of the soluble lysate was loaded to appreciate global translational effects (input), while the rest was used for immunoprecipitations with anti-flag antibodies specific for tagged isg20 and trex1 proteins. samples were then loaded onto an sds-page gel for phosphor imager analysis. the graph presents means and sem of three to five independent experiments. � ; p�0.05 between the indicated mutant and control, according to a student t test. f) correlation between antiviral and translation inhibitory properties of individual isg20 mutants. the ability of these mutants to inhibit translation from ectopically expressed genes was assessed either directly by 35 s metabolic labeling, or by wb (fig 2e and s7b fig, using trex1 or gfp as examples of exogenous genes, respectively). under these conditions, the m2 and m5 mutants behaved as wt and strongly reduced the rate of protein synthesis from the exogenous reporter genes, while in contrast the m3, m4 and m2-1 mutants behaved as the catalytically inactive m1-isg20 and did not significantly affect protein translation, highlighting a near perfect correlation between antiviral abilities and translation inhibition of non-self material ( fig 2f) . with the exception of the catalytically inactive m1, all mutants exhibited robust rnase activities in vitro (s7c and s7d fig) . interestingly, two of the three mutations that altered the properties of isg20 were spatially close and exposed at the surface of the crystal structure of isg20 [5] , potentially defining a region that may be involved either directly or indirectly in the regulation of the protein's functions (s7e fig). self mimicry allows the escape of target genes' mrnas from the effects of isg20 vsv infection and ectopic dna or rna transfection do not bear much in common apart from the fact that both represent artificial injections into the cell of non-self genetic material from without. to determine whether isg20 could be an ifn-induced mechanism of defense acting at the translational level, we determined the effects of isg20 on exogenous expression cassettes that mimicked cellular host genes. to this end, retroviral-mediated gene transduction was used to stably integrate into the host genome the identical cmv-gfp expression cassette used in fig 2a. after a three weeks selection, cells were transiently transfected with wt isg20, lysed and analyzed by wb (fig 3a) . in this case, isg20 did not modify the amount of produced gfp, indicating that, when expressed from the cell's genome, the gfp-coding mrna is isg20-resistant and suggesting that cellular mrnas mimicry (meant here as the ability to behave as, or similarly to, cellular genes) could constitute an escape mechanism from isg20. some dna viruses as the epstein-barr virus (ebv, herpesviridae) can be stably maintained in an extrachromosomal latent form in the nucleus of target cells (referred to as episomes) using the cellular dna machinery for their duplication and segregation into dividing cells. although both transiently transfected dna and the ebv genome are extrachromosomal, only the latter presents a chromatin status identical to the host genome [19, 20] . to determine whether this, rather than integration per se, could be a determining factor in the susceptibility to isg20, hek293t cells bearing stable ebv-gfp episomes were transiently transfected with dnas coding for wt isg20, in addition to a control plasmid coding for hiv-1 gag (that served as a control for non-self genetic material within the same cells, fig 3a) . upon cell lysis and wb analysis, isg20 did not affect the amount of gfp expressed from the ebv genome, whereas it did affect the amount of gag. to further strengthen this point, the effect of isg20 on ebv infection was determined in hone-1 cells upon the reactivation of the viral lytic cycle with tpa/ba (fig 3b) [21]. isg20 did not affect the production of several ebv proteins that typically initiate its lytic cycle indicating that ebv mrnas were, as cellular ones, largely resistant to the action of isg20. also, isg20 did not modify the extent of ebv infection, when virion particles produced under these conditions were used to challenge raji cells (s8 fig) . overall, these results indicate that isg20 discriminates mrnas based on their self/non-self genesis and also underline the fact that certain viruses, as ebv, may have devised mechanisms to resist this inhibition due to their specific modes of replication. to determine whether isg20 could affect translation through modifications in either splicing or transport of mrnas from the nucleus to the cytoplasm, its activity on intron-or intronlessluciferase based constructs was assessed [22] and the accumulation of target mrnas was determined in cytoplasmic and nuclear fractions (s9a and s9b fig) . isg20 exerted similar inhibitory effects on both intron and intronless constructs and it did not appreciably modify the distribution of target mrnas in cytoplasm and nucleus, overall suggesting that isg20 does not affect the nucleo-cytoplasmic transport of mrnas. lastly, isg20 could not be detected in crude ribosomal fractions, indicating a lack of stable association with ribosomes (s9c fig). a series of constructs was used to determine whether isg20 affected translation initiation by comparing more specifically its effects on cap-dependent versus internal ribosomal entry site (ires)-translation (fig 3c, through the use of bicistronic vectors), as well as its effects on rnas with different 5' untranslated regions (5' utrs) that require or not eif factors to initiate translation (fig 3d) . gene mimicry of self through integration into the host genome, or ebv-mediated episomal maintenance, allows escape from isg20 translational targeting. a) hek293t cells were first stably transduced with a retroviralbased vector bearing a cmv-gfp expression cassette and then transiently transfected with an isg20-expressing dna vector. cells were analyzed forty-eight hours after by wb. similarly, hek293t cells carrying a stable ebv-gfp episomal genome were transiently transfected with dnas coding for isg20 and hiv-1 gag, used here as a reporter of the activity of isg20 on ectopic expression. b) hone-1 cells that bear a stable latent episomal ebv genome were transfected with isg20 prior to stimulation with 12-o-tetradecanoylphorbol-13-acetate (tpa) and butyric acid (ba) that reactivates the life cycle of ebv via a pkc-dependent mechanism. cells were lysed twenty-four hours later and analyzed by wb for the indicated ebv proteins. panels present typical results obtained. c) hek293t cells were transfected with isg20 along with the indicated bicistronic dna vectors. the first cistron bears the 5'utr of the cellular gene globin (glo). the second cistron is under the control of several ireses derived from the cellular gene myc (myc), polyomavirus (pv), or the encephalomyocarditis virus (emcv). mrna levels have been normalized to controls (no isg20 condition) and luciferase activities are reported after mrna normalization. d) translationcompetent mrna generated in vitro were transfected in isg20 expressing cells, prior to cell lysis one hour afterwards and analyses by rt-qpcr or luciferase assays. the 5'utrs of these mrnas varied with respect to the requirement of specific eifs and were derived from the cellular gene myc (myc), the cricket paralysis virus (crpv) and vsv. the graphs present means and sem of 3 (monocistronic) and 8 (bicistronic) independent experiments. � ; p�0.05 between control and isg20, as indicated, according to a student t test. isg20 was capable of inhibiting both cap-and ires-dependent translation from bicistronic mrnas in the absence of mrna degradation and interestingly this occurred on ires sequences in which translation initiation required ribosomal scanning along the mrna or not (fig 3c, ireses obtained from c-myc, polyomavirus-pv, or the encephalomyocarditis virus-emcv, respectively). to further determine whether isg20 acted on translation initiation and more specifically through specific initiation factors (eifs), capped and polyadenylated mrnas coding luciferase under the control of several 5' untranslated regions (5' utrs) were generated in vitro and directly transfected in isg20-expressing cells (fig 3d) . under these conditions, isg20 was able to inhibit translation of target rnas containing either the 5' utr of c-myc, that requires the complete set of eifs, or of the cricket paralysis virus (crpv) that requires none in the absence of mrna degradation. we noted that in the case of direct mrna transfection the extent of isg20 inhibition was lower than in the case of dna transfections, but we believe this is mainly due to the shorter time frame elapsing between transfection and luciferase assay (one hour, as opposed to overnight). as expected, the catalytically inactive m1-isg20 mutant lost its ability to inhibit translation from these reporter constructs (s10 fig) . overall, these analyses underline the fact that the translational interference of isg20 proceeds in the absence of overt mrna degradation in a manner that appears independent from the presence of specific initiation factors during 5' cap loading. when expressed in two clonal mouse embryonic fibroblast (mef) cell lines, isg20 has been recently described to drive type i interferon responses resulting in the expression of different interferon-sensitive genes among which the interferon-induced protein with tetratricopeptide repeats 1 (ifit1) [11] . ifit1 recognizes viral uncapped rnas (often defined as non-self, although the definition used in our study includes more broadly all foreign genetic elements) and has been extensively associated to translation inhibition [23-26], this work raised the possibility that isg20 could inhibit translation indirectly via the induction of ifit1. to address this possibility, we analyzed ifit1 expression in both ectopically transfected hek293t cells, as well as in stable hek293t and u937 cells, a myeloid cell line highly sensitive to ifn (fig 4a) . when hek293t cells were ectopically transfected with increasing amounts of isg20-coding dnas, ifit1 remained undetectable unless ifnα was provided (1.000 u/ml). similarly, isg20 induction by doxycycline in stable hek293t and u937 cells did not induce a concomitant ifit1 production, indicating that isg20 does not induce ifit1 expression under the experimental conditions used here. to more generally appreciate whether isg20 induced an ifn program, we measured the levels of type-i ifns potentially secreted upon induction of isg20 in the supernatants of u937 cells that are more responsive to ifn stimulation than hek293t. to this end, isg20 expression was induced via doxycycline in stable u937 cells and 24 hours later supernatants were harvested and incubated with reporter hek293t cells bearing an integrated expression cassette responsive to type i interferons (6-16:luc). reporter cells were then lysed twenty-four hours after and luciferase measured. the levels of type-i ifn measured via this assay remained below the threshold of detection (30 u/ml, in our hands), indicating the absence of major ifn up-regulation following isg20 induction (fig 4b) . lastly, we wished to determine whether ifit1 could exert similar activities than isg20. to this end, hek293t cells were transiently transfected with dna coding these proteins along with gfp as non self genetic material (fig 4c) . under these conditions, isg20 but not ifit1 inhibited the accumulation of gfp, while both inhibited vsv infection (s11 fig). ifit1 has been described to act in complex with other ifit proteins [27] . given that its ectopic expression alone is sufficient to inhibit vsv replication, we believe that other ifits are expressed at a basal level under the conditions used here and that ifit1 may be a limiting component of the complex. on the whole, our results indicate that ifit1 is not involved in the mechanism of translation inhibition by isg20 given that: 1) isg20 does not induce the expression of ifit1, at least under the experimental conditions used in this study and 2) that isg20, but not ifit1, is capable of inhibition of translation from non-self genetic material. isg20 is largely distributed throughout the entire cell [3, 10] , although certain studies reported a more specific association with nuclear bodies (pml nuclear bodies, nucleoli and cajal bodies [1, 2] ). to determine whether this was the case, we employed a commonly used technique to reveal protein association to nuclear bodies. cells were transiently transfected in duplicate with isg20 (transfection rates �80%) and while one sample was immediately fixed, the other was previously treated with detergent prior to fixation to reveal proteins strongly associated to in parallel, ifit1 expression levels were analyzed by wb in hek293t and u937 stable-isg20 cells stimulated for twenty-four hours with different concentrations of doxycycline, or with ifnα2 (1.000 u/ml). b) to measure the possible presence of ifn subtypes in the supernatant of cells expressing isg20, u937 cells that express the most readily detectable levels of ifit1 were stimulated as indicated and the supernatant was then transferred to reporter cells expressing luciferase under the control of the ifn-inducible 6-16 promoter. luciferase production from reporter cells was then determined twenty-four hours later. the linearity and limit of detection of the assay (30 u/ml in our hands) are routinely determined with external dilutions of ifn. c) hek293t cells were transiently transfected with plasmids coding for either isg20 or ifit1 along with a gfp-coding plasmid followed by wb analysis. the graph present results obtained from 2 independent experiments, while the wb panels display representative results obtained from 3 independent experiments. https://doi.org/10.1371/journal.ppat.1008093.g004 nuclear bodies. under these conditions, partial co-localization of isg20 was observed with splicing speckles (sc35 nuclear bodies, s12a fig), but the signal was essentially lost upon detergent pre-extraction, suggesting that the association between isg20 and nbs may be very weak. instead, we noted a partial colocalization between isg20 and both the endogenous forms of the trinucleotide repeat containing 6a protein (tnrc6a) and the dead-box helicase 6 (ddx6), two well-established markers of p bodies [28, 29] (fig 5a for hek293t cells expressing isg20 and s12b fig for the complete panels of control cells) both in unstimulated cells, as well as in cells treated for one hour with puromycin, drug that induces translational stress and increases both the proportion of cells exhibiting p bodies, as well as their number on a per cell basis. however, isg20 did not affect the proportion of cells presenting or not p bodies over controls, suggesting that isg20 does not induce p bodies by itself. to study the correlation between the antiviral phenotype of isg20 and p bodies, hek293t cells expressing the different isg20 mutants were examined by confocal microscopy after ectopic expression of a destabilized version of tnrc6a, that allows for an easier visualization of p bodies (fig 5b for representative pictures and graph for comprehensive quantification of the proportion of p bodies within cells that scored positive for isg20). under these conditions, wt isg20 was present in 70% of p bodies in double-positive cells, a proportion similar to the functional isg20 m2 and m5 mutants (62% and 64%, respectively). in contrast, the proportion of isg20 present in p bodies dropped substantially in all the non-functional mutants examined (m1, m3 and m4, with rates of 12%, 22% and 19%, respectively). thus, these results indicate isg20 co-localizes with p bodies. a) hek293t isg20-expressing cells were analyzed by confocal microscopy along with two markers of p bodies, tnrc6a and ddx6 in the presence or absence of a one-hour incubation with 100 μg/ml of puromycin that induces a translational stress known to increase the number of cells expressing p bodies as well as the number of p bodies on a per cell basis [45] . control cells not expressing isg20 are shown only as a zoomed overlay, while the complete panel are presented in s12b fig. the graph presents the proportion of cells exhibiting detectable p bodies in the presence or absence of puromycin and/or isg20. b) the different isg20 mutants were similarly analyzed by confocal microscopy with the exception that an ha-tagged, destabilized form of tnrc6a was also concomitantly expressed by transfection to increase the extent of p bodies accumulation. representative pictures and distributions (out of >100 cells per condition/mutant in two to three independent experiments) are shown. the graph presents the proportion of p bodies with or without isg20 in double-positive cells. that p bodies and/or p bodies localization may be critical for translation inhibition and antiviral properties of isg20. murine and human isg20 are highly conserved and display more than 80% identity at the amino acidic level (s13 fig) and a recent report indicated that murine isg20 was also endowed with anti-viral properties [11] . to determine whether the remaining functions of isg20 described here were also similarly conserved, murine isg20 (misg20) was ectopically expressed in hek293t cells and compared to its human counterpart (hisg20). under these conditions, misg20 was able to inhibit vsv spread through the culture (fig 6a) , it impaired expression from transfected dna coding for a luciferase reporter (fig 6b) and was also able to cluster along with tnrc6a bodies (fig 6c) . therefore, these results indicate that the key functions of isg20 described here are mainly conserved between these two animal species. to determine the importance of isg20 during viral control in vivo, isg20 knockout mice were generated by crispr mediated gene editing (fig 7a, left panel) and the functional ablation of isg20 was controlled upon in vitro stimulation of bone-marrow derived macrophages (bmdm, fig 7a, right panel) . isg20-/-mice were viable and displayed the expected mendelian frequency. when animals were challenged intraperitoneally with vsv, the survival rates of isg20-/-mice were severely compromised with respect to age-and sex-matched control groups (fig 7b) , indicating that isg20 plays an important controlling role during viral containment in vivo. the higher replicative capacity of vsv was evident in different organs and correlated with higher infiltration of inflammatory cells as observed upon hematoxylin and eosin staining (fig 7c and 7d , lung sections). as expected, peritoneal macrophages (pem) isolated from isg20-/-mice exhibited an increased susceptibility to infection (fig 7e, in both percentage of infected cells as well as in their mfi), thus confirming a primary role for isg20 in intracellular innate defenses. overall, these results indicate that isg20 is an important contributor of viral replication control in vivo, corroborating similar results obtained in a previously published animal model [11] . in this study, we determine that isg20 acts as an important antiviral factor in vitro, as well as in vivo, given that mice genetically deficient in isg20 exhibit higher mortality following viral challenge. the antiviral properties of isg20 are not linked to its ability to directly degrade viral rna, but rather to its capacity to interfere with the process of translation. this mechanism of translational regulation targets and is able to distinguish viral rnas as well as rna originated from ectopically transfected dna (that we globally refer to as non-self, for mrnas originated from non-self genetic elements) from cellular mrnas (or self) that are instead resistant to isg20, under the experimental conditions used here. finally, we determine that resistance to isg20 can be achieved via integration into the host genome, or through ebv-like maintenance in stable episomes through what can be defined as self mimicry. isg20 is a potent and non-specific rna exonuclease in vitro and this property had long been hypothesized to underpin a broad mechanism of inhibition via the direct degradation of the rna genome of a large spectrum of rna viruses, albeit not all [2, 3, [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . however, a few studies failed to observe a decline in viral rna levels in the presence of isg20 [10, 11] and evidence of direct binding of target rnas to isg20 in cells is lacking. the data presented in our study also argue against overt degradation of viral rna and point instead to an interference with the process of translation that not only targets viral rnas, but more generally distinguishes mrnas according to their self/non-self origins (according to the broader definition used in this study). despite the fact that the catalytically inactive mutant m1 loses both its exonuclease and translation inhibition activities, whether the former is required for translation inhibition remains unclear. indeed, mutations in the dedd residues may potentially exert multiple effects on the protein functions in addition to inhibiting its rnase activity and the in vitro assays used in the field (as well as in this study) do not seem to recapitulate the stringency of action of isg20 in cells. it is important to note that although our results do not support a mechanism based on the direct degradation of target mrnas by isg20, they do not exclude that its exonuclease activity may be turned against a cellular rna component, itself involved in translation. rather, we favor this hypothesis in which isg20 may attack ribosomal rnas or small nucleolar rnas that are required for ribosomal rna methylation, possibly affecting the ribosomes' behavior. given that cellular rnas continue to be translated, this implies that isg20 may affect some, but not all ribosomes. we do not know whether this occurs here, however recent genome-wide studies have clearly highlighted that ribosomes are not only heterogeneous in composition, but also in function [30-33], so that it is possible that the activity of isg20 is directed only against a specific subset of ribosomes. the ability to discriminate rnas according to the global definition of self/non-self origins we use here during translation is an interesting concept and sting has also been recently shown to inhibit the translation of viral, as well as of ectopically expressed mrnas through a novel ifn-independent activity [34]. notable differences exist however with the mechanism described here, as sting seems to act on the phase of translation initiation, thus sparing mrnas that require no initiation factors (such as crpv) [34], while on the contrary isg20 targets also these mrnas, strongly pointing in this case to a mechanism of regulation at a step other than translation initiation. a previous study also suggested that isg20 could interfere with viral translation in the absence of target rna degradation [11] . in this study however, this inhibition was linked to the ability of isg20 to induce ifn when expressed in mef cell lines and more specifically the interferon-protein with tetratricopeptide repeats 1 protein (ifit1, [11] ), a known translational modulator that impairs uncapped rnas [25, 35, 36]. however, we have failed to obtain evidence of ifn and of ifit1 upregulation in our experimental system and since ifit1 cannot target the translation of mrnas expressed from plasmid dnas its involvement in the isg20-mediated inhibition we describe here is unlikely. furthermore, if isg20 expression resulted in the direct upregulation of an ifn program, isg20 would be expected to inhibit far more viruses than currently reported. it is therefore possible that this aspect of the biology of isg20 can be observed solely under very particular conditions. if translation initiation does not seem to be the main step affected by isg20, our results highlight a tight correlation between the ability of the different isg20 mutants to inhibit translation and their localization in p bodies, which is in line with the key role of these cytoplasmic structures in translational silencing and which would suggest rna re-routing by isg20. p bodies are involved in numerous aspects of rna metabolism and although initially described as sites of rna degradation [37], their functions have been further complexified by studies indicating their role in rna storage as well as in rna translational repression in the absence of rna degradation [38] [39] [40] [41] [42] . thus, the results we have obtained at this stage support the hypothesis that p bodies play an instrumental role in the action of isg20. however, it remains possible that these structures form as a consequence of the translational block itself [43] [44] [45] , so that additional work will be required to firmly establish the functional role of these structures during isg20-mediated translation inhibition. our data indicate that the main determinant of susceptibility to isg20 is the self/non-self nature of the genetic element from which the mrnas originate and we hypothesize that the chromatin status of the transcribed elements may be a determining factor of this phenomenon. specific co-or post-transcriptional modifications themselves linked to the chromatin status (as for example, n 6 -methyladenosine, m 6 a, reviewed in [46, 47] ) are known to modulate different steps of mrna biology and it is possible that a major difference between genetic elements embedded in the host chromatin and the others resides in the identity of marks deposited on the mrna. lack of such signals may expose the mrna to isg20 and to its translational regulation, providing an interesting level of regulation of foreign elements. an alternative and non-mutually exclusive hypothesis may be that the levels -and not the identity-of post-transcriptional modifications may be lower in elements that are either present in high number of copies or that are expressed at high levels, simply because the system is overrun, thus exposing the rna to the action of isg20. this hypothesis is also of interest and may be viewed as a basic threshold control mechanism of transcription/translation that is automatically turned on when genetic elements that replicate (or transcribe/translate) rapidly at high levels, as is the case during viral infection or following ectopic dna transfection. this hypothesis may certainly explain the different isg20-susceptibility of rna viruses with respect to cellular genes, because most rna viruses replicate to exponential levels in cells in a very brief period of time, but it may also explain the isg20-susceptibility of ectopically transfected dnas/genes, as their chromatin status is distinct from the one of the host genome [19, 20, 48, 49] and their expression levels are generally much higher when compared to integrated cellular genes. in this respect, integration into the host genome or latent episomal maintenance may constitute a self mimicry defense strategy used by certain viruses, as we have shown here for ebv, to escape isg20 surveillance. from a more general perspective, it will be of high interest to determine the extent of the translational control activity of isg20 on viruses such as retroviruses, retroelements, as well as more broadly on different dna viral families. ifn responses rely on a complex network of more than 1.000 isgs. along with its multiple effects on the cell physiology, a major antiviral block of type i ifn responses takes place during translation initiation, by protein kinase r (pkr)-mediated inactivation of the eukaryotic initiation factor alpha (eif2α, [50, 51] ). given that the effects of ifn on translation are not absolute and vary according to the cell type, the fact that isg20 targets a step downstream translation initiation is of high interest, because it suggests the existence of a second layer of translational control in which isg20 would target rnas that have escaped the eif2 α -block. by extension, this may suggest the existence of yet additional layer of translational control during ifn responses all of which concur in limiting viral replication. overall, our study reveals that isg20 is an important modulator of antiviral responses in vitro and in vivo and points to a novel function through which this protein is able to distinguish self from non-self mrnas at translation. n-terminal flag-tagged human isg20 mutants (gene id: 3669) were produced by overlapping pcr from an original pcdna5/frt-flag-isg20 plasmid obtained from ju-tao guo (baruch s. blumberg institute, doylestown, pennsylvania, usa) and cloned into pcdna3 (invitrogen), or pretrox-tight-puro backbones by standard molecular biology techniques (clontech). the latter is a murine leukemia virus (mlv)-based genome suitable for retroviral gene transduction and it allows for stable selection of recipient cells in which expression of the gene of interest is under the control of doxocycline of a modified e. coli tet repressor coded by a second mlv-based vector (pretrox-tet-on, rtta-advanced, clontech). murine isg20 (gene id: 57444) was obtained following the same cloning scheme. the remaining vectors were obtained as follows: prrl-cmv-destabilized gfp (hiv-1-based lentiviral vector genome bearing an expression cassette for destabilized gfp, described in [52] and clontech); flag-trex1 (in pcdna3, this study), hiv-1 gag (in [53] , respectively), ha-ubiquitin (gift of pierre jalinot, ens-lyon), vsv-tagged samhd1 (in prrl-cmv, this study); ha-destabilized tnrc6a was engineered by standard molecular biology techniques from the double-tag pfrt/to/ flag/ha-dest-tnrc6a (gift from thomas tuschl, addgene, #19883, [54] ). an n-terminal ha tagged ifit1 (gene id: 3434) was cloned by standard molecular biology techniques into pcdna3.1 (invitrogen). the following bicistronic vectors were used to examine the role of different ireses and were described in [55] and [22]: bi-glo-fluc-cmyc -rluc; bi-glo-fluc-emcv-rluc and bi-glo-fluc-pv-rluc. the following constructs code for a renilla luciferase gene under the control of the beta-globin promoter and contain or lack an intron: pglobin-intron-rluc and pglobin-rluc (described in [22] , gift from fabrice mure, ciri, lyon, france). the following vectors have been used for in vitro production of rna and they allow upon linearization the t7-based synthesis of a polyadenylated mrna that is then 5' capped: phcv-rluc, pcrpv-rluc, pcmyc-rluc. the pvsv-rluc was constructed by standard molecular biology techniques by placing the region 1-63 upstream of the n gene of vsv (ef197793) in front of the renilla luciferase. upon synthesis, rnas are dnase treated, precipitated and used for cell transfection. antibodies directed against the following proteins were as follows: tubulin (t0198), actin (a5441), flag (f7425 and f3165), gfp (g1544), ha (h3663), vsv-g (c7706), tnrc6a (hap015305) from sigma; msin3a (ab129087); donkey anti-rabbit igg (alexa fluor 594 conjugate, a-21207) and donkey anti-mouse igg (alexa fluor 488 conjugate, a-21202) from invitrogen; creb (4820) and akt (9272) from cell technology; ddx6 (bet1300-460a) from ozyme; isg20 (22097-1-ap, in our hands this antibody is highly unreliable on human isg20 but is more efficient on murine isg20) from proteintech; sc35 (nb100-1774) from novus biologicals; anti bmrf1 (mab8186, merck millipore); anti bmlf1, anti brlf1 and bzlf1 were described in [56, 57] ; anti-ifit1 (pa3-848, life technologies). the anti-gag/p24 antibody (clone 183-h5c) was obtained from the aids reagents program of the nih. human embryonic kidney 293t (hek293t) and human cervix epithelial hela cells (hela) were maintained in complete dmem media supplemented with 10% fcs, while human peripheral blood t lymphocyte jurkat (jurkat), human peripheral blood monocytic thp-1 cells (thp-1) and u937 were maintained in complete rpmi1640 medium with 10% fcs. cells were obtained from the atcc (crl-3216; ccl-2; tib-152; tib-202, crl-1593.2 respectively). hek293t-ebv [58] , hone [21] and raji cells were obtained from the laboratory of oncogenic herpesviruses of henri gruffat (ciri, lyon) and were maintained in complete dmem or rpmi1640, respectively. ebv reactivation was induced in hone cells upon incubation with 12-o-tetradecanoylphorbol 13-acetate (tpa) and butyric acid (ba) provided at 50 ng/ml and 3 mm, respectively (sigma). when indicated the proteasome inhibitor mg132 was added at a final concentration of 10 μg/ml (sigma). hl116 cells, derivative of ht1080 cells that bear an integrated luciferase under the control of the interferon alpha-inducible protein 6 promoter (6-16:luc) were obtained from gilles uze (university of montpellier, montpellier, france [59] ) and were maintained in complete dmem. primary human blood monocytes were obtained by negative depletion as described in [52] (monocyte isolation kit ii, catalogue n˚130-091-153, miltenyi) prior to their differentiation in macrophages or dendritic cells upon incubation for 4 to 6 days in m-csf or gm-csf and il4 (at 100 ng/ml, 01-a0220-0050, pcyt-221 and pcyt-211, eurobio). when indicated, cells were incubated for further 24 hours with 1.000 u/ml of human ifnα (catalogue n˚11100-1, tebu bio), or in the case of murine bmdm with 1.000 u/ml of murine ifnβ (50708-m02h, sino biological inc.). primary mouse bone marrow derived macrophages (bmdms) were prepared from isg20-/and isg20+/+ mice, as described before [60] . briefly, bone marrow cells were isolated from mice femurs and tibias, washed with pbs and filtered through a 70 μm cell strainer to remove cell clumps. the single-cell suspension was then cultured in rpmi1640 medium containing 10% fbs and 20ng/ml of m-csf (416-ml, r&d systems) for 7 days with daily media replenishment. differentiation was monitored by flow cytometry analysis of cd11b+f4/80+ cells (bd) and reached proportion higher than 90%. pools of stable cell lines allowing for doxycycline (dox)-inducible isg20 expression were generated upon mlv-based retroviral gene transduction based on the pretrox-tight system of clontech. briefly, the corresponding mlv vectors were produced by transient dna transfection of hek293t cells with plasmids coding the structural mlv protein gag-prol-pol, the pantropic vesicular stomatitis virus g protein (vsvg), pretrox-tight-puro and pretrox--tet-on vectors that code for a miniviral genome bearing expression cassettes for a resistance gene (puromycin and neomycin, respectively) and either isg20, or the teto transcriptional rtta activator (ratios 8:4:4:4, respectively, for a total of 20 μg of transfected dna for a 10-cm dish). virion particles released in the supernatant of transfected cells were filtered, titered by exogenous rt activity [60] and used to challenge the indicated target cells. pools of transduced cells were obtained upon selection with puromycin and g418 (p9620 and g418-ro, sigma). isg20 expression was induced by incubation with doxycycline (631311, clontech). for mrna transfection, 10 5 hek293t cells were electroporated in quadruplicates with 200 ng of in vitro transcribed capped and polya mrna in a total volume of 10 μl (settings of 1150v, 20ms, 2 pulses, neon electroporator, invitrogen). cells were then transferred into 500 μl pure rpmi 1640 medium and cultured for 1 hour prior to cell lysis and luciferase analysis (see below). mrnas were generated by an in-house in vitro transcription reaction and then capped. briefly, 1 μg of linearized dna was incubated for 2 hours at 37˚c with 20u of t7 rna polymerase, 40 u of rnasin (both from promega) and 1.6 mm for each ribonucleotide triphosphate, 3 mm dtt, 40mm tris-hcl (ph 7.9), 6 mm mgcl 2 , 2 mm spermidine, and 10 mm nacl. to induce mrna cap addition, the rgtp concentration was reduced to 0.32 mm, and 1.28 mm of m 7 gpppg cap analog (new england biolabs) was added. rnas were then precipitated and finally resuspended in rnase-free water. dna transfections were carried out generally by calcium/phosphate transfection, or lipofectamine 2000 for confocal microscopy (invitrogen). generally, for transient dna transfections a ratio of 1:1 between reporters and isg20 was used (from 1:1 to 1:8 in the dose curve presented in s4 fig) not surpassing an overall amount of 3 μg of isg20 per well of 6 well plate to avoid cytotoxic effects. cells were analyzed in triplicates and lysed between eighteen and twenty-four hours after dna, or 1 hour after rna transfection. one fifth of the cell lysates was used for firefly or renilla luciferase assays (luciferase assay system or renilla luciferase assay system, promega; mithras lb 940 multimode microplate reader, berthold), while the remaining fractions were used for rna extraction and quantitative real-time pcr assay. the luciferase activities are normalized for the amount of rna present in each sample. cells were starved in 1 ml/well of met-/cys-free dmem medium (21013024, gibco) for 1 hour and then medium was replaced with 500 μl of the same medium containing 20 μci 35 s met/cys (amersham) for a pulse of 1 hour. labeled cells were then lysed thoroughly with ripa buffer (150 mm nacl, 1% np40, 0.5% deoxycholate, 0.1% sds, 50 mm triscl ph 8.0) in the presence of protease inhibitors (roche), by vigorous pipetting and 3 cycles of freezethawing. lysates were centrifuged at 12.000 g for 10 minutes to remove insoluble material and then either directly analyzed by sds-page gels or incubated with 20 μl anti-m2-flag beads for 4 hours to overnight at 4˚c. after extensive washing, material was separated by sds-page gel and upon gel fixation in methanol and drying, radioactivity was quantified by phosphor imager (typhoon 8600 system, ge healthcare). stable or transfected cells were directly grown on coverslips for at least twenty-four hours prior to fixation in cold methanol for 10 min at -20˚c (hela or hek293t cells, respectively). when indicated, a prior permeabilization step was carried out to evidence the strong association of isg20 to nuclear bodies. in this case, cells were transfected in duplicate and while one aliquot was immediately fixed, the other was first washed in cold wash buffer (pbs, 0.2% tween 20) and further permeabilized in cold psb containing 0.5% triton-x100 for 5 min on ice. cells were then fixed and incubated with the indicated antibodies (see relevant section above). dapi fluoromount g mounting medium was used (0100, southern biotech). images were acquired using a spectral zeiss lsm710 or lsm800 confocal microscopes and analyzed using the fiji software. for p bodies analyses, cells were fixed first in 4% paraformaldehyde, then in 70% methanol (10 min each), followed by permeabilization in cold psb containing 0.5% triton-x100 for 5 min on ice. cells either stably or transiently expressing isg20 were lysed in pre-cooled buffer sd-150 (150 mm nacl, 50 mm tris ph7.4, 0.5% triton x100, 0.5 mm mncl 2 and 1× roche protease inhibitor cocktail), sonicated and the soluble supernatants were incubated with pre-washed anti-flag antibody-conjugated agarose beads (a2220, sigma, 20μl per 10-cm plate dish) for 4 hours at 4˚c with constant nutation. beads were then extensively washed in the same buffer and either analyzed by wb or incubated with 0.5-1 μg of exogenously added nucleic acids for 60 minutes at 37˚c in a total volume of 30 μl of sd-150 buffer, prior to agarose gel migration and densitometric quantification by multi gauge v3.0 software (fujifilm). sequences of the complementary dna/rna oligonucleotides are as follows: dna oligonucleotides d20 and d15 were respectively: acatgtacaggatgcatttg and tacaggatgcatttg. rna oligonucleotides r20u, r20d and r20d were respectively: caaaugcauccuguacau gu, acauguacaggaugcauuug, uacaggaugcauuug. hybrids were obtained by mixing the desired oligonucleotides at a concentration of 1 μg in sd-150 buffer, prior to denaturation at 95˚c and annealing at 50˚c for 5 minutes each. poly i:c and yeast trnas were purchased from invivogen and sigma, respectively. ssrna was a luciferase based mrna generated by in vitro transcription of approximatively 1700 nucleotides. ssdna and dsdna were respectively a long chemically-synthesized oligonucleotide available in the laboratory for other purposes (eurogentec, cgcggaa ttcaccatggattacaaggatgacgacga taagggtggtggttcagccccactggatgccgccctccac) and double-stranded pcdna3 plasmid (invitrogen) linearized with psti. the levels of type i ifns secreted in the supernatant of cells expressing isg20 was determined according to a well established procedure using hl116 reporter cells [59] . briefly, isg20-stable u937 cells were stimulated for 24 hours with doxycyclin to induce isg20 expression, then supernatant was harvested, filtered and added to hl116 cells in serial dilution (2.10 4 cells in 96-well plate). twenty hours afterwards, cells were lysed and the extent of luciferase accumulation was measured (see above, luciferase assays section). hl116 cells stimulated with serial dilutions of ifnα were used in parallel to ensure the linearity of the assay. the limit of detection of this assay is in our hands of 30 u/ml. rnas were extracted by the trizol reagent (15596018, ambion), or by nucleospin rna (740955, macherey-nagel) according to the manufacturers' instructions. when indicated, cytoplasmic and nuclear rnas were separated and purified, as previously described (ricci ep, pmid: 19528074) . the extracted rna was treated with rq1 dnase to remove contaminant dna (m6101, promega) and then reverse-transcribed by qscript cdna supermix using the manufacturer's instructions and either oligo-dt or random primers (quanta biosciences; the enzyme was instead omitted in the no-rt controls). when indicated, the amount of transfected dna was quantified after the addition of rq1-dnase to the cell culture media to remove extracellular dna (twice for two hours at 37˚c at 20 u/ ml). after extensive cell washing, cells were lysed with the following solution (50 mm kcl, 10 mm tris hcl ph 8.3, 2.5 mm mgcl 2, 0.5% np-40, 0.5% tween 20) in the presence of 10 μg/ml of proteinase k (p2308, sigma) for one hour at 60˚c. dna was then purified through phenol/chloroform extraction and dna precipitation. qpcrs were performed on a stepone plus real-time pcr system (applied biosystems) using the faststart universal sybr green master mix (roche). primers were as follows (forward-f and reverse-r from 5' cytoplasmic and nuclear fractions were obtained from transfected hek293t cells. briefly, 0.5-1.10 6 cells were collected and resuspended in 0.2 ml of cold rnla buffer (10 mm of tris hcl ph 8.1, 10 mm nacl, 3 mm mgcl 2 , 0.5% np-40, 1 mm dtt and 100 u/ml of rnasin plus-promega) for 5 minutes on ice, then centrifugated at 1.200 g for 5 minutes at 4˚c. the supernatant (cytoplasmic fraction) was transferred to a new tube, while the pellet (nuclear fraction) was washed twice with 0.2 ml of cold rnla buffer to eliminate residual cytoplasm and discarded. the pellet was then resuspended with 0.2 ml of cold rnla buffer, sonicated on ice for 10 seconds. an aliquot of each fraction was stored for wb analysis, while the rest was used for rna extraction (trizol, thermo fisher scientific). results are presented on normalized cytoplasmic and nuclear fractions. ribosomal fractions were purified as described [61] . briefly, cells at less than 80% confluency were washed in cold pbs, gently pelleted and lysed for 10 minutes in hypothonic buffer (10 mm kcl, 0.5 mm mgcl 2 , 10 mm tris-hcl ph 7.4). nuclei were pelleted by centrifugation at 800 g for 10 minutes and the supernatant was then transferred centrifuged again to remove mitochondria (12.000 g for 10 minutes). supernatant was then harvested, adjusted to 0.5 m of kcl and placed onto a sucrose cushion (34.2% w/v) prior to ultracentrifugation at 75 000 rpm on a tl100 ultracentrifuge (for two hours at 4˚c). after ultracentrifugation, the supernatant constitutes the cytoplasmic fraction remaining after migration of the ribosomes at the bottom of the tube. the replication-competent vsv-gfp (indiana serotype, [18] ) contains an additional viral transcriptional unit coding gfp between m and g and was kindly provided by joanna brunel and denis gerlier (ciri, ens-lyon). infections of isg20 expressing cells were carried out generally mois comprised between 0.001 and 0.01, twenty-four hours after dox-induction of isg20. the percentage of gfp-positive cells was determined at different time points postinfection by fluorescence-activated cell sorting (facs). isg20-knockout mice were generated by clustered regularly interspaced short palindromic repeats (crispr)-associated systems (crispr/cas9). briefly, a single isg20 sgrna was designed to target the mouse isg20 orf (5'-gatcactaatacgactcactataggtggg cctcaaagggtgaggttttagagctagaaat-3'). in vitro transcription t7 kit (6140, takara) was used for transcription of both sgrna and cas9, the rna products were purified and injected into c57bl/6 mice zygotes [60] . screening of knockout mice was carried out by pcr on genomic dna from f0 and f1 mice followed by pcr products cloning and sequencing (ta cloning kit, takara: f: tttctgagggtcgccaa and r, tgtacttgtcataca ggact) for in vivo vsv replication studies, age-and sex-matched groups of littermate mice were intraperitoneally infected with 10 8 plaque-forming units (pfu) of wt-vsv (indiana serotype, but in this case lacking gfp, [60] ). mice were then either sacrificed twelve hours after infection for tissue analysis and in the case of lung sections for staining with hematoxylin and eosin that marks preferentially inflammatory cells or kept for prolonged periods of time for survival studies. pems were harvested by peritoneal washes from mice treated 3 days before via an intraperitoneal injection of 3 ml of 4% sterile thioglycollate (sigma t9032). the cell suspension was filtered through a 70-μm cell strainer (falcon 352350) to remove cell clumps and plated at 0.5 million per well in 24-well plate in complete 1640 medium to force cell adhesion to plastics and then cells were extensively washed to remove non-adherent cells. vsv infections were performed the next day at an moi of 0.5 prior to flow cytometry analysis twenty-four hours later. primary blood cells were obtained from the blood of healthy donors (efs-lyon) as discarded "leukopacks". these were obtained anonymously so that gender, race, age of donors, inclusion of women, minorities or children cannot be determined. this research is exempt from approval and was therefore not submitted to institutional review board approval, although written informed consent was obtained from blood donors to allow use of their cells for research purposes. all animal experiments described in this study were performed according to the regulations of the association for assessment and accreditation of laboratory animal care in shanghai and the animal investigation committee of east china normal university (document approval n˚m20130105). c57bl/6 wild-type mice were purchased from the shanghai laboratory animal company. mice were housed in a temperature and humidity-controlled room (21˚c +/-1˚c and 55% +/-10%, respectively) with a 12-hour light/12-hour dark cycle. throughout the protocol, animals were weighed and observed twice daily for clinical signs of infection. animals that reached the end of the experiment or lost more than 20% of initial body weight were sacrificed. supporting information s1 fig. expression levels and cytotoxicity of isg20. a) the levels of isg20 mrnas were quantified by rt-qpcr in the different cell types indicated. monocyte-derived macrophages and dendritic cells (mdm and dcs, respectively) were generated upon incubation of primary blood monocytes with m-csf or gm-csf and il4, respectively for 4 to 6 days as described in [60] . when indicated, cells were incubated for twenty-four hours with 1.000 u/ml of interferon α, prior to cell lysis. the expression levels of isg20 obtained in dox-inducible stable cell lines generated upon retroviral mediated gene transduction are also shown (dox concentrations of 0, 1 and 10 μg/ml). b) the cytotoxicity of isg20 was evaluated in stably transduced cell lines in which isg20 induction is dox-sensitive. cells were counted daily upon induction of isg20 with dox concentrations of 0,1 and 10 μg/ml. the graph presents data obtained from three to four independent experiments, while the wb panels present typical results obtained. fig 1a. � , p�0 .05 according to a student t test comparing control and isg20 conditions. (tif) s4 fig. exonuclease activities and specificities of isg20 in vitro and in cells. a and b) hek293t cells transiently transfected in 10 cm plates with isg20 coding dna were lysed and isg20 immunoprecipitated via anti-flag antibodies conjugated to agarose beads. after washing, beads bound material was incubated with the indicated nucleic acids (1 μg), prior to loading on agarose gels and densitometry quantification. nucleic acids were as follows: ssrna, in vitro transcribed single-stranded rna, length of approximatively 1700 nucleotides; ssdna, single-stranded dna, oligonucleotide of 76 nucleotides; dsdna, double stranded pcdna3 plasmid (invitrogen) linearized with psti; poly i:c and yeast trnas, self-explicatory. in b, the indicated rna and dna forms were generated as described in the methods section. c) hek293t cells transiently transfected as above were directly lysed and the amount of small nuclear rnas (u1, u2, u3 and u6) or of total rna was evaluated by rt-qpcr and agarose migration, respectively. d) as in c but cells were also transfected with 200 ng of in vitro transcribed mrna containing the 5' utr of the hepatitis c virus (hcv) and bearing the firefly luciferase. upon cell lysis the amount of transfected hcv-luc mrna was determined by rt-qpcr. a schematic representation of the rna target and the position of the pcr amplicon is provided. the graphs present means and sem of independent experiments (n = 2 to 3 depending on target for b; n = 3 for c; n = 4 for d). the panels present typical results obtained. � , p� 0.05, following a student t test. hek293t cells transiently transfected with isg20 and gfp coding plasmids (as in fig 2b) were analyzed by wb twenty-four hours afterwards. to better appreciate the linearity and magnitude of the defect in gfp accumulation by wb, the control sample was serially diluted. c) as above, but cells were analyzed by flow cytometry to appreciate the decrease in the percentage of gfp-positive cells (displayed in the panels), as well as their mfi. d) hek293t cells were transiently transfected with a fixed dose of gfp reporter and increasing concentrations of isg20 followed by cell analysis by wb (from 1:1 to 1:8). e) hek293t cells were transfected with dnas coding for indicated proteins using either calcium phosphate or lipofectaminebased dna transfection, prior to wb analysis. reporter. the amount of gfp reporter was then determined by densitometry after wb. c) the different isg20 mutants were immunoprecipitated from transfected hek293t cells and incubated with a single-stranded rna oligo (r20). upon migration on an agarose gel, the amount of intact rnas were measured by densitometry. d) correlative analysis between the rnase and antiviral properties of individual isg20 mutants. the graph present means and sem of four individual experiments (two for panel b). � , p� 0.05 following a student t test. e) spatial positioning of the indicated mutations on the crystal structure of isg20. (tif) s8 fig. isg20 does not affect the production of infectious virion particles of ebv. hone cells containing a latent ebv genome bearing gfp were transfected with isg20 and ebv reactivated from latency upon tpa/ba treatment as in the legend to fig 2d. to determine whether isg20 could affect the translation of the plethora of virion products required for the production of infectious virion particles, supernatants were syringe-filtered and virion infectivity was measured after challenge of target raji cells and flow cytometry analysis three days later. (tif) s9 fig. isg20 does not influence nucleocytoplasmic rna transport and is not detectably associated to ribosomes. a) hek293t cells were cotransfected with isg20 along with two constructs coding for a firefly luciferase reporter expressed from a globin promoter and containing or not an intron, prior to cell lysis and luciferase measurement. b) hek293t cells were transiently transfected with isg20 and gfp-coding plasmids and subsequently lysed to obtain nuclear and cytoplasmic fractions. upon normalization of the two fractions by volume, samples were analyzed by wb and rt-qpcr (gfp). the distribution of two small nuclear rnas known to be enriched in the nucleus (u1 and u3 by rt-qpcr) was also included as control for fractionation. c) hek293t cells transfected with isg20 were lysed, the ribosomal fraction purified, followed by wb analysis. the graphs present data obtained from two (luc) and five (pcrs) independent experiments and panels present typical results. � , p� 0.05, following a student t test. (tif) s10 fig. the catalytically inactive m1-isg20 mutant is devoid of translation inhibition activity. hek293t cells were transfected with the indicated reporters along with wt or m1-isg20 mutant prior to cell lysis and luciferase assay measurement. the graph presents data obtained from two and four independent experiments. glo, globin; pv, polyomavirus; myc, c-myc. the nomenclature 1 st and 2 nd refers to the position of the cistron in bicistronic vectors. (tif) s11 fig. isg20 and ifit1 inhibit vsv infection. hek293t cells transfected with plasmids coding the above-mentioned proteins were challenged twenty-four hours afterwards with vsv-gfp at an moi of 0.01 prior to flow cytometry analyses twenty four hours later. the graph presents results obtained with three independent experiments. � , p� 0.05, following a student t test between control cells and the indicated condition. (tif) s12 fig. isg20 displays a weak association to nuclear bodies and does not modify the extent of p bodies formation in the cell. a) hek293t cells were transiently transfected in duplicate with plasmids coding isg20 (routine transfection rates �80%). twenty-four hours afterwards, one aliquot was immediately fixed while the second was first permeabilized with detergent prior to fixation. both were then similarly processed and analyzed by confocal microscopy using antibodies specific for isg20 (flag), as well as for the nuclear speckles marker sc35. this procedure is commonly used to study nuclear bodies and associated proteins that resist detergent extraction prior to fixation. representative pictures and relative distributions are show here (>80 cells scored per condition). b) control cells were analyzed by confocal microscopy with the endogenous p bodies markers tnrc6a and ddx6 (isg20-expressing cells and a portion of the overlay of control cells depicted here are presented in fig 5a) . molecular cloning of a new interferon-induced pml nuclear body-associated protein the exonuclease isg20 mainly localizes in the nucleolus and the cajal (coiled) bodies and is associated with nuclear smn protein-containing complexes isg20, a new interferoninduced rnase specific for single-stranded rna, defines an alternative antiviral pathway against rna genomic viruses exoribonuclease superfamilies: structural analysis and phylogenetic distribution crystal structure of human isg20, an interferon-induced antiviral ribonuclease the human interferon-and estrogen-regulated isg20/ hem45 gene product degrades single-stranded rna and dna in vitro identification and characterization of interferon-induced proteins that inhibit alphavirus replication identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis c virus identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections antiviral activities of isg20 in positivestrand rna virus infections the interferon-induced exonuclease isg20 exerts antiviral activity through upregulation of type i interferon response proteins. msphere interferon-induced exonuclease isg20 exhibits an antiviral activity against human immunodeficiency virus type 1 interferon-stimulated gene of 20 kda protein (isg20) degrades rna of hepatitis b virus to impede the replication of hbv in vitro and in vivo influenza a virus-induced expression of isg20 inhibits viral replication by interacting with nucleoprotein interferon-inducible ribonuclease isg20 inhibits hepatitis b virus replication through directly binding to the epsilon stem-loop structure of viral rna a robust cell culture system supporting the complete life cycle of hepatitis b virus interferon-stimulated gene (isg)-expression screening reveals the specific antibunyaviral activity of isg20 cell-type-specific growth restriction of vesicular stomatitis virus polr mutants is linked to defective viral polymerase function the epstein-barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group a proteins altered histone h1 stoichiometry and an absence of nucleosome positioning on transfected dna p-bodies react to stress and nonsense movement of eukaryotic mrnas between polysomes and cytoplasmic processing bodies processing bodies require rna for assembly and contain nontranslating mrnas quantifying mrna targeting to p-bodies in living human cells reveals their dual role in mrna decay and storage p-bodies and stress granules: possible roles in the control of translation and mrna degradation p-bodies: composition, properties, and functions p-body purification reveals the condensation of repressed mrna regulons p-body formation is a consequence, not the cause, of rna-mediated gene silencing adenosine methylation as a molecular imprint defining the fate of rna chemical modifications in the life of an mrna transcript micrococcal nuclease digestion of nuclei reveals extended nucleosome ladders having anomalous dna lengths for chromatin assembled on non-replicating plasmids in transfected cells chromatin profiling of epstein-barr virus latency control region phosphorylation of initiation factor elf-2 and the control of reticulocyte protein synthesis regulation of protein synthesis: activation by double-stranded rna of a protein kinase that phosphorylates eukaryotic initiation factor 2 apobec3a is a specific inhibitor of the early phases of hiv-1 infection in myeloid cells inactivation of the human immunodeficiency virus type 1 inhibitory elements allows rev-independent expression of gag and gag/protease and particle formation molecular characterization of human argonaute-containing ribonucleoprotein complexes and their bound target mrnas mirna repression of translation in vitro takes place during 43s ribosomal scanning protein kinase ck2 phosphorylation of eb2 regulates its function in the production of epstein-barr virus infectious viral particles the enhancer factor r of epstein-barr virus (ebv) is a sequence-specific dna binding protein propagation and recovery of intact, infectious epstein-barr virus from prokaryotic to human cells inquiring into the differential action of interferons (ifns): an ifn-alpha2 mutant with enhanced affinity to ifnar1 is functionally similar to ifn-beta extracellular udp and p2y6 function as a danger signal to protect mice from vesicular stomatitis virus infection through an increase in ifn-beta production purification of ribosomes from human cell lines we thank serge bouaziz (university paris descartes, paris), rosa bernardi (division of experimental oncology, irccs san raffaele scientific institute) and emiliano ricci (ens-lyon) for counseling on structure analyses, murine cells and ribosome purification, respectively. we are indebted to denis gerlier, joanna brunel and to fabrice mure (ciri, france, lyon) for providing the vsv-gfp virus and intron/less globin luciferase reporters, respectively. we also acknowledge ju-tao guo from the baruch s. blumberg institute, doylestown, pennsylvania, usa for sharing material. key: cord-307067-cpc1yefj authors: van doremalen, neeltje; haddock, elaine; feldmann, friederike; meade-white, kimberly; bushmaker, trenton; fischer, robert j.; okumura, atsushi; hanley, patrick w.; saturday, greg; edwards, nick j.; clark, madeleine h. a.; lambe, teresa; gilbert, sarah c.; munster, vincent j. title: a single dose of chadox1 mers provides protective immunity in rhesus macaques date: 2020-06-10 journal: sci adv doi: 10.1126/sciadv.aba8399 sha: doc_id: 307067 cord_uid: cpc1yefj developing a vaccine to protect against the lethal effects of the many strains of coronavirus is critical given the current global pandemic. for middle east respiratory syndrome coronavirus (mers-cov), we show that rhesus macaques seroconverted rapidly after a single intramuscular vaccination with chadox1 mers. the vaccine protected against respiratory injury and pneumonia and reduced viral load in lung tissue by several orders of magnitude. mers-cov replication in type i and ii pneumocytes of chadox1 mers–vaccinated animals was absent. a prime-boost regimen of chadox1 mers boosted antibody titers, and viral replication was completely absent from the respiratory tract tissue of these rhesus macaques. we also found that antibodies elicited by chadox1 mers in rhesus macaques neutralized six different mers-cov strains. transgenic human dipeptidyl peptidase 4 mice vaccinated with chadox1 mers were completely protected against disease and lethality for all different mers-cov strains. the data support further clinical development of chadox1 mers. coronaviruses (cov) pose a continuous emerging virus threat, as demonstrated by the emergence of three previously unidentified corona viruses in the past 18 years. severe acute respiratory syndrome coronavirus (sars-cov) was first detected in 2003 and went on to infect >8000 people, resulting in 774 fatalities (1) . in 2012, middle east respiratory syndrome coronavirus (mers-cov) was first detected. mers-cov continues to infect humans from the dromedary camel host and has thus far infected >2500 people, resulting in 866 fatalities (2). an outbreak of pneumonia with an unknown cause in wuhan, china was first reported in december 2019. although information is still limited, we now know that this outbreak is caused by a third emerging cov. currently, >200,000 cases are associated with this outbreak (3) . the clinical spectrum of mers-cov infection in humans varies from asymptomatic to severe respiratory disease and death. patients present with influenza-like symptoms such as a fever and shortness of breath. thereafter, they may develop pneumonia, which can require mechanical ventilation and support in an intensive care unit (2). human-to-human transmission of mers-cov is relatively limited and occurs mainly in nosocomial settings but has been reported in local communities as well (1) . in 2015, a traveler from the middle east to south korea caused an outbreak involving 186 people and 38 fatalities (4). the nosocomial outbreak lasted from may to july, and 16,752 people were isolated with mers-cov-like symptoms. at least three superspreaders were identified in the outbreak, who infected 28, 85, and 23 patients, respectively (5) . the introduction and spread of mers-cov in south korea underscore the potential of this virus to cause epidemics outside of the arabian peninsula. mers-cov has continued to cause disease in humans, and in 2019, 152 cases have been reported in the kingdom of saudi arabia (ksa), of which 33% were fatal (2). the ongoing circulation of mers-cov and subsequent outbreaks in the human population highlight the need for an efficient mers-cov vaccine. mers-cov is mainly prevalent in the arabian peninsula, with the majority of cases occurring in ksa (84%) (6) . however, phylogenetically diverse mers-cov strains have been isolated from africa and the middle east (7) (8) (9) , and antigenic differences have been reported between spike (s) proteins, the main antigen used in mers-cov vaccines, from the middle east and africa (8) . thus, it is important that a mers-cov vaccine is protective against a variety of diverse mers-cov strains. thus far, only four vaccines have been tested in rhesus macaques. these are a dna vaccine (10); a vaccine based on the receptor-binding domain of the mers-cov s adjuvanted with alum (11); a combination of s plasmid dna and s1 protein (12) ; and virus-like particles based on mers-cov s, matrix, and envelope proteins (13) . of the four vaccine studies, only three studies included challenge studies, all of which used mers-cov isolates from 2012. to date, there have been no reports of efficacy of a single-dose mers-cov vaccine in nonhuman primates (nhps) . we recently demonstrated that vaccination of mice with a replication-deficient simian adenovirus vaccine vector (chadox1) encoding full-length mers-cov s protein (chadox1 mers) elicited high-titer mers-cov-neutralizing antibodies and a robust cd8 + t cell response against the s protein (14) . in addition, chadox1 mers vaccination resulted in full protection of human dipeptidyl peptidase 4 (hdpp4) transgenic mice against a lethal challenge with mers-cov (15) . chadox1 mers vaccination of dromedary camels was immunogenic and reduced mers-cov shedding after challenge in a highly stringent natural transmission model with multiday exposure to infectious mers-cov (16) . chad-vectored vaccines against malaria (17) , hiv (18) , influenza (19) , hepatitis c (20), tuberculosis (21) , ebola (22) , and others show an excellent immunogenicity and safety profile in humans. in the current manuscript, we show that a single dose of chadox1 mers vaccine protects rhesus macaque model against a mucosal challenge with hcov-emc/2012. serum obtained from vaccinated rhesus macaques was able to neutralize six diverse mers-cov strains. furthermore, a single dose of chadox1 mers vaccine protects hdpp4 transgenic mice against all evaluated mers-cov strains. three groups of six animals each were vaccinated with chadox1 mers via a prime-boost regimen [−56 and −28 dpi (days post infection)] or prime-only regimen (−28 dpi) or with chadox1 green fluorescent protein (gfp) (−56 and −28 dpi). animals were then challenged with 7 × 10 6 tcid 50 (median tissue culture infectious dose) of hcov-emc/2012 on 0 dpi via combined intratracheal, intranasal, oral, and ocular route (23) . blood samples were taken at −56, −42, −28, −14, and 0 dpi (fig. 1a) . the humoral immune response to vaccination was examined by enzyme-linked immunosorbent assay (elisa) using s protein and by virus neutralization assay. s proteinspecific immunoglobulin g (igg) antibodies were detected as early as 14 days after vaccination in both chadox1 mers-vaccinated groups. all animals in these groups had s protein-specific antibodies at 0 dpi, whereas no antibodies against s protein were found before vaccination or at any time in the chadox1 gfp-vaccinated group. a significant difference in elisa titers was observed between primeboost and prime-only animals at 0 dpi (fig. 1b) . neutralizing antibodies were detected in 11 of 12 chadox1 mers-vaccinated animals at the time of challenge. one animal in the prime-only vaccination group did not have detectable neutralizing antibodies at this time and had the lowest antibody titer as measured by elisa (1600). a significant difference in vn (virus-neutralizing) titers was observed between prime-boost and prime-only animals at 0 dpi (fig. 1c) . a second chadox1 mers vaccination at −28 dpi resulted in a statistically significant increase in s protein-specific elisa titer (geometric mean titer −28 dpi = 1600; 0 dpi = 10,159; p < 0.0001) and neutralizing antibody titer (geometric mean titer −28 dpi = 24; 0 dpi = 148; p < 0.0001) as determined via two-tailed t test, although neutralizing antibodies against the chadox1 vector could be detected at the time of the second vaccination ( fig. s1a ). this suggests that the presence of neutralizing antibodies against chadox1 does not prevent the vaccine vector from boosting the immune response. vaccination with chadox1 mers reduces disease severity animals vaccinated with chadox1 gfp and challenged with mers-cov showed similar clinical signs as previously reported (23), such as a decreased appetite and increased respiratory rate. clinical signs were scored using a standard nhp scoring sheet, focusing on areas such as general appearance, nasal discharge, and food intake. on average, chadox1 mers-vaccinated animals had a lower clinical score than chadox1 gfp-vaccinated animals ( fig. 2a) . all animals underwent exams on 0, 1, 3, 5, and 6 dpi. no significant changes in weight or body temperature were observed for the duration of the study. peripheral capillary oxygen saturation (spo 2 ) measurements supported a decrease in oxygen saturation from the baseline in chadox1 gfp-vaccinated animals, but not in chadox1 mersvaccinated animals (fig. 2b ). ventrodorsal and lateral thoracic radiographs were collected on all exam days. all animals vaccinated with chadox1 gfp showed moderate to severe pulmonary interstitial infiltrations, whereas animals vaccinated with chadox1 mers showed only mild signs of respiratory disease. a severe collapsed lung was observed on 3 dpi in two animals in the prime-boost group, likely caused by the bronchoalveolar lavage (bal) performed on 1 dpi. full details of all observed signs can be found in table s2 ( fig. 3a and table s2). all lung lobes (right cranial, right middle, right caudal, left cranial, left middle, and left caudal) of each individual animal were scored for severity of disease signs for each day radiographs were taken, and average scores were compared. scores obtained from animals vaccinated with chadox1 gfp were significantly higher from animals vaccinated with a prime-boost regimen of chadox1 mers on 3, 5, and 6 dpi (fig. 3b) . upon necropsy on 6 dpi, gross lung lesions were more prevalent in animals vaccinated with chadox1 gfp than in animals vaccinated with chadox1 mers (fig. 3 , c and d). animals vaccinated with chadox1 gfp showed focally extensive areas of consolidation in all lung lobes and lungs generally failed to collapse. mediastinal lymph nodes were often edematous and enlarged. animals that received a vaccination with chadox1 mers, either via a prime-boost or primeonly regimen, had either no lesions or limited small multifocal areas of consolidation and congestion. an increased lung:body weight ratio is an indicator of pulmonary edema. animals in the control group had significantly higher lung:body weight ratios compared to chadox1 mers-vaccinated animals, and there was minimal difference between prime-boost and prime-only chadox1 mers-vaccinated animals (fig. 3f) . . statistical significance between −28 and −14 dpi in the prime-boost group was determined via one-tailed paired student's t test. statistical significance between prime-boost and prime-only groups on 0 dpi was determined via two-tailed unpaired student's t test. **p < 0.01; ***p < 0.001. lung tissue sections were stained with hematoxylin and eosin or with mers-cov-specific antibodies. all slides were evaluated by a board-certified veterinary pathologist blinded to study group allocations. in animals that received a vaccination with chadox1 mers, either prime-boost or prime-only, a minimal to mild bronchointerstitial pneumonia was present, characterized by mild thickening of the alveolar septae by lymphocytes and macrophages. pulmonary vessels were bound by moderate numbers of lymphocytes. in stark contrast, in lung tissue obtained from animals vaccinated with chadox1 gfp, moderate to marked bronchointerstitial pneumonia was present throughout the lung lobes characterized by thickening of the alveolar septae by lymphocytes and macrophages and edema and fibrin. alveoli contained abundant edema and fibrin and moderate to abundant numbers of alveolar macrophages, neutrophils, and necrotic debris. inflammation often surrounded bronchioles and pulmonary vasculature, and type ii pneumocyte hyperplasia was prominent. in addition, the presence of mers-cov antigen by immunohistochemistry was found only in lungs of animals vaccinated with chadox1 gfp within type i and ii pneumocytes and was not found in lung tissue of chadox1 mers-vaccinated animals. severity of bronchointerstitial pneumonia, type ii pneumocyte hyperplasia and hemorrhages, edema, and fibrin deposits was scored. statistically significant differences between animals vaccinated with chadox1 mers or chadox1 gfp were found for all three categories ( fig. 3 , e, f, and h). thus, vaccination with chadox1 mers, either via a prime-boost or prime-only regimen, significantly decreased the severity of pulmonary pathology and protected rhesus macaques against bronchointerstitial pneumonia. bal was performed on all animals on 1, 3, 5, and 6 dpi, and the amount of viral rna, mrna, and infectious virus was determined. in the prime-boost group, infectious virus was only detected at 1 dpi (n = 3) after inoculation, and in the prime-only group at 1 dpi (n = 6) and 3 dpi (n = 3) but not thereafter. in contrast, infectious virus was detected on all days in bal fluid from the control group (fig. 4a ). viral mrna in bal fluid of animals in the prime-boost group was only detected at 1 dpi (n = 5) and 3 dpi (n = 1). in contrast, viral mrna in bal fluid from animals that received a single vaccination with chadox1 mers mrna was detected on all days, but the amount was reduced compared to animals vaccinated with chadox1 gfp (fig. s1b ). viral rna as measured by upe quantitative reverse transcription polymerase chain reaction (qrt-pcr) assay could be detected in all groups up to 6 dpi. however, the number of genome copies per milliliter detected was lower in animals vaccinated with chadox1 mers compared to animals vaccinated with chadox1 gfp (fig. 4a) . a significant association was found between higher elisa titer or vn titer and lower levels of viral rna, mrna, or infectious virus in bal fluid for all days, except 6 dpi for infectious virus (spearman's rank correlation coefficient; table s2). all animals were euthanized on 6 dpi, and tissues were analyzed for the presence of viral rna, mrna, or infectious virus. infectious virus titers were only found in nasal turbinate tissue (n = 1) and lung lobe tissue (n = 4) from control animals. no viral mrna was found in all tissues obtained from animals that received a prime-boost regimen. in tissue from animals that received a prime-only regimen, limited viral mrna could be found in upper and lower lung lobe tissues (n = 4). in contrast, mrna could be found in respiratory tract tissues of all control animals and in conjunctiva (fig. 4b ). viral rna was detected in tissues from all groups but was mainly found in lung lobes and bronchi. viral load was higher for lower respiratory tract tissue obtained from animals vaccinated with chadox1 gfp (n = 6) than from animals receiving a prime-only (n = 6) or a prime-boost regimen of chadox1 mers (n = 2) (fig. 4b ). the presence of 23 cytokines was evaluated in lung tissue. several cytokines were up-regulated in animals vaccinated with chadox1 gfp compared to animals vaccinated with chadox1 mers, including interleukin-2 (il-2), il-6, il-8, il-18, monocyte chemotactic protein-1 (mcp-1), macrophage inflammatory protein-1a (mip-1), and transforming growth factor- (tgf-), although only il-2 and mcp-1 were significantly different in tissue obtained from control animals compared to vaccinated animals. a significant difference was observed in il-1ra levels in lung tissue obtained from the prime-only and prime-boost groups. it is not clear what the clinical significance of this difference is ( fig. s2 ). overall, these results show local increased immune activity in animals vaccinated with chadox1 gfp, but not in animals vaccinated with chadox1 mers, 6 days after challenge with mers-cov. the likely explanation for this is that vaccinated animals have controlled the infection rapidly, whereas in the control animals, more viral replication has taken place followed animal vaccinated with chadox1 gfp shows focally extensive area of increased pulmonary opacity and deviation of the cardiac silhouette, highlighted in the circle located in the middle and caudal lung lobes. (b) thoracic radiographs of each animal were scored per lung lobe, resulting in a maximum score of 18. values were averaged per group per day (d), and mean with sd is shown (see table s2 for more details). (c) gross pathology of lungs shows no pathologic changes in chadox1 mers-vaccinated animals and focally extensive areas of consolidation in left cranial, middle, and caudal lung lobes in control animals (asterisks). (d) gross lung lesions were scored for each lung lobe, ventral and dorsal. values were averaged per group, and mean with sd is shown. (e) lung tissue sections were stained with hematoxylin and eosin. moderate numbers of lymphocyte accumulation around pulmonary arterioles (asterisks) and mild thickening of alveolar septae by lymphocytes and macrophages (arrows) in lung tissue of animals vaccinated with chadox1 mers. marked bronchointerstitial pneumonia with abundant pulmonary edema and fibrin (asterisks), type ii pneumocyte hyperplasia (arrows), and increased numbers of alveolar macrophages (arrowheads) in lung tissue of control animals. magnification, ×200. (f) lung-to-body weight (bw) ratio was determined for all animals at necropsy. mean with sd is shown. (g) lung tissue sections were stained with antibody against mers-cov antigen, which is visible as a red-brown staining. no immunoreactivity was found in chadox1 mers-vaccinated animals, whereas multifocal immunoreactivity of type i and ii pneumocytes could be found in lung tissue of chadox1 gfp-vaccinated animals. (h) lung tissue sections were scored on severity of lesions (0, no lesions; 1, 1 to 10%; 2, 11 to 25%; 3, 26 to 50%; 4, 51 to 75%; and 5, 76 to 100%) and averaged per group. mean with sd is shown. a, bronchointerstitial pneumonia; b, type ii pneumocyte hyperplasia; c, hemorrhages, edema, and fibrin deposits. statistical significance between groups was determined via two-tailed unpaired student's t test. *p < 0.025; **p < 0.01; ***p < 0.001; ****p < 0.0001. photo credit: neeltje van doremalen, niaid/nih. chadox1 mers is based on the s protein from camel/qatar/2/2014, and we currently do not have access to an isolate. we thus already show cross-protection of chadox1 mers against hcov-emc/2012 (99.78% s protein amino acid identity). here, we extend that analysis to five other strains. we selected six different strains of mers-cov ( fig. s2 ). s protein identity for all strains to the vaccine s protein was >99.3%. amino acid identity was lowest for camel/burkina faso/ cirad-hku785/2015 (99.33%) and highest for hu/korea/seoul/ snu1-035/2015 (99.85%). we tested neutralizing capability of serum obtained at 0 dpi. strains were selected on the basis of geographical location (ksa, south korea, and burkina faso), host (dromedary camel or human), and time of isolation (2012 to 2018). all six strains were neutralized by antibodies elicited by chadox1 mers vaccination. although we were not able to detect neutralizing ability of serum obtained from animal 12 against hcov-emc/2012, antibodies in the serum were able to neutralize four of six tested mers-cov strains (table 1) . to investigate whether vaccination with chadox1 mers vaccination provides protection against a variety of different mers-cov strains, we vaccinated balb/c mice transgenic for hdpp4 with chadox1 mers or chadox1 gfp 28 days before challenge with 10 4 tcid 50 of one of six diverse mers-cov strains ( fig. s3 ) via the intranasal route. all mice vaccinated with chadox1 mers survived challenge with mers-cov, independent of the challenge virus used, whereas most control mice were euthanized because of >20% weight loss or poor body condition (fig. 5a) . four animals per group were euthanized at 3 dpi, and infectious mers-cov titers in lung tissue were evaluated. whereas infectious virus could be found in lung tissue of control animals, we were unable to find infectious virus in lung tissue of chadox1 mers-vaccinated animals (fig. 5b) . thus, chadox1 mers protects against a variety of different mers-cov strains in hdpp4 transgenic mice. (24) . mers is associated with a high case-fatality rate (34.5%), and human-tohuman transmission is a major contributor to patient infections (1). currently, no mers-cov vaccine is available. ideally, such a vaccine would only require a single administration and would protect against a wide variety of different mers-cov strains. several studies have evaluated different types of mers-cov vaccines in animal models, but few have taken these vaccines into nhps. in the current study, we show the efficacy of chadox1 mers in rhesus macaques. unlike other nhp vaccine studies (10-13), we investigate vaccine efficacy after a single dose. animals that received a single dose of chadox1 mers showed an induction of a neutralizing antibody response associated with mostly normal clinical parameters, showing no breathing irregularities or reduced lung function by spo 2 values, limited evidence of infiltration by radiograph analysis after challenge, and no signs of gross pathological lesions. vaccination reduced viral rna load in tissues collected at 6 dpi compared to chadox1 gfp-vaccinated animals by several logs. in contrast to the abundant presence of viral mrna and infectious virus in control animals, we found no evidence of infectious virus, and only a limited presence of viral mrna, in the lungs of prime-only vaccinated macaques. viral mrna and infectious virus was completely absent from animals vaccinated with a prime-boost regimen of chadox1 mers. although both vaccine regimens protected rhesus macaques from the clinical symptoms of mers-cov inoculation, we detected lower virus replication in animals that received a prime-boost regimen compared to animals receiving a prime-only regimen, suggesting that the prime-boost regimen is a superior therapeutic approach. it should be noted that in the current model, animals are inoculated with a high dose of virus (7 × 10 6 tcid 50 per animal). this is likely a higher inoculum than most humans the study was not designed to determine correlates of protection, which must be determined separately for each vaccine candidate, but it is of interest that here, one animal was protected despite not having detectable neutralizing antibodies against hcov-emc/2012, and in general, the neutralizing antibody titer was not high. in clinical trials, chadox1-vectored vaccines prime strong t cell responses against the vaccine antigen (17) (18) (19) (20) (21) (22) . in a clinical study of patients that recovered from mers infection, some had strong cd8 + t cell responses without detectable antibodies (25) . further studies addressing correlates of protection for chadox1 mers should assess cd8 + t cell responses. following the successful induction of protective immunity after vaccination as demonstrated here, the duration of immunity and ability to induce the development of memory b and t cells should be assessed. the phenotype of memory cells induced by vaccination may not necessarily mimic that induced by infection with the pathogen. in the first clinical trial of chadox1 mers (clinicaltrials.gov identifier: nct03399578) both humoral-and t cell-mediated responses were assessed at multiple times and persisted up to 1 year after vaccination. further work will be required to assess memory b and t cell phenotypes. we are currently planning studies to look at long-term protection by chadox1 mers vaccination in our hdpp4 mouse model. a variety of different mers-cov strains have been isolated from dromedary camels and humans over the past 8 years of mers-cov emergence (24) . dromedary camels are distributed throughout africa, the middle east, asia, and australia (26) . although mers-cov has not been detected in dromedary camels in australia (27) , strains have been isolated from africa (9) and the middle east (7), and seropositive dromedary camels have been found in asia (28, 29) . phylogenetic analyses show a clustering of mers-cov by geographical location (8, 9) , and analysis of 219 complete mers-cov genomes, which only included one african strain, showed the presence of two clades, with human isolates in both clades (30) . notably, antigenic differences have been reported between s proteins from the middle east and africa (8), potentially affecting the efficacy of a vaccine based in conclusion, we show that a single vaccination with chadox1 mers results in protection against disease progression and virus replication associated with mers-cov challenge in the rhesus macaque, and a prime-boost regimen reduced viral replication further. furthermore, chadox1 mers vaccination protected against a diverse panel of contemporary mers-cov strains in hdpp4 mice. this is the first time that broad protection after a single vaccination has been shown for any mers-cov vaccine. last, chadox1 vaccines can be produced rapidly, have been shown to be safe in human patients, and are protective against mers-cov in rhesus macaques and hdpp4 mice. we conclude that the chadox1 platform is ideal for the development of vaccines against novel emerging coronaviruses, such as hcov-19/sars-cov-2. animal experiment approval was provided by the institutional animal care and use committee at rocky mountain laboratories. all animal experiments were executed in an association for assessment and accreditation of laboratory animal care-approved facility by certified staff, following the guidelines and basic principles in the national institutes of health (nih) guide for the care and use of laboratory animals, the animal welfare act, the u.s. department of agriculture, and the u.s. public health service policy on humane care and use of laboratory animals. rhesus macaques were housed in individual primate cages allowing social interactions, in a climatecontrolled room with a fixed light-dark cycle (12/12 hours). rhesus macaques were monitored a minimum of twice daily throughout the experiment. commercial monkey chow, treats, and fruit were provided twice daily by a trained personnel. water was available ad libitum. environmental enrichment consisted of a variety of human interaction, commercial toys, videos, and music. the institutional biosafety committee (ibc) approved work with infectious mers-cov virus strains under bsl3 conditions. all sample inactivation was performed according to ibc-approved standard operating procedures for the removal of specimens from high containment. the s protein gene from mers-cov strain camel/qatar/2/2014 (genbank accession no. kj650098.1) was codon optimized for humans and synthesized by geneart (thermo fisher scientific). the synthesized s gene was cloned into a transgene expression plasmid comprising a modified human cytomegalovirus immediate early promoter (cmv promoter) with tetracycline operator sites and the polyadenylation signal from bovine growth hormone. the resulting expression cassette was inserted into the e1 locus of a genomic clone of chadox1 using site-specific recombination (31) . the virus was rescued and propagated in t-rex-293 cells (invitrogen). purification was by cscl gradient ultracentrifugation, and the virus was titered, as previously described (32) . doses for vaccination were based on infectious units (ius) (33) . eighteen adult rhesus macaques (17 males and 1 female) were purchased from morgan island and randomly divided into three groups of six animals each. the group 1 was vaccinated with chadox1 mers at −56 and −28 dpi, the group 2 was vaccinated with chadox1 mers at −28 dpi, and the group 3 was vaccinated with chadox1 gfp at −56 and −28 dpi. all vaccinations were done with 3.9 × 10 8 iu per animal per vaccination. blood samples were obtained before vaccination and 14 days thereafter. animals were challenged with mers-cov strain hcov-emc/2012 on 0 dpi with administrations of 4 ml intratracheally, 1 ml intranasally, 1 ml orally, and 1 ml ocularly of 10 7 tcid 50 /ml virus solution. clinical exams were performed on −56, −42, −28, −14, 0, 1, 3, and 5 and 6 dpi; animals were euthanized at 6 dpi. all exams existed of the following: weight and temperature measurements, radiographs, spo 2 measurements using pulse oximetry, and blood sampling. bal was performed on 1, 3, 5, and 6 dpi by insertion of an endotracheal tube and bronchoscope into the trachea, then past the third bifurcation, and subsequent installation of 10 ml of sterile saline. manual suction was applied to retrieve the bal sample. necropsy was performed on 6 dpi. radiographs were evaluated and scored by a board-certified veterinarian who was blinded to the group assignment of the animals according to the following criteria: 0, normal examination; 1, mild interstitial pulmonary infiltrates; 2, moderate interstitial infiltrates, perhaps with partial cardiac border effacement and small areas of pulmonary consolidation (alveolar patterns and air bronchograms); and 3, pulmonary consolidation as the primary lung pathology, seen as a progression from grade 2 lung pathology (34) . veroe6 cells were maintained in dmem supplemented with 10% fetal calf serum, 1 mm l-glutamine, penicillin (50 u/ml), and streptomycin (50 g/ml). virus titrations were performed by endpoint titration in veroe6 cells, which were inoculated with 10-fold serial dilutions of virus. after 1-hour incubation at 37°c and 5% co 2 , tissue homogenate dilutions were removed, and cells were washed twice with phosphate-buffered saline (pbs) and incubated in 100 l of 2% dmem. cytopathic effect was scored at 5 dpi, and the tcid 50 was calculated from four replicates by the spearman-karber method (33, 35) . sera were heat inactivated (30 min, 56°c), and twofold serial dilutions were prepared in 2% dmem. hereafter, 100 tcid 50 of mers-cov was added. after 60-min incubation at 37°c, virus:serum mixture was added to veroe6 cells and incubated at 37°c and 5% co 2 . at 5 dpi, the cytopathic effect was scored. the virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum, which still inhibited virus replication (33) . chimpanzee adenovirus chadox1-specific neutralizing antibody titers were assessed using a secreted placental alkaline phosphatase (seap) quantitation assay. briefly, griptite msr 293 cells (invitrogen, catalog no. r795-07) were cultured as per manufacturer's instructions and were seeded at 3 × 10 4 cells per well in a 96-well plate the day before starting the assay (24 ± 2 hours). cells were infected with the test sera dilutions (fourfold dilution series) at 1:18, 1:72, 1:288, 1:1152, and 1:4608 in phenol red-free 0% fbs dmem (life technologies, catalog no. 31053028) and the chadox1-seap reporter virus in a 1:1 mixture (pre-incubated for 1 hour to allow any neutralization to occur) for 1 hour before replacing with phenol redfree 10% fbs dmem for a further 24 hours (±2 hours). sample dilutions were tested in duplicate lanes. for each sample, seap concentration was assessed in 50 l aliquots of culture supernatant, with cpsd as an indicator substrate (tropix phospha-light chemiluminescent assay kit, life technologies, catalog no. t1017), using a minor variant of the manufacturer's instructions; luminescence intensity was measured using a varioskan flash luminometer (thermo fisher scientific). serum dilution neutralization titers were measured by linear interpolation of adjacent values (to 50% inhibition) to determine the serum dilution required to reduce seap concentration by 50% compared to wells with virus alone. tissues (30 mg) were homogenized in rlt buffer, and rna was extracted using the rneasy kit (qiagen) according to the manufacturer's instructions. rna was extracted from bal fluid using the qiaamp viral rna kit (qiagen) on the qiaxtractor. the upe mers-cov (36) or mrna (37) detection assay was used for the detection of mers-cov viral rna. five microliters of rna was tested with the rotor-gene probe kit (qiagen) according to the instructions of the manufacturer. dilutions of mers-cov virus stock with known genome copies were run in parallel. genome copies were determined using droplet digital pcr (bio-rad) and the corresponding qrt-pcr. a soluble, trimeric recombinant s protein of mers-cov (isolate ca/jeddah/d42/2014) incorporating amino acids 1 to 1273 and a c-terminal trimerization domain was produced in chinese hamster ovary cells (expicho; thermo fisher scientific) and purified by immunoaffinity chromatography. maxisorp plates (nunc) were coated overnight at room temperature with 5 g of s protein per plate in pbs. plates were blocked with 100 l of casein (thermo fisher scientific) for 90 min at room temperature. serum (2× serial dilution in casein starting at 100× dilution) was incubated at room temperature for 2 hours. antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat anti-monkey igg (seracare, 074-11-021) in casein and 3,3′,5,5′-tetramethylbenzidine (tmb) two-component peroxidase substrate (seracare) and read at 450 nm. all wells were washed three times with pbst (pbs-tween 0.05%) in between steps. threshold for positivity was set at 3× optical density (od) value of negative control (serum obtained from nhps before the start of the experiment). samples for analysis of cytokine/chemokine levels were inactivated with -radiation (2 mrad) according to the standard operating procedures. concentrations of granulocyte colony-stimulating factor (g-csf), granulocyte-macrophage csf, interferon-, il-1, il-1 receptor antagonist, il-2, il-4, il-5, il-6, il-8, il-10, il-12/23 (p40), il-13, il-15, il-17, il-18 mcp-1, and mip-1, mip-1, soluble cd40 ligand (scd40l), tgf-, tumor necrosis factor-, and vascular endothelial growth factor were measured on a bio-plex 200 instrument (bio-rad) using the non-human primate cytokine milliplex map 23-plex kit (millipore) according to the manufacturer's instructions. necropsies and tissue sampling were performed according to ibcapproved protocols. lungs were perfused with 10% formalin and processed for histologic review. harvested tissues were fixed for a minimum of 7 days in 10% neutral-buffered formalin and then embedded in paraffin. tissues were processed using a vip-6 tissue-tek (sakura finetek, usa) tissue processor and embedded in ultraffin paraffin polymer (cancer diagnostics, durham, nc). samples were sectioned at 5 m, and the resulting slides were stained with hematoxylin and eosin. specific anti-cov immunoreactivity was detected using mers-cov nucleocapsid protein rabbit antibody (sino biological inc.) at a 1:4000. the tissues were processed for immunohistochemistry using the discovery ultra automated ihc/ish staining instrument (ventana medical systems) with a discovery red (ventana medical systems) kit. all tissue slides were evaluated by a board-certified veterinary anatomic pathologist blinded to study group allocations. tukey's multiple comparison test or a two-tailed unpaired student's t test was conducted to compare differences between vaccine groups and the control 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fascicularis) beitrag zur kollektiven behandlung pharmakologischer reihenversuche detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction growth and quantification of mers-cov infection diversity of middle east respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing competing interests: s.c.g. is a board member of vaccitech and named as an inventor on a patent covering use of chadox1-vectored vaccines. the other authors declare that they have no competing interests. data and materials availability: all data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary materials. chadox1 mers can be provided by the jenner institute, university of oxford pending scientific review and a completed material transfer agreement a single dose of chadox1 mers provides protective immunity in rhesus macaques key: cord-102184-8u73adnk authors: li, jinzhi; mahoney, brennan dale; jacob, miles solomon; caron, sophie jeanne cécile title: visual input into the drosophila melanogaster mushroom body date: 2020-05-02 journal: biorxiv doi: 10.1101/2020.02.07.935924 sha: doc_id: 102184 cord_uid: 8u73adnk the ability to integrate input from different sensory systems is a fundamental property of many brains. yet, the patterns of neuronal connectivity that underlie such multisensory integration remain poorly characterized. the drosophila melanogaster mushroom body — an associative center required for the formation of olfactory and visual memories — is an ideal system to investigate how different sensory channels converge in higher-order brain centers. the neurons connecting the mushroom body to the olfactory system have been described in great detail, but input from other sensory systems remains poorly defined. here, we use a range of anatomical and genetic techniques to identify two novel types of mushroom body input neuron that connect visual processing centers — namely the lobula and the posterior lateral protocerebrum — to the dorsal accessory calyx of the mushroom body. together with previous work that described a pathway conveying visual information from the medulla to the ventral accessory calyx of the mushroom body (vogt et al., 2016), our study defines a second, parallel pathway that is anatomically poised to convey information from the visual system to the dorsal accessory calyx. this connectivity pattern — the segregation of the visual information into two separate pathways — could be a fundamental feature of the neuronal architecture underlying multisensory integration in associative brain centers. sensory systems use different strategies to detect specific physical features of the outside world. for instance, the olfactory system contains many different types of sensory neuron that are each specialized in detecting a specific class of volatile chemicals. through only two neuronal layers, 35 olfactory information -the identity of an odor and its concentration -is relayed to higher brain centers (leinwand and chalasani, 2011) . in contrast, the visual system contains far fewer types of sensory neuron but, through numerous neuronal layers, it relays a range of highly processed information -for instance, color, brightness, motion and shape -to higher brain centers (baden et al., 2020) . thus, higher brain centers have to integrate different types of processed information, 40 bind that information into a coherent representation of the outside world, and use such representations to guide behavior (yau et al., 2015) . how higher brain centers achieve this feat remains largely unknown. this gap in our knowledge mainly stems from the fact that higher brain centers are formed by a large number of neurons, and that the projection neurons conveying information from different sensory systems to these centers often remain poorly characterized. 45 this makes it difficult to understand whether there are specific patterns of neuronal connectivity that enable multisensory integration and what the nature of these patterns are. deciphering the fundamental neuronal mechanisms that underlie multisensory integration requires a model system such as the drosophila melanogaster mushroom body, which consists of a relatively small number of neurons whose connections can be charted reliably. 50 the drosophila mushroom body is formed by ~2,000 neurons -called the kenyon cells -and has long been studied for its essential role in the formation of olfactory associative memories (aso et al., 2014; hige, 2018) . the identity of the projection neurons that connect the olfactory system to the mushroom body -and the way kenyon cells integrate input from these 55 neurons -has been characterized in great detail, highlighting fundamental connectivity patterns that enable this higher brain center to represent olfactory information efficiently (bates et al., 2020; caron et al., 2013; tanaka et al., 2012a tanaka et al., , 2012b zheng et al., 2018) . evidence in drosophila melanogaster shows that the mushroom body is more than an olfactory center, as it is also required for the formation of visual and gustatory associative memories (liu et al., 1999; masek 60 and scott, 2010; vogt et al., 2014) . however, the identity of the neurons that connect the mushroom body to other sensory systems remains poorly characterized. thus, a first step towards understanding how the mushroom body integrates multisensory information is to identify such non-olfactory mushroom body input neurons and the genetic tools necessary to manipulate these neurons. 65 the mushroom body receives its input through its calyx and sends its output through its lobes. the calyx -a morphologically distinct neuropil containing the synapses formed between projection neurons and kenyon cells -can be divided into four, non-overlapping regions: one main calyx as well as three accessory calyces named the dorsal, lateral and ventral accessory 70 calyces (aso et al., 2014; yagi et al., 2016) . the five output lobes -the a, a', b, b' and g lobes -contain the synapses formed between kenyon cells, mushroom body output neurons and dopaminergic neurons (aso et al., 2014) . with respect to these input and output regions, kenyon cells can be divided into seven distinct types (aso et al., 2014) . of these seven types, five types -the a/bc, a/bs, a'/b'ap, a'/b'm and gmain kenyon cells -extend their dendrites only into the main 75 calyx and their axons along one or two lobes. most of the neurons that project to the main calyx emerge from the antennal lobe, the primary olfactory center in the drosophila brain. thus a/bc, a/bs, a'/b'ap, a'/b'm and gmain kenyon cells receive input primarily from the olfactory system (caron et al., 2013; aso et al., 2014; zheng et al., 2018) . in contrast, the two other classes of kenyon cells do not extend their dendrites into the main calyx. instead, the a/bp kenyon cells extend their dendrites into the dorsal accessory calyx -avoiding completely the main, lateral and ventral accessory calyces -and their axons along the a and b lobes. likewise, the gd kenyon cells extend their dendrites exclusively into the ventral accessory calyx and their axons along the g lobe (aso et al., 2014; vogt et al., 2016) . thus, both 85 the a/bp and gd kenyon cells are anatomically poised to receive non-olfactory input. there is evidence suggesting that the ventral accessory calyx receives input from the medulla, a region of the optic lobe that specializes in processing brightness and color (morante and desplan, 2008; vogt et al., 2016) . furthermore, a recent study suggests that the dorsal accessory calyx is a multisensory center that integrates input from multiple sensory pathways including the olfactory, 90 gustatory and visual systems (yagi et al., 2016) . here, we report a strategy that uses a combination of genetic tools -including transgenic lines that drive expression in few neurons and a photo-labelling technique used to identify individual neurons and their presynaptic partners -to characterize the input neurons of the a/bp 95 kenyon cells. we identify two novel types of mushroom body input neuron that, together, form about half of the total input the a/bp kenyon cells receive in the dorsal accessory calyx. the first neuronal type -henceforth referred to as lopns -consists of a neuron that projects from the lobula, a region of the optic lobe specialized in detecting visual features such as shape and motion. the second type of neuron -henceforth referred to as plppns -consists of projection 100 neurons that emerge from the posterior lateral protocerebrum, a brain region that receives input from the optic lobe (otsuna and ito, 2006; keleş and frye, 2017; wu et al., 2016) . interestingly, lopn and plppns do not project to the ventral accessory calyx and do not connect to the gd kenyon cells. based on these findings, we conclude that there are two parallel pathways that convey visual information to the mushroom body: a pathway projecting from the medulla to the gd kenyon 105 cells and another pathway projecting from the lobula and posterior lateral protocerebrum to the a/bp kenyon cells. neurons projecting to the dorsal accessory calyx emerge from different brain regions the dorsal accessory calyx is a neuropil formed from the synapses connecting ~90 a/bp kenyon cells to their input neurons ( figure 1a ; (aso et al., 2014) ). using transgenic lines that drive expression specifically in the a/bp kenyon cells (the r85d07-gal4dbd, transgenic line also known as the mb371-gal4) and transgenic lines that express a photoactivatable form of gfp or pa-gfp (a combination of the uas-c3pa-gfp and uas-spa-gfp transgenic lines, henceforth referred to as uas-pa-gfp), we photo-labelled individual a/bp kenyon cells similarly as described in a previous study (aso et al., 2014; datta et al., 2008; ruta et al., 2010) . we found that individual a/bp kenyon cells extend on average 5 ± 1 claw-shaped 120 dendritic terminals (n = 13) exclusively into the dorsal accessory calyx ( figure 1b , d) and project their axons along the a and b lobes (not depicted). the overall morphology of a/bp kenyon cells is similar to the morphology of other types of a/b kenyon cell; for instance, individual a/bs kenyon cells extend on average 6 ± 1 claw-shaped dendritic terminals (n = 12) exclusively in the main calyx ( figure 1c ) and project their axons along the a and b lobes (not depicted). it is worth noting 125 that the claws formed by the a/bp kenyon cells are much smaller in diameter (1.8 ± 0.4 µm; n = 9 claws) than the claws formed by the a/bs kenyon cells (3.0 ± 0.4 µm; n = 10 claws; figure 1b -c (white arrows), e). it is also worth noting that the border of the dorsal accessory calyx is looser than the compact and well-defined circular border of the main calyx. individual a/bp kenyon cells can extend dendrites further away from the core of the dorsal accessory calyx, resulting in an 130 irregularly shaped calyx ( figure 1d) . thus, the a/bp kenyon cells resemble a/bs and a/bc kenyon cells but also differ from them in one major way: the dendrites of the a/bs and a/bc kenyon cells exclusively innervate the main calyx -a region known to receive most of its input from the olfactory system -whereas dendrites of the a/bp kenyon cells exclusively innervate the dorsal accessory calyx -a poorly characterized region of the mushroom body. 135 to identify neurons that project to the dorsal accessory calyx and connect to the a/bp kenyon cells, we used a targeted photo-labeling technique that was adapted from previously published techniques (aso et al., 2014; datta et al., 2008; ruta et al., 2010) . in order to only photo-label the neurons projecting to the dorsal accessory calyx, and not the a/bp kenyon cells, 140 we used a combination of transgenes that, in concert, drive the expression of pa-gfp in all neurons except the kenyon cells (the n-synaptobrevin-gal4, mb247-gal80 and uas-pa-gfp transgenes); instead, kenyon cells were labeled with the red fluorescent protein dsred using the mb247-dsred transgene ( figure 2a ). the brains of adult flies carrying all the aforementioned transgenes were dissected and imaged using two-photon microscopy ( figure 2b -c). guided by 145 the expression of dsred, we targeted the dorsal accessory calyx -which is clearly distinct from the main calyx -with high-energy light in order to photo-convert pa-gfp specifically in the neurons projecting to that area ( figure 2c , white dashed outline). upon photo-labeling, the somata of the neurons that express pa-gfp, and that project either dendrites or axons into the dorsal accessory calyx, were labeled ( figure 2d -e). on average, 71 ± 22 neurons (n = 22) were 150 photo-labeled. the somata of these neurons are located in seven distinct clusters that are distributed across the brain (figure 2a , d-e). on the anterior side of the brain, we found three clusters: one located near the antennal lobe (cluster al containing 3 ± 3 neurons), one located in the optic lobe (cluster ol containing 1 ± 2 neurons) and one located near the anterior ventral lateral protocerebrum (cluster avlp containing 6 ± 3 neurons) ( figure 2d , f). on the posterior 155 side of the brain, we found four clusters: one located in the superior medial protocerebrum (cluster smp containing 4 ± 2 neurons), one located in the superior lateral protocerebrum (cluster slp containing 20 ± 8 neurons), one located in the lateral horn (cluster lh containing 34 ± 12 neurons) and one located in the posterior lateral protocerebrum (cluster plp containing 3 ± 2 neurons) ( figure 2e -f). altogether, these results suggest that the dorsal accessory calyx receives input 160 from a diverse and distributed collection of projection neurons that can be divided into seven clusters. while en masse photo-labeling of the neurons projecting to the areas within and around the dorsal accessory calyx gives a good approximation of the number and identity of neurons possibly connecting to the a/bp kenyon cells, this technique cannot confidently identify true pre-synaptic partners. additionally, the amount of photo-activated pa-gfp within individual neurons varies 170 significantly between trials, and because a large number of neurons project to the dorsal accessory calyx, it is difficult to resolve the morphology of individual neurons using this technique. we, therefore, sought to identify gal4 transgenic lines that drive expression specifically in these neurons. to identify such lines, we carried out an anatomical screen using the flylight collection of gal4 transgenic lines ( figure 3a ; (jenett et al., 2012) ). as a first step, we screened through 175 the flylight database -an online catalogue that reports the expression patterns of ~7,000 gal4 driver lines -and selected 267 transgenic lines which that highlighted neuronal processes in the dorsal accessory calyx ( figure 3b -c). as a second step, we specifically labeled neurons that project to the dorsal accessory calyx 180 using the same technique as described above but with a different combination of transgenes (the r_line-gal4, uas-pa-gfp and mb247-dsred transgenes). the brains of adult flies that carry this combination of transgenes were dissected and imaged using two-photon microscopy ( figure 3d , f). guided by the expression of dsred, we targeted the dorsal accessory calyx with high-energy light in order to convert pa-gfp specifically in the neurons that project to that region ( figure 3d , 185 white dashed outline). we screen through the 267 pre-selected transgenic lines and identified 10 lines that we chose to investigate further based on two criteria. first, we selected lines that showed a strong signal after photo-labeling and clear pre-synaptic terminals near or in the dorsal accessory calyx ( figure 3e , g; table 1 ). possible pre-synaptic terminals -bouton-shaped ( figure 3e , white arrow) -were distinguished from possible post-synaptic terminals -mesh-shaped -based on 190 the recovered photo-labeling signal. second, we selected lines based on the strength and specificity of their expression patterns: lines expressing at high level in a few neurons led to clear photolabeling signals, making it easier to characterize these putative input neurons further ( figure 3g ). finally, we determined whether the neurons identified with these lines are pre-synaptic 195 partners of the a/bp kenyon cells. to this end, we used an activity-dependent version of the gfpreconstitution across synaptic partners (grasp) technique ( figure 4a ) (feinberg et al., 2008; macpherson et al., 2015) . in this technique, the green fluorescent protein (gfp) is split into two complementary fragments -the gfp1-10 and gfp11 fragments -that do not fluoresce when expressed alone; the gfp1-10 fragment is tagged to an anchor that specifically targets the pre-200 synaptic membrane (syb::spgfp1-10), whereas the gfp11 fragment is tagged to an anchor that targets the membrane (spgfp11::cd4). when both fragments are in close contact -in this case, when the syb::spgfp1-10 fragment is released at an active synapse -gfp molecules can be reconstituted and recover their fluorescence. the reconstitution of gfp molecules, as indicated by the presence of fluorescent gfp speckles, suggests that the neurons expressing the two 205 complementary fragments form functional synapses (macpherson et al., 2015) . in our study, the syb::spgfp1-10 fragment was expressed in the putative input neurons using the different transgenic lines identified in our screen as well as two transgenic lines that a previous study found to drive expression in putative dorsal accessory calyx input neurons ( figure 4b -m, table 1 ; (yagi et al., 2016) ). the expression of the spgfp11::cd4 fragment was driven in most of the kenyon 210 cells using the mb247-lexa transgenic line. we look for gfp speckles in the core of the dorsal accessory calyx, which is clearly visible in the red channel as an irregularly shaped structure anterior to the main calyx ( figure 4b , white dashed outline). we detected a large number of gfp speckles in the core of the dorsal accessory calyx for four of the ten lines that were identified in the screen as well as for the two transgenic lines identified by the 215 4l-m and table 1 ). additionally, we detected a small number of gfp speckles in the same region for three other transgenic lines ( figure 4f -h and table 1 ). it is worth noting that some of the gfp speckles lay outside of the red signal; most likely these speckles are formed by kenyon cells that extend dendrites outside of the core of the dorsal accessory calyx ( figure 4b -h, 4l-m). finally, we did not detect any gfp speckles for the remaining three lines ( figure 4i -k and table 1) . 220 altogether, our screen identified at least nine transgenic lines that drive expression in neurons that form synapses with the a/bp kenyon cells of the dorsal accessory calyx. to characterize further the morphology of the projection neurons that connect to the a/bp kenyon cells, we photo-labeled individual neurons using the transgenic lines recovered from our screen that showed reliable grasp signal. to this end, neurons were photo-labeled in the dissected brains of flies carrying the r_line-gal4 and uas-pa-gfp transgenes; these brains were then immuno-stained and imaged using confocal microscopy. we first focused our attention on the 230 transgenic lines in which we had found the strongest grasp signal ( figure 4b -e and table 1 ). photo-labeling of neurons that project to the dorsal accessory calyx using the r44h11-gal4 and r72d07-gal4 transgenic lines revealed a single neuron ( figure 5a ). in each line, a neuron projecting from the lobula to the dorsal accessory calyx was clearly photo-labeled ( figure 5b -c). therefore, we named this type of neuron "lopn". the soma of lopn is located in cluster plp, a 235 region medial to the optic lobe ( figure 2e , 5d-e). lopn extends its dendrites to the lobula and projects its axon to the superior lateral protocerebrum and the dorsal accessory calyx, completely avoiding the main calyx and the ventral accessory calyx ( figure 5d -e). it is worth noting that lopn is very similar to the olct1 neuron that has been previously described using a different transgenic line, r95f09-gal4; accordingly, we recovered a strong grasp signal for that 240 transgenic line ( figure 4l , table 1 ; (yagi et al., 2016) ). we then set out to confirm whether lopn does indeed connect to a/bp kenyon cells, as surmised from the grasp results. a previous study has demonstrated that a technique, which combines photo-labeling and dye-labeling tools, can be used to identify the complement of input 245 that individual kenyon cells receive from the antennal lobe, and the frequency at which individual projection neurons connect to kenyon cells (caron et al., 2013) . we have modified this technique in order to directly measure the connectivity rate between a given projection neuron and kenyon cells. in this modified version of the technique, we used a combination of transgenic lines to photolabel the projection neuron of interest -using the same protocol that we described in the previous 250 section -and to dye-label a single kenyon cell ( figure 6a ). we then assessed how many of the dendritic claws formed by the dye-labeled kenyon cell are connected to a given projection neuron. as a proof of principle, we measured the connectivity rate of the dc3 glomerulus projection neurons to the olfactory kenyon cells that innervate the main calyx. the connectivity rate of the dc3 projection neurons to the olfactory kenyon cells has been previously approximated to be 255 5.1%; this means that there is a 5.1% chance that a given olfactory kenyon cell claw receives input from the dc3 projection neuron (caron et al., 2013) . using the modified technique, we found that the dc3 projection neurons connect to kenyon cells at a connectivity rate of 4.3% (n = 30), a value well within the range measured previously (data not shown). we measured the connectivity rates of lopn to a/bp kenyon cells using the two transgenes we identified in our 260 screen. using the r44h11-gal4 driver line, we found that 4.1 % (n = 27) of the claws formed by the a/bp kenyon cells connect to lopn ( figure 6b -c, h and table 1 ). likewise, using the r72h07-gal4 driver line, we found that 3.0 % (n = 27) of the claws formed by the a/bp kenyon cells connect to lopn ( figure 6h and table 1 ). altogether, based on the results obtained using both the grasp technique and the dye/photo-labeling technique, we conclude that lopn is a true input 265 neuron of the a/bp kenyon cells and that it conveys information from the lobula, a visual processing center, to the dorsal accessory calyx. photo-labeling of neurons that project to the dorsal accessory calyx using the r19h07-gal4 and r20g07-gal4 transgenic lines revealed a group of neurons ( figure 7a ). in each line, neurons that project from the posterior lateral protocerebrum to the dorsal accessory calyx were photolabeled ( figure 7b -c). therefore, we named this type of neuron "plppns". the somata of the plppns are located in cluster lh, a region near the lateral horn ( figure 7d ). we could identify 13 275 ± 4 plppns (n = 21) per hemisphere using the r19h07-gal4 transgenic line ( figure 7d ). the r20g07-gal4 transgenic line drives expression at a much lower level, making it difficult to analyze the plppns in great detail ( figure 7c ). plppns extends projections into many brain centers, including the posterior lateral protocerebrum, the superior clamp, the superior lateral protocerebrum and the dorsal accessory calyx, avoiding completely the main calyx and the ventral 280 accessory calyx ( figure 7d ). using the dye/photo-labeling technique described above, we measured the connectivity rate between plppns and a/bp kenyon cells. we found that the plppns identified using the r19h07-gal4 transgenic line connect at a high rate: we found that 14.1 % (n = 24) of the claws formed by the a/bp kenyon cells connect to the plppns ( figure 6d , e, h). we also measured the connectivity rate of individual plppns using the same transgenic line and found 285 that, on average, an a/bp kenyon cell claw has 1.90% (n = 32) probability of connecting to a plppn ( figure 6h and table 1 ). to determine whether the plppns identified in the r19h07-gal4 and r20g07-gal4 transgenic lines are the same or different group of neurons, we used the split-gal4 technique 290 (luan et al., 2006) . in this technique, the gal4 transcription factor is split into two complementary fragments -the gal4ad and gal4dbd domains -that are both transcriptionally inactive when expressed alone; when both fragments are expressed in the same cell, gal4 can be reconstituted and recovers its transcriptional activity. plppns were visible in flies that carry the r20g07-gal4dbd and r19h07-gal4ad transgenes, thus confirming that both lines are expressed in the 295 same group of plppns ( figure 7e -g). this result revealed, even more clearly, that individual plppns project in many different brain centers ( figure 7g ). in order to distinguish the axonal terminals from the dendritic arbors, we used the split-gal4 combination of transgenic lines (the r20g07-gal4dbd and r19h07-gal4ad transgenic lines) to drive the expression of the presynaptic marker synaptotagmin in the plppns ( figure 7h ). we found that the projections 300 extending into the superior lateral protocerebrum, superior clamp and dorsal accessory calyx contain presynaptic terminals. similarly, when the expression of the postsynaptic marker denmark was driven specifically in the plppns, we found that all of the projections made by the plppns contain postsynaptic terminals ( figure 7i ). however, the projections extending into the posterior lateral protocerebrum are the only projections formed by plppns that contain only post-305 synaptic terminals and no pre-synaptic terminals ( figure 7h -i). to identify the neurons that project to the post-synaptic terminals formed by the plppns in the posterior lateral protocerebrum, a poorly characterized visual processing center, we used the targeted photo-labeling technique described above ( figure 8a ). in short, we used a combination 310 of transgenes that, in concert, drive the expression of pa-gfp in all neurons (the n-synaptobrevin-qf and quas-pa-gfp transgenes) and the expression of tdtomato in the plppns (using the r19h07-gal4ad, r20g07-gal4dbd and uas-tdtomato transgenes). guided by the expression of tdtomato, we targeted the post-synaptic terminals formed by plppns in the posterior lateral protocerebrum with high-energy light ( figure 8b , c). upon photo-labeling, two types of 315 neuron were clearly photo-labeled: the plppns -showing that the photo-activation was specific to these neurons -and neurons projecting from the ventral medulla ( figure 8d ). these photolabeled neurons project into deeper layers of the medulla ( figure 8e ). altogether, from this set of experiments, we conclude that plppns are one of the major input neurons of the a/bp kenyon cells and that they convey information from the posterior lateral protocerebrum, and possibly from the 320 ventral medulla, to the dorsal accessory calyx. photo-labeling using the transgenic lines that displayed weak grasp signal -these are the 325 r30e11-gal4, r31c03-gal4 and r53b06-gal4 lines -or no grasp signal -these are the r11f07-gal4, r11f08-gal4 and r12c04-gal4 lines -led to the identification of a third type of neuron (figure 9 ). the somata of these neurons are all located in al cluster, a region near the antennal lobe. although each of these neurons has a distinct overall morphology, they all project from the antennal lobe to the superior lateral protocerebrum and extend their axons in a region 330 near the dorsal accessory calyx. therefore, we named this type of neuron "alpns". the neurons photo-labeled using the r30e11-gal4 and r31c03-gal4 driver lines show a nearly identical morphology: in each line, a single neuron that extends its dendrites into the posterior antennal lobe and project its axons into the superior lateral protocerebrum was visible 335 ( figure 9a ). we named this neuron "alpn1". it is worth noting that alpn1 is very similar to the thermosensitive ac neuron that has been previously described (shih and chiang, 2011) . using a combination of photo-labeling and dye-tracing techniques, as described above, we measured the connectivity rate between alpn1 and a/bp kenyon cells. we could not detect any connections between alpn1 and a/bp kenyon cells (n = 26), suggesting that alpn1 is not a dorsal accessory 340 calyx input neuron ( figure 6h , table 1 ). similarly, the neuron photo-labeled using the r53b06-gal4 line extends its dendrites into the column region of the posterior antennal lobe, a region known to be activated by high humidity, and the sub-esophageal ganglion, a gustatory processing center ( figure 9b ). we named this neuron "alpn2". again, we could not detect any connections between alpn2 and a/bp kenyon cells (n = 30), suggesting that alpn2 is not a dorsal accessory 345 calyx input neuron ( figure 6f -h, table 1 ). thus, alpn1 and alpn2 are most likely not contributing major input to the a/bp kenyon cells. we extended our analysis to the transgenic lines that displayed no grasp signal. the neurons photo-labeled using the r11f08-gal4 and r12c04-gal4 driver lines show an overall 350 similar morphology: in each line, a single neuron that extends its dendrites into the arm region of the posterior antennal lobe, a region known to be activated by low humidity, was visible ( figure 9d ). we named this neuron "alpn3". not surprisingly, we could not detect any connections between alpn3 and a/bp kenyon cells (n = 26) suggesting that alpn3 does not provide input into the dorsal accessory calyx (table 1) . similarly, the neuron photo-labeled using the r11f07-gal4 355 line extends its dendrites broadly throughout the anterior antennal lobe, an olfactory processing center ( figure 9f ). we named this neuron "alpn4". again, we could not detect any connections between alpn4 and a/bp kenyon cells (n = 25), suggesting that alpn4 does not provide input into the dorsal accessory calyx (table 1) . thus, we could confirm that the transgenic lines that displayed no grasp signal do not appear to provide major input to the a/bp kenyon cells. 360 altogether, these results show that the third type of neuron identified in our screen -alpnsproject to a region close to the dorsal accessory calyx but are most likely not presynaptic to the a/bp kenyon cells. our results suggest that the lopn and plppns that we identified in our screen connect to the a/bp kenyon cells and that, together, these two types of projection neuron represent a large fraction of the input neurons of the dorsal accessory calyx. our results also suggest that the alpns do not connect to the a/bp kenyon cells, as another study suggested (yagi et al., 2016) . thus, we 370 conclude that the dorsal accessory calyx is anatomically poised to receive information primarily from the lobula and the posterior lateral protocerebrum, two visual processing centers. we verified whether these findings corroborate with the recently released drosophila hemibrain connectome (xu et al., 2020) . using the neuprint 1.0.1 platform, we focused our attention on the 60 reconstructed a/bp kenyon cells that have been fully traced. altogether, these a/bp kenyon cells 375 receive input from a large number of neurons but most of these synapses are most likely axoaxonic synapses as they are located along the a and b lobes. these input neurons are primarily other kenyon cells, various dopaminergic neurons as well as neurons known to connect broadly to all kenyon cells such as the apl neuron and the dpm neuron (data not shown). we focused our attention on the 141 input neurons that connect to the a/bp kenyon cells in the dorsal 380 accessory calyx. not surprisingly, in accordance with the results obtained from our en masse photo-labeling experiment, the cell bodies of these input neurons can be divided into seven different clusters and the ratio of neurons belonging to a given cluster is largely consistent between both analyses ( figure 10a ). interestingly, the lh cluster is the most numerous cluster: our study identified 34 ± 12 neurons (n = 22) in this cluster -including the 13 ± 4 plppns (n = 21) -whereas the connectome shows a total of 61 input neurons with cell bodies located in the lateral horn ( figure 10a ). among these neurons, we identified 13 that are morphologically highly similar to plppns ( figure 7j -k, table 2 ). we found that these plppn-like neurons connect to 59 of the 60 reconstructed a/bp 390 kenyon cells, representing 49.40% of the input all a/bp kenyon cells receive in the dorsal accessory calyx ( figure 10b and table 2 ). plppns-like neurons connect mostly to a/bp kenyon cells but also form a few connections to gd and gmain kenyon cells, reinforcing the observation that the dorsal accessory calyx input neurons and the ventral accessory calyx input neurons form two parallel pathways ( figure 10c ). in our study, we identified neurons connecting the ventral medulla 395 to the plppns (figure 8 ) but, because the hemibrain connectome does not include the medulla, we could not identify fully traced neurons similar to these plppns input neurons. additionally, our study identified 1 ± 2 neurons (n = 22) in the ol cluster -including lopn -whereas the connectome shows a total of 4 input neurons with cell bodies located in the optic lobe ( figure 10a ). among these neurons, we identified a neuron very similar to lopn (figure 5f -g). we found 400 that this lopn-like neuron connects to 11 of the 60 reconstructed a/bp kenyon cells, representing 2.66% of the input all a/bp kenyon cells receive in the dorsal accessory calyx ( figure 10b and table 2 ). as we found in our study, this lopn-like neuron does not connect to any other types of kenyon cell, forming a pathway parallel to the one conveying visual information to the ventral accessory calyx. 405 finally, both studies identified a number of neurons with cell bodies located near the antennal lobe: using en masse photo-labeling, we identified a total of 3 ± 3 neurons (n = 22) in the al cluster, whereas nine such neurons are found in the connectome. among these neurons, we recognized three of the four alpns we characterized: an alpn2-like neuron and an alpn4-like 410 neuron that represents, respectively, 0.39% and 0.54% of the input that a/bp kenyon cells receive in the dorsal accessory calyx ( figure 9c , g and table 2 ). we could not recognize an alpn1-like neuron in the connectome but we identified an alpn3-like neuron ( figure 9e , table 2 ). the alpn3like neuron does not connect to any kenyon cells. together, the nine antennal lobe input neurons -including the alpn2-like and alpn4-like neurons -represent less than 2% of the input that 415 a/bp kenyon cells receive in the dorsal accessory calyx ( figure 10b ). interestingly, most of the remaining input a/bp kenyon cells receive in the dorsal accessory calyx (22.93% of the total input a/bp kenyon cells receive in the dorsal accessory calyx) is from a single neuron projecting from many brain regions including the main calyx and the pedunculus, two regions of the mushroom body, as well as the superior lateral protocerebrum (table 2) . altogether, these observations 420 confirm that lopn-like and plppn-like neurons are the major input neurons that connect to the a/bp kenyon cells in the dorsal accessory calyx. thus, the dorsal accessory calyx receives most of its input from visual processing centers -the posterior lateral protocerebrum and the lobula -and is thus anatomically poised to process mostly, if not strictly, visual information. in this study, we identified and characterized neurons projecting to the dorsal accessory calyx of the mushroom body and show that these neurons are presynaptic to the a/bp kenyon cells ( figure 11 ). using a combination of genetic and anatomical techniques, we could distinguish two different 430 types of projection neuron: lopn projecting from the lobula -an area of the optic lobe processing visual features such as shape and motion -and the plppns projecting from the posterior lateral protocerebrum. although the posterior lateral protocerebrum remains poorly characterized in d. melanogaster, evidence from other insects shows that this brain region receives input from the optic lobe (paulk et al., 2008 (paulk et al., , 2009 ). interestingly, we found that the dendrites formed by the 435 plppns in the posterior lateral protocerebrum are in close proximity to neurons that project from the ventral medulla. based on our results -and considering insights from the connectome -we estimate that lopns and plppns account for half of total input that a/bp kenyon cells receive in the dorsal accessory calyx. lopns and plppns do not extend axonal terminals into the ventral accessory calyx, the other calyx known to receive visual input, but rather extend axonal terminals 440 into the dorsal accessory calyx and into the superior lateral protocerebrum. likewise, the a/bp kenyon cells do not connect to the visual projection neurons that are associated with the ventral accessory calyx (vogt et al., 2016) . these findings suggest that the visual system is connected to the mushroom body via two parallel pathways: the a/bp kenyon cells receive input from the lobula and the posterior lateral protocerebrum, whereas the gd kenyon cells receive input directly 445 from the medulla. further functional studies are necessary to determine what kind of visual information is processed by the a/bp kenyon cells. in drosophila melanogaster, the mushroom body has long been studied as an olfactory processing center. however, evidence from many insects, including the honeybee apis mellifera, 450 shows that the mushroom body integrates sensory information across different modalities. in honeybees, the input region of the mushroom body, also called the calyx, is divided into different layers and each layer receives input from either the olfactory or visual system (gronenberg, 2001) . because the dendrites of kenyon cells are also restricted to specific layers, it has been suggested that, in the honeybee, multisensory integration does not occur at the level of individual 455 kenyon cells, but rather at the population level (ehmer and gronenberg, 2002) . although the honeybee mushroom body differs greatly from the drosophila mushroom body -it contains about a hundred times as many kenyon cells and its input region is divided in multiple complex layers -it appears that both mushroom bodies share a common fundamental connectivity principle: the segregation of input based on sensory modality. this connectivity mechanism is immediately 460 apparent in the structural organization of the drosophila melanogaster mushroom body: the kenyon cells receiving input from the olfactory system all extend their dendrites into the main calyx, whereas as the kenyon cells receiving input from the visual system extend their dendrites either in the dorsal accessory calyx or the ventral accessory calyx. many studies have demonstrated that the kenyon cells that process olfactory information -those associated with 465 the main calyx -integrate input broadly across the different types of olfactory projection neuron (caron et al., 2013; zheng et al., 2018) . interestingly, it appears that the kenyon cells that process visual information are wired differently. we have a thorough understanding of how olfactory kenyon cells integrate input from the 470 antennal lobe: most kenyon cells receive, on average, input from seven projection neurons and the projection neurons connecting to the same kenyon cell share no apparent common features (caron et al., 2013; zheng et al., 2018) . theoretical studies have shown that this random-like connectivity pattern enables the mushroom body to form sparse and decorrelated odor representations and thus maximizes learning (litwin-kumar et al., 2017) . randomization of 475 sensory input is a connectivity pattern that is well suited for representing olfactory informationas an odor is encoded based on the ensemble of olfactory receptors it activates -and might not be suitable for representing visual information. indeed, our results suggest that that specific visual features -the signals processed by the medulla and the ones processed by the lobula and the posterior lateral protocerebrum -need to be represented by two separate subpopulations of 480 kenyon cells. this observation mirrors anatomical studies of the honeybee brain: the neurons projecting from the lobula terminate in a different layer than the neurons projecting from the medulla (ehmer and gronenberg, 2002) . this arrangement might be essential to preserve distinct visual features when forming associative memories. functional and behavioral studies are required to determine whether indeed the mushroom body represents multisensory stimuli in this 485 manner. flies (drosophila melanogaster) were raised on standard cornmeal agar medium and maintained in an incubator set at 25°c, 60% humidity with a 12 hours light / 12 hours dark cycle (percival scientific, inc.). crosses were set up and reared under the same conditions, but the standard cornmeal agar medium was supplemented with dry yeast. the strains used in this study are 495 described in the table below. two to six-day-old flies were used for all photo-labeling experiments. the protocol was largely based on the one developed in a previous study (aso et al., 2014) . in short, brains were dissected in saline (108 mm nacl, 5 mm kcl, 5 mm hepes, 5 mm trehalose, 10 mm sucrose, 1 mm nah2po4, 4 mm nahco3, 2 mm cacl2, 4 mm mgcl2, ph≈7.3), treated for 1 minute with 2 mg/ml 505 collagenase (sigma-aldrich) and mounted on a piece of sylgard placed at the bottom of a petri dish. photo-labeling and image acquisition were performed using an ultima two-photon laser scanning microscope (bruker) with an ultrafast chameleon ti:saphirre laser (coherent) modulated by pockels cells (conotopics). for photo-labeling, the laser was tuned to 710 nm with an intensity of 5-30 mw; for image acquisition, the laser was tuned to 925 nm with an intensity of 510 1-14 mw (both power values were measured behind the objective lens). a 60x water-immersion objective lens (olympus) was used for both photo-labeling and image acquisition. a 40x waterimmersion objective lens (olympus) was used for image acquisition in some experiments (the ones described in figure 2 and figure 3) . a gaasp detector (hamamatsu photonics) and pmt detector were used for measuring green and red fluorescence, respectively. photo-labeling and 515 image acquisition files were visualized on a computer using the prairie view software version 5.4 (bruker). image acquisition was performed at a resolution of 512 by 512 pixels, with a pixel size of 0.39 μm (60x lens) or 0.582 μm (40x) and a pixel dwell time of 4 μs. each pixel was scanned 8 times. for photo-labeling of the dorsal accessory calyx (or the posterior lateral protocerebrum), a volume spanning the entire neuropil was divided into eight to 12 planes with a step size of 2 μm. the mask function of the prairie view software was used to mark the targeted region in every plane, and the boundaries of the mask were determined based on the red fluorescent protein dsred expressed by the a/bp kenyon cells. the photo-labeling step was performed using a pixel 525 size of 0.019 μm and a pixel dwell time of 2 μs. each pixel was scanned four times. each plane was scanned 30 times with 30-second interval. the entire photo-labeling cycle was repeated two to three times, with a 10-minute resting period between cycles. the entire brain was imaged before and after photo-labeling using the 40x water-immersion objective lens (olympus, japan). the number of cell bodies recovered after the photo-labeling was measured using the multi-point 530 tool function of the fiji software (schindelin et al., 2012) . for the photo-labeling of single neurons, a slightly modified protocol was used. instead of a volume, a single square plane of 1.0 μm by 1.0 μm centered on the soma of a neuron cell was scanned 70 to 100 times with a 10 seconds interval between scans. photo-labeled brains were 535 fixed in 2% paraformaldehyde diluted in 1x phosphate bovine saline (pfa) for 45 minutes at room temperature, washed five times in 0.3% triton x-100 diluted in 1x phosphate bovine saline technologies) were used. on the following day, brains were washed four times in pbst and mounted on a slide (fisher scientific) using the mounting media vectashield (vector laboratories inc.). immuno-stained brains were imaged using an lsm 880 confocal microscope. (zeiss). the neuropils innervated by the input neurons were identified by comparing the confocal images with the adult brain template jfrc2 available on virtual fly brain 565 (https://v2.virtualflybrain.org/) (jenett et al., 2012) . one to three-day-old flies were used when mapping the connectivity rate between input neurons 570 and α/βp kenyon cells. brains were dissected in saline, treated for one minute with 2 mg/ml collagenase (sigma-aldrich) and mounted on a piece of sylgard placed at the bottom of a petri dish. the imaging protocol is the same as described above but the photo-labeling protocol is different. each of the input neurons was photo-labeled using a single plane centered on either its soma or its projection and by scanning the plane three to five times. each pixel was scanned eight 575 times with a pixel size of 0.019 μm and a pixel dwell time of 4 μs. a fire-polished borosilicate glass pipette (0.5 mm i.d., 1.0 mm o.d., 10 cm length; sutter instruments) was pulled using the p-2000 micropipette puller (sutter instruments) and backfilled with texas red dye (lysine-fixable 3000 mw; life technologies) dissolved in saline. the tip of the pipette was positioned next to the cell body of a randomly chosen α/βp kenyon cell under the two-photon microscope. the dye was electroporated 580 into the cell body using three to five 1-5 millisecond pulses of 20-50 v. the dye was allowed to diffuse within the kenyon cell for 5 minutes before the brain was imaged. all confocal images were collected on lsm880 confocal microscope (zeiss). for imaging whole brain, each sample was imaged twice using a plan-apochromat 40x/1.3 oil m27 objective lens. first, the entire brain was divided into four tiles, each tile was imaged separately (voxel size = 0.46 μm by 0.46 μm by 2 μm, 764 by 764 pixels per image plane) and then stitched together using the stitch function of the zen microscope software (zeiss). for imaging specific neuropil regions, 590 the same objective lens was used, but with higher resolution (voxel size = 0.09 μm by 0.09 μm by 1 μm, 2320 by 2320 pixels per image plane). for imaging brains manipulated using the grasp technique, a plan-apochromat 63x/1.4 oil m27 objective lens was used in combination with the rgb airyscan mode. images were processed using the airyscan function of the zen microscope software. all confocal images were analyzed using the fiji software (schindelin et al., 2012) . all 595 figure panels are maximum intensity projection of confocal stacks or sub-stacks. (a) a schematic of the drosophila brain shows how the frequency of connections between the α/βp kenyon cells (red) and a given input neuron (green), here lopn, was measured. (b-g) a 905 given input neuron was photo-labeled (bright green) and a randomly chosen a/bp kenyon cell was dye-filled (red). the total number of claws formed by the dye-filled kenyon cell was counted and claws connecting to the axonal terminals of the photo-labeled neuron were detected (arrows). such connections were found for lopn (b-c), plppns (d-e) but not for the other neurons identified in this study such as alpn2 (f-g). (h) the frequency of connections between α/βp kenyon cells 910 and a given input neuron was calculated by dividing the number of connections detected (for instance arrows in c and e) by the total number of claws sampled for that particular input neuron. the following genotypes were used in this figure: yw/yw;uas-c3pa-gfp unknown /uas-spa-gfp attp40 ; r13f02-gal4ad vk00027 ,r85d07-gal4dbd attp2 /r_line(as indicated in the panel) the neuronal architecture of the mushroom body provides a logic for associative learning understanding the retinal basis of vision across species complete connectomic reconstruction of olfactory projection neurons in the fly brain random convergence of olfactory inputs in the drosophila mushroom body the drosophila pheromone cva activates a sexually dimorphic neural circuit segregation of visual input to the mushroom bodies in the 635 honeybee (apis mellifera) gfp reconstitution across synaptic partners (grasp) defines cell contacts and 640 synapses in living nervous systems subdivisions of hymenopteran mushroom body calyces by their afferent supply systematic analysis of the visual projection neurons of drosophila melanogaster: lobula-specific pathways what can tiny mushrooms in fruit flies tell us about learning and memory? 650 a gal4-driver line resource for drosophila neurobiology object-detecting neurons in drosophila olfactory networks: from sensation to perception context generalization in drosophila visual learning requires the mushroom bodies refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression dynamic labelling of neural connections in multiple colours by trans-synaptic fluorescence complementation connectomics analysis reveals first, second, and third order thermosensory and hygrosensory neurons in the adult drosophila brain limited taste discrimination in drosophila the color-vision circuit in the medulla of drosophila visual processing in the central bee brain the processing of 695 color, motion, and stimulus timing are anatomically segregated in the bumblebee brain a dimorphic pheromone circuit in drosophila from sensory input to descending output fiji: an open-source platform for biological-image analysis. 705 organization of antennal lobe-associated neurons in adult drosophila melanogaster brain dye fills reveal additional olfactory tracts in the protocerebrum of wild-type drosophila direct neural pathways convey distinct visual information to drosophila mushroom bodies shared 720 mushroom body circuits underlie visual and olfactory memories in drosophila visual projection neurons in the drosophila lobula link feature detection to distinct 725 behavioral programs a connectome of the adult drosophila central brain convergence of multimodal sensory pathways to the mushroom body calyx in drosophila melanogaster dissecting neural circuits for multisensory integration and crossmodal processing a naturally monomeric infrared fluorescent protein for protein labeling in vivo a complete electron microscopy volume of the brain of adult drosophila 760 melanogaster r19h07-gal4) or weakly (c, r44h11-gal4) in neurons projecting to or from the dorsal accessory calyx (white dashed outline) were selected for further investigation. these images were obtained from the flylight website. (d, e) the dorsal accessory calyx (white 850 dashed outline) of the mushroom body (red) was visualized in the selected lines, here r19h07-gal4, and targeted for photo-activation by designing a mask that exposed the outlined region to high energy light; transgenic lines driving expression in a few neurons extending clear axonal terminals in the dorsal accessory calyx (e, arrow) were selected for further investigation. (f, g) before photo-labeling the following genotype was used in the d-g panels: yw/yw;mb247-dsred unknown ,uas-c3pa-gfp unknown /uas-c3pa-gfp attp40 b-c) lopn (bright green) was identified in the screen using two different transgenic lines (b: r44h11-gal4 and c: r72d07-gal4); the neurons photo-885 labeled in each line show an overall similar morphology. (d-e) lopn was photo-labeled using either the r44h11-gal4 (d) r72d07-gal4 (e) transgenic lines; the samples were fixed, immuno-stained (nc82 antibody, blue) and imaged. the photo-labeled neurons show an overall similar morphology: their somata are located near the optic lobe; they extend dendritic terminals in a small region of the lobula (lo); they extend axonal terminals in the dorsal accessory calyx 890 (daca) and the superior lateral protocerebrum (slp). (f-g) a lopn-like neuron was identified in the hemibrain connectome: (f) this neuron projects from the lobula to the dorsal accessory calyx and the superior lateral protocerebrum; (g) its axonal terminals innervate the dorsal accessory calyx (daca), but not the main calyx (ca) or the ventral accessory calyx (vaca) e) yw/yw;mb247-dsred unknown ,uas-c3pa-gfp attp40 /uas-c3pa-gfp unknown b-c) a group of plppns (bright green) were identified in the screen using two different transgenic lines (b: r19h07-gal4 and c: r20g07-gal4). (d) plppns were photolabeled using the r19h07-gal4 transgenic line; the sample was fixed, immuno-stained (nc82 antibody, blue) and imaged. the photo-labeled neurons show an overall similar morphology: their 925 somata are located near the lateral horn; they extend projections in the posterior lateral protocerebrum (plp), the superior clamp (sc), the superior lateral protocerebrum (slp) and the dorsal accessory calyx (daca). (e-f) the expression patterns observed for each of the gal4 lines are broad and include many neurons; (e) the r19h07-gal4 line drives expression strongly in many neurons, including the plppns, (f) whereas the r20g07-gal4 line drives expression 930 weakly in fewer neurons, including the plppns. these images were obtained from the flylight website. (g) a split-gal4 j) these neurons project from the posterior lateral protocerebrum to the dorsal accessory calyx, the superior lateral protocerebrum and the superior clamp; (k) their axonal terminals innervate the dorsal accessory calyx (daca), but not the main calyx (ca) or the ventral accessory calyx (vaca). the following genotypes were used in this figure: (b, d) yw/yw; mb247-dsred unknown , uas-c3pa-940 gfp unknown /uas-c3pa-gfp attp40 c) yw/yw; uas-c3pa-gfp unknown /cyo;uas-c3pa-gfp attp2 , r20g07-gal4 attp2 /mkrs; (g) yw/yw; mb247-dsred unknown , uas-c3pa-gfp unknown /cyo; uas-c3pa-gfp attp2 unknown ; (i) yw/yw;uas-denmark 2 /cyo the authors declare that no competing interests exist. key: cord-330176-1ugzkf22 authors: weeratunga, prasanna; uddin, md bashir; kim, myun soo; lee, byeong-hoon; kim, tae-hwan; yoon, ji-eun; ma, jin yeul; kim, hongik; lee, jong-soo title: retracted article: interferon-mediated antiviral activities of angelica tenuissima nakai and its active components date: 2016-01-05 journal: j microbiol doi: 10.1007/s12275-016-5555-4 sha: doc_id: 330176 cord_uid: 1ugzkf22 angelica tenuissima nakai is a widely used commodity in traditional medicine. nevertheless, no study has been conducted on the antiviral and immune-modulatory properties of an aqueous extract of angelica tenuissima nakai. in the present study, we evaluated the antiviral activities and the mechanism of action of an aqueous extract of angelica tenuissima nakai both in vitro and in vivo. in vitro, an effective dose of angelica tenuissima nakai markedly inhibited the replication of influenza a virus (pr8), vesicular stomatitis virus (vsv), herpes simplex virus (hsv), coxsackie virus, and enterovirus (ev-71) on epithelial (hek293t/hela) and immune (raw264.7) cells. such inhibition can be described by the induction of the antiviral state in cells by antiviral, ifnrelated gene induction and secretion of ifns and pro-inflammatory cytokines. in vivo, angelica tenuissima nakai treated balb/c mice displayed higher survivability and lower lung viral titers when challenged with lethal doses of highly pathogenic influenza a subtypes (h1n1, h5n2, h7n3, and h9n2). we also found that angelica tenuissima nakai can induce the secretion of il-6, ifn-λ, and local iga in bronchoalveolar lavage fluid (balf) of angelica tenuissima nakai treated mice, which correlating with the observed prophylactic effects. in hplc analysis, we found the presence of several compounds in the aqueous fraction and among them; we evaluated antiviral properties of ferulic acid. therefore, an extract of angelica tenuissima nakai and its components, including ferulic acid, play roles as immunomodulators and may be potential candidates for novel anti-viral/anti-influenza agents. viral diseases range from trivial infections to plagues that alter the course of history. because of the enormous variations in viruses and in their epidemiology and pathogenesis, there is no single, magic-bullet approach to control. each virus presents its own set of problems. as an example, influenza viruses are highly infectious and constitute a major causative agent for recurrent epidemics and pandemics. on average, approximately 10% of the world's population is infected by the virus annually, resulting in an estimated 250,000 deaths, hence posing a serious health threat (rajasekaran et al., 2013) . moreover, new and re-emerging infectious viral diseases are a rising global health threat, and the risk of spreading these viruses between continents and countries is even greater. hiv/aids, severe acute respiratory syndrome (sars) and the 2009 h1n1 influenza pandemic and mers epidemic are only a few of many examples of emerging infectious diseases in the modern world (morens and fauci, 2013; who, 2015) . several preventive and therapeutic measures, including biosecurity, vaccination and antiviral drugs, are routinely used or tried to prevent and treat viral diseases. vaccines are the basis of prevention of many viral infections; however, there are considerable drawbacks. because vaccination requires regular monitoring to confirm matching between the vaccines and the circulating virus strains, time-consuming generation processes limit its availability. failures of influenza vaccines have been widely documented, and in the elderly populationin which most mortality occurs-influenza vaccines are only approximately 50% effective (reichert et al., 2012) . in the eventuality of a pandemic infection with a new strain, antiviral drugs also represent the first line of defense. owing to their metabolic properties, viruses are difficult to control, and the limited availability, associated side effects, and rapid development of antiviral resistance have limited the usefulness of these drugs. therefore, within this evolving environment, groundbreaking strategies and responses are required to reduce the economic and human health risks associated with viral diseases. the development of safe, effective and inexpensive antiviral drugs is among the top global priorities in drug development. currently, there is a large and ever-expanding global population base that prefers the use of natural products for preventing and treating medical conditions (gandhiraja et al., 2009 ) and many pharmaceutical companies are attempting to produce new antimicrobial formulations extracted from plants or herbs. plants are an important sources of lead compounds; up to 40% of modern drugs are derived from plant material (pan et al., 2013) . for the viral diseases, empirical evidence of the ethno-medical benefits of plants, coupled with bioassay-guided fractionation and isolation, has the potential to identify novel antiviral drugs. in our study, approximately 400 natural oriental herbal extracts were screened for antiviral effects. of these, extracts from angelica tenuissima nakai (atn) showed considerable therapeutic promise because of its broad spectrum of antiviral activities and immune-enhancing properties. angelica tenuissima nakai belongs to the family apiaceae and grows in certain areas in china and rocky slopes in the korean peninsula (ka et al., 2005) . it is widely used to treat headache, diarrhea, epilepsy and rheumatic arthralgias in traditional oriental medicine (nam et al., 2014) . however, the antiviral activity or immune-modulatory potential of the crude plant extract of angelica tenuissima nakai have not been reported in detail. hence, we evaluated the antiviral activities of total aqueous extracts from this herb against a wide array of viruses in vitro. we also examined the effects of angelica tenuissima nakai on innate immune responses. in addition, we used highperformance liquid chromatography (hplc) analysis to determine the active molecules present in the aqueous fraction. commercial dried bark of the angelica tenuissima nakai was obtained and verified by professor ki hwan bae at the college of pharmacy, chungnam national university. the water extract of angelica tenuissima nakai was prepared by vitabio corporation. further, the extract quality was assured by herbal medicine improvement research center, korea institute of oriental medicine, daejeon, republic of korea. hundred gram (100 g) of the dried bark was mixed with 1 l of distilled water and extracted by heating for 2.5 h at 105°c using a medical heating plate (gyeongseo extractor cosmos-600). then, angelica tenuissima nakai was filtered using a filter paper (0.45 μm) (millex ® ). the extract was next centrifuged at 12,000 rpm for 15 min and the supernatant was collected, and the ph was adjusted to 7.0. the total aqueous extract was then filtered through a syringe filter (0.22 μm) and lyophilized. the final concentration was adjusted to 0.1 mg/ml with phosphate buffered saline (pbs) and kept at 4°c until utilization. raw264.7 (atcc ® tib-71 tm ), hek293t (atcc ® crl-11268 tm ), hela (atcc ® ccl-2 tm ), mdck (atcc ® ccl-34, nbl-2), and a549 (atcc ® ccl-185 tm ) cells were cultured in dulbecco's modified eagle's medium (dmem) (invitrogen) supplemented with 10% fetal bovine serum (fbs; gibco) and 1% antibiotic-antimycotic solution (gibco) in a humidified incubator at 37°c and 5% co 2 . green fluorescent protein (gfp)-fused viruses such as pr8-gfp, vsv-gfp, h3-gfp, and ev-71 were kindly provided by dr. jae u. jung, department of molecular microbiology and immunology, university of southern california, usa. challenge viruses were provided by dr. y. k. choi, chungbuk national university, cheongju, republic of korea. antiviral assays in angelica tenuissima nakai pretreated raw264.7, hek293t, and hela cells raw264.7 cells were cultured in 12-well tissue culture plates (8 × 10 5 cells/well) and incubated at 37°c for 12 h. hek293t and hela cells were cultured in six-well tissue culture plates (1 × 10 6 cells/well) under similar conditions. antiviral assays were performed according to moon et al. (2012) , with some modifications. dmem alone (untreated and virus-only groups), dmem with 1,000 u/ml of recombinant mouse/human interferon (ifn)-β (positive control, sigma) and dmem with 1.0 μg/ml (10 μl/ml or 1% v/v) of angelica tenuissima nakai were incubated in different wells. at 12 hpt (hour post treatment), all the wells were gently washed with pbs once and raw264.7 cells were infected with either vsv-gfp (moi = 1.0) or pr8-gfp (moi = 1.0), hek293t cells were infected with vsv-gfp (moi = 0.2) or hsv-gfp (moi = 3.0) and hela cells were infected with h3-gfp (moi = 3.0) or ev-71 (moi = 0.5) viruses using dmem containing 1% fbs. two hours post-infection (hpi), the culture medium was renewed, and incubated for an additional 24 h. gfp expression was observed was quantified with the glomax multi-detection system (promega). gfp expression, virus titrations and cell viabilities were determined at 12 and/or 24 hpi. in vitro pro-inflammatory cytokine inducing effect of angelica tenuissima nakai was tested using commercial elisa kits. in raw264.7 cells, murine interleukin (il)-6 and ifn-β were measured, as previously described (wadsworth and koop, 1999) . raw264.7 cells were treated with recombinant murine ifn-β (sigma-aldrich) and 1.0 μg/ml (10 μl/ml or 1% v/v) angelica tenuissima nakai and cultured supernatants were collected at 0, 12, and 24 hpt, clarified by centrifugation at 2,500 × g for 10 min at 4°c and dispensed into murine ifn-β (pbl interferon source) elisa plates or murine il-6 or murine tnf-α (bd bioscience) capture antibody-coated elisa plates. in the case of hek293t cells, recombinant human ifn-β (sigma-aldrich) was used as the positive control and the clarified supernatant was dispensed into human ifnβ (tfb, inc.) and human il-6 (invitrogen) elisa plates. murine ifn-β, human ifn-β, and human il-6 elisa were performed in duplicate, and other elisa's was performed in triplicate. interferon-mediated antiviral activities of angelica tenuissima nakai 59 determination of the effect of angelica tenuissima nakai on type i ifn-related protein phosphorylation in raw264.7 cells by immunoblot analysis raw264.7 cells were grown in 6-well tissue culture plates and incubated at 37°c. after 12 h the cells were treated with dmem containing 10% fbs alone (negative control), dmem with 100 ng/ml lps (positive control), or dmem with 1.0 μg/ml (10 μl/ml or 1% v/v) angelica tenuissima nakai, and the cells were harvested at 0, 8, 12, and 24 hpt. the cell pellets were lysed in lysis buffer for immunoblot analysis; loaded on sds-page and then transferred onto a polyvinylidene difluoride membrane (bio-rad) for 2 h. the membranes were blocked for 1 h in tris-buffered saline containing 0.05% tween-20 (tbst) and 5% bovine serum albumin. then incubations were performed at 4°c overnight with indicated primary antibody such as anti-irf3 (abcam, #ab25950), antiphopho-irf3 (ser396), anti-p65, anti-phopho-p65, anti-stat1, anti-phospho-stat1, anti-tbk1, or anti-phospho-tbk1, anti-p38, anti-phopho-p38 (cell signaling technology), or anti-β-actin (santa cruz sc#47778) antibodies. horseradish peroxidase-conjugated secondary antibody (sigma) was used to visualize the respective proteins by means of an enhanced chemiluminescence detection system (ecl-ge healthcare, uk) using a las-3000 mini lumino image analyzer. raw264.7 cells were grown in 6-well tissue culture plates (1 × 10 6 cells/well) and incubated at 37°c. the cells were treated with dmem containing 10% fbs alone (negative control), dmem with 1000 u/ml of recombinant murine/ human ifn-β, dmem with 1.0 μg/ml (10 μl/ml or 1% v/v) angelica tenuissima nakai, and the cells were harvested at 0, 4, 8, 12, and 24 hpt. the total rna from the cells was isolated using the rneasy mini kit (qiagen), and cdna was synthesized using reverse transcriptase (toyobo). the different levels of cdna were quantified by real-time polymerase chain reaction (pcr) using a quantitect sybr green pcr kit (qiagen) on a mygenie96 thermal block (bioneer). the pcr primers are listed in tables 2 and 3 . fifty two female, five-week-old balb/c mice were separated into four experimental sets, with two groups per set. of the four sets, one had two groups with 11 mice each (six for lung virus titration at 3 and 5 day's post-infection [dpi]). the other three sets had two groups containing 5 mice each. the mice were orally inoculated 0.1 mg/ml angelica tenuissima nakai at a total volume of 200 μl (20 μg/head) 1, 3, and 5 days before infection. the control groups were orally inoculated 200 μl of pbs. all mice were intra-nasally infected (20 μl) with five times the 50% mouse lethal dose (mld50) of h1n1, h5n2, h7n3 or h9n2. mice showing a more than 25% body weight loss were regarded to have reached the experimental end point and were humanely killed. the body weight and survival were recorded up to 13 dpi. at 3 and 5 dpi, three mice from each of the two groups from the h1n1infected set were randomly sacrificed to measure the lung virus titers. lung tissues were collected from euthanized mice aseptically, and virus titers were measured by 50% tissue culture infectious dose (tcid50) (quan et al., 2007) . first, lung tissues were mechanically homogenized in 500 ml of pbs solution added with 1% antibiotic/antimycotic solution, centrifuged (15 min, 12,000 × g and 4°c) and stored at -80°c. madin-darby canine kidney (mdck) cells cultured in 96-well microtiter plates were infected with 10-fold serial dilutions (in dmem with 1% fbs) of lung homogenate (50 μl/well) in quadruplicate and incubated at 37°c. at 1 hpi, the media was removed and renewed with medium containing l-1-tosylamido-2-phenylethyl chloromethyl ketone (tpck) trypsin (thermo scientific) and incubated for 72 h. viral cytopathic effects (cpe) were monitored daily and the titers were determined by the hemagglutination assay (ha). the virus titer was calculated by the reed and muench method (zhao et al., 2010) and expressed as log10 tcid 50 /lung. twenty four, five-week-old balb/c mice were divided into two groups with 12 mice each. in one group, mice were orally administered 0.1 mg/ml angelica tenuissima nakai at a total volume of 200 μl (20 μg/head) 1, 3, and 5 days before the collection of samples. the control group was orally administered 200 μl of pbs. three mice from each group were randomly selected at 6, 12, 24, and 36 hpt of angelica tenuissima-nakai or pbs, and their bronchoalveolar lavage fluid (balf) and small-intestinal fluids (sif) were collected as described below. balf: balf was collected as previously described with some modifications (viana et al., 2002) . briefly, lungs of mice were lavaged four times with 1 ml of hank's balanced salt solution (hbss), collected and immediately stored at -20°c and later subjected to elisa (murine iga, il-6, ifn-λ). sif: small intestines of mice were collected as previously described (lefrançois and lycke, 2001) . briefly, mice were euthanized by cervical dislocation; midline incision was performed and retracted the skin. small intestine was cut ~1 cm above cecum to separate it from others. then, the intestine was flushed very carefully with 500 μl of hbss (sigma-aldrich), centrifuged and fluids were immediately stored at -20°c. the sif supernatants were further assayed by the elisa (murine iga). a reversed-phase high performance liquid chromatography (hplc) system was an agilent technologies 1260 infinity (agilent technologies co.) having g1311c quaternary pump, g1329b auto-sampler, g1316a column oven, g1365c multiple wavelength detector a pump l-2130. the column was carried out on a zorbax eclipse xdb-c18 column (150 × 4.5 mm i.d. s-5 um) and the column oven temperature was kept at 30°c. the mobile phase consisted of 1% formic acid (solvent a) and acetonitrile (solvent b) in the gradient mode as: 0-12 min 0-20% b; 12-15 min 20-50% b; 15-20 min 50-58% b; 20-25 min 58-100% b; 25-30 min 100-0% b. the flow rate was 1.0 ml/min, and the elution was monitored at 280 nm. fraction (fr)-5 (later identified and confirmed as ferulic acid) was purified by a semi prep-hplc agilent technologies 1260 infinity (equipped with a fraction collector, fc203b, gilson) using ymc-pack ods-a (250 × 10 mm i.d. s-5 μm, 12 nm) (ymc) at a flow rate of 3 ml/min, with constant temperature of 30°c. purified fractions were further subjected to antiviral assays and cytokine inducing ability in immune (raw264.7) cells using an effective dose of 5.0 μg/ml. antiviral activity against pr8-gfp in raw264.7 cells, virus titration and induction of cytokine secretion were determined according to the protocols described in 'materials and methods.' the viral titers were determined by standard plaque assays using vero cells (coil and miller, 2004) . to assay the viral replication, culture supernatants were collected from vsv-gfp and h3-gfp infected cells both at 12 and 24 hpi and viral titer was determined in vero cells. in the case of pr8-gfp titration, cells were collected at 24 hpi and subjected to five cycles of freezing at -70°c and thawing at rt and titer was determined in vero cells. viral titers for ev-71, brv, and hrv were measured by the median tissue culture infectious doses (tcid 50 ) using hela cells (wilden et al., 2009 ) with some modifications. briefly, supernatants collected from the infected cells were used to infect hela cells cultured in 96-well microtiter plates with 10-fold serial dilutions (50 μl/well). after 2 hpi, dmem (10% fbs) containing tpck trypsin (thermo scientific) was added to the infected wells and incubated for additional 2 days. viral cpe were observed daily and titers were determined by cpe-tcid50. raw264.7, hek293t and hela cells were cultured in 96well plates (2.5 × 10 4 cells/well, 2 × 10 4 cells/well and 2 × 10 4 cells/well, respectively) and incubated at 37°c in a 5% co 2 atmosphere. after 12 h, two-fold serially diluted angelica tenuissima nakai (50 μl/well) was added. at 12 hpt, the cells were infected using dmem containing 1% fbs. raw264.7 cells were infected with pr8-gfp (moi = 1.0) or vsv-gfp (moi = 1.0), hek293t cells were infected with vsv-gfp (moi = 0.2) or hsv-gfp (moi = 3.0) and hela cells were infected with coxsackie-gfp (moi = 3.0) or ev-71 (moi = 0.5) viruses. at 2 hpi, the culture medium was renewed. gfp expression was measured at 24 hpi with the glomax multi-detection system (promega). the ec 50 values were then calculated as the angelica tenuissima nakai concentration yielding 50% gfp expression. the experiments were performed in triplicate. the cc 50 was assessed in a cell viability assay through the trypan blue exclusion (strober, 2001) . the assay was performed using 72-well tissue culture plates. increasing concentrations (1-160 μl/ml or 0.1-16 μg/ml) of the water extract were added to confluent raw264.7, hek293t and hela cell monolayers. clarified cells were stained with 0.4% trypan blue stain (invitrogen) (ratio: 1:1) at 24 hpt and mounted to a hemocytometer to get the percentages of viable cells. cc50 was calculated as the concentration of the extract resulting in 50% cell viability. the experiment was performed in triplicate. all the experiments were repeated in triplicate and the data were assessed as the means ± standard deviation (sd). statistical significance was evaluated using a student's t-test or one-way analysis of variance (anova), and was considered significant with p<0.05 (*p<0.05 and **p<0.01). results for percent initial body weight were also compared by student's t-test. comparison of survival was done by log-rank test using graphpad prism 6 version. to determine the antiviral effects of angelica tenuissima nakai in epithelial cells, we checked the antiviral activity in hek293t and hela cells. in the hek293t cells, the antiviral activity was determined with gfp-fused vsv or hsv upon pre-treatment with the extract (1.0 μg/ml [10 μl/ml or 1% v/v]). upon pre-treatment ( fig. 1a and b), gfp expression was significantly reduced in hek293t cells in comparison with the untreated groups and showed reduced viral titers by nearly 6-fold or 5-fold against vsv-gfp and hsv-gfp, respectively at 24 hpi. moreover, a significant reduction in cell death was observed in angelica tenuissima nakai extract-treated hek293t cells compared with the untreated cells. also, we examined the antiviral activity of angelica tenuissima nakai in the hela cells against gfp-fused coxsackie virus (h3-gfp) and enterovirus-71 (ev-71). pre-treated cells with angelica tenuissima nakai exhibited markedly reduced h3-gfp expression and reduced viral titers by nearly 4.6fold at 24 hpi, together with a significant reduction in cell death (fig. 1c ). in addition, ev-71-induced cpes were markedly inhibited upon pre-treatment with angelica tenuissima nakai (fig. 1d ), resulting in a low level of viral replication. collectively, these results indicate that the total aqueous extract of angelica tenuissima nakai reduces the replication of rna and dna viruses in epithelial cell lines. to investigate the antiviral effects in immune cells, we first assessed the replication of divergent gfp-expressing viruses that were treated or untreated with cytotoxic-free (data not shown) angelica tenuissima nakai in raw264.7 cells. a total aqueous extract of angelica tenuissima nakai-treated (1.0 μg/ml [10 μl/ml or 1% v/v]) raw264.7 cells exhibited markedly reduced gfp expression; however, the untreated groups had high levels of gfp expression for vsv ( fig. 2a) and pr8 (fig. 2c) . when quantitated, a significant reduction in the gfp expression was observed in the extract-treated cells compared to untreated groups ( fig. 2b and d middle panel). these findings further correlated with the observed viral titers from vsv-gfp or pr8-gfp infected cells (fig. 2b and d right panel) . interestingly, the angelica tenuissima nakai-treated cells had a ≥75% cell viability within 24 hpi for all the tested viruses compared with the untreated cells, which were shown to have significantly higher cell death following virus infection ( fig. 2b and d left panel) . consequently, these results also suggest that the aqueous extract of angelica tenuissima nakai could significantly inhibit the replication of vsv and pr8 viruses in immune cells. we then developed an improved gfp assay to determine the ec 50 values of angelica tenuissima nakai against divergent viruses in vitro using hek293t and raw264.7 cells (magadula and suleimania, 2010; lin et al., 2014) . the ec50 is the extract concentration that results in a 50% reduction in virus, and the extract concentration that results in 50% cell viability is the cc50. because we primarily used gfp-fused viruses, a 50% reduction in gfp expression was considered equivalent to a 50% reduction in virus titer. in the case of ev-71, a 50% reduction in cpe was considered equivalent to a 50% reduction in virus titer. the aqueous extract of angelica tenuissima nakai inhibited the replication of vsv-gfp (moi=0.2) and hsv-gfp (moi=3.0) by 50% at an ec50 of 0.34±0.04 μg/ml and 0.41±0.12 μg/ml, respectively in hek293t cells (table 1 ). in raw264.7 cells, vsv-gfp (moi=1.0) and pr8-gfp (moi =1.0) by 50% at an ec50 of 0.58±0.08 μg/ml and 0.44±0.12 μg/ml, respectively (table 1 ). in addition, the observed ec 50 values against coxsackie-gfp (moi=3.0) and ev-71 (moi =0.5) were 0.31±0.03 μg/ml and 0.24±0.02 μg/ml, respectively, in hela cells. given its effectiveness and convenience during the experiments, 1.0 μg/ml has been chosen as the optimal dose of angelica tenuissima nakai for further in vitro antiviral assays, observed from ec 50 values. following treatment with various concentrations, a cell viability test was performed to assess the cytotoxicity of angelica tenuissima nakai extract. the extract had a cc50 of 3.47±0.24 μg/ml, 4.34±0.62 μg/ml, and 5.77±0.14 μg/ml in hek293t, raw264.7 and hela cells, respectively (table 1) . the selection indexes (sis) of angelica tenuissima nakai for vsv and hsv on hek293t cells were 10.2 and 8.5, respectively; those for vsv and pr8 on raw264.7 cells were 7.4 and 9.8, respectively; and those for coxsackie and ev-71 on hela cells were 18.6 and 24.0, respectively. notably, the sis of angelica tenuissima nakai were several magnitudes higher for the tested viruses in their respective cell lines, a clear indication of the extract's broad prophylactic and therapeutic potential. because angelica tenuissima nakai possesses antiviral activity, we hypothesized that angelica tenuissima nakai might be involved in the type i ifn signaling pathway. to test this hypothesis, we first evaluated the levels of interferon-β (ifnβ) and the pro-inflammatory cytokines in the extract-treated cell supernatant of hek293t or raw264.7 cells (fig. 3) . angelica tenuissima nakai (1.0 μg/ml [10 μl/ml or 1% v/v]) induced high levels of secreted ifn-β and il-6 in hek293t cells (fig. 3a) and tnf-α, il-6, and ifn-β in raw264.7 cells at both 12 hpt and 24 hpt (fig. 3b) . importantly, the secreted cytokine amounts were significantly higher in hek-293t cells, although the secreted level was not as high as the levels observed in the raw264.7 cells in comparison with the non-treated groups. these results suggest that the aqueous extract of angelica tenuissima nakai can induce the secretion of ifns and pro-inflammatory cytokines, which can stimulate the cellular antiviral state for inhibition of viral replication. further, we checked the phosphorylation of interferon-related signal molecules or p65 molecule related to nf-kb activation to observe correlation of above results with the ifninducing signaling pathway. for this study, immunoblot analyses were performed using whole-cell lysates of the angelica tenuissima nakai-treated (1.0 μg/ml [10 μl/ml or 1% v/v]) raw264.7 cells. as shown in fig. 3c , we found that the significant upregulation of type i interferon or nf-kb related signal molecules (irf3, stat1, tbk1, p65, and p38) in angelica tenuissima nakai-treated cells such as lps-treated cells. taken together, these results clearly indicate that the aqueous extract of angelica tenuissima nakai can induce the secretion of ifns and pro-inflammatory cytokines through the activation of signal molecules in the type i ifn and nf-kb signaling pathway. given that the angelica tenuissima nakai extracts were able to secrete ifn-β and pro-inflammatory cytokines, we further confirmed an interaction between angelica tenuissima nakai and the transcriptional levels of antiviral genes or interferonstimulatory genes (isgs) in vitro. as confirmed by real-time pcr assay, the mrna expression levels of various antiviral and interferon stimulatory genes in the cells treated with angelica tenuissima nakai (1.0 μg/ml [10 μl/ml or 1% v/v]) were up-regulated to levels similar to those found with the ifn-β-treated positive controls (fig. 4) . also, to determine the transcription levels of various antiviral genes in the angelica tenuissima nakai-treated hek293t cells (fig. 4a) , an ifn-β real-time pcr assay was performed from 0 to 24 hpt to monitor the time-dependent mrna changes. interestingly, the highest fold inductions of cellular transcriptional levels were observed at 12 hpt for all the tested primers (the primers are listed in table 2 ). in hek293t cells, upon treatment of angelica tenuissima nakai extract, transcriptional levels for ifn-β, mx-1, gbp-1, tnf-α, il-8, and il-6 were up-regulated by 7.64-fold, 5.6-fold, 4.3-fold, 10.1-fold, 2.6-fold, and 6.9-fold, respectively (fig. 4a) . similarly, transcriptional activation patterns were also observed in the angelica tenuissima nakai-treated raw264.7 cells. the results showed that the extract-treated cells displayed a nine-fold increase in the level of ifn-β mrna at 8 hpt and a thirteen-fold induction at 12 hpt compared with the untreated cells (fig. 4b) . hence, we performed a pcr assay for other genes of interest at 0, 8, and 12 hpt using specific primers (the primers are listed in table 3 ) in raw264.7 cells. we found that various antiviral gene tran-scriptional levels of were to be up-regulated by angelica tenuissima nakai at 8 hpt, including mx1, oas-16, pml, pkr, il-6, isg-20, and isg-56, to levels that were 3.1-fold, 3.9fold, 5.4-fold, 6.5-fold, 8.2-fold, 3.2-fold, and 3.3-fold, respectively, higher than those of the control (fig. 4b) . thus, overall results suggests that angelica tenuissima nakai extracts were able to up-regulate the transcriptional levels of ifn-β, isgs, and various antiviral related genes in hek293t and raw264.7 cells which can induce antiviral state. to study the angelica tenuissima nakai induced prophylactic interferon-mediated antiviral activities of angelica tenuissima nakai 65 after challenge, the control (pbs) mice groups were suffered severe illnesses and the body weights loss significantly. by approximately 3-4 dpi, most of the infected mice in the con-trol groups displayed severe clinical signs of respiratory disease, including labored respirations and respiratory distress. moreover, the control groups succumbed to death by 11 dpi for all of the viruses tested. however, the angelica tenuissima nakai-inoculated mice showed a ≤20% body weight loss between 4 and 7 dpi and had begun to regain by 8 dpi, returning to their normal state by 12-13 dpi (fig. 5) . besides, all the angelica tenuissima nakai-inoculated groups had higher survival rates: 100% survival for the groups infected with h5n2 (fig. 5b) , h7n3 (fig. 5c) , and h1n1 (fig. 5a) ; and 80% survival infected with h9n2 (fig. 5d ). the surviving mice in these groups did not show obvious clinical signs, except for negligible weight loss. because influenza virus is transmitted primarily by aerosols via the respiratory system of mice and replicated most . 6 . induction of immune state by angelica tenuissima nakai in balb/c mice. five-week-old female balb/c mice were orally administered 0.1 mg/ml angelica tenuissima nakai (atn) at a total volume of 200 μl (20 μg/head) at 1, 3, and 5 days before the collection of samples. control groups were orally administered with 200 μl of pbs. three mice from each group were randomly selected at 6, 12, 24, and 36 hpt of angelica tenuissima nakai or pbs, and their bronchoalveolar lavage fluid (balf) and small-intestinal fluid (sif) were collected and analyzed for the presence of secreted murine (a) il-6 or ifn-λ, (b) immunoglobulin a (iga). the test was performed in duplicate for iga and ifn-λ and triplicate for il-6. the data shows representative means ± sd of each cytokine measured over time (*p<0.05 indicates a significant difference between groups compared by student's t-test). efficiently in lungs (bouvier and lowen, 2010) , it is important to examine the ability of angelica tenuissima nakai extracts to inhibit viral replication in lungs. to evaluate the ability of angelica tenuissima nakai to inhibit viral replication in the h1n1 infected lung tissues, three mice from each group were sacrificed, and their lungs were collected at 3 and 5 days post-infection for viral titration. in comparison of control groups, h1n1 virus replicated efficiently in the lungs with a viral titer of 4.33 log tcid 50 /lung and 5.35 log tcid 50 /lung on 3 and 5 dpi, respectively (fig. 5e) . comparatively, the viral loads in the angelica tenuissima nakai-treated groups were significantly lower: 2.69 log tcid 50 /lung and 2.05 log tcid50/lung, at 3 dpi and 5 dpi, respectively (fig. 5e ). consequently, inoculation of angelica tenuissima nakai extracts induce the sufficiently strong inhibition of viral replication and endorsed the survival of mice against diverse influenza a subtypes lethal infections. to observe the immune status in balf of angelica tenuissima nakai inoculated balb/c mice, we measured the level of the cytokines and iga in the balf and sif. after inoculation with angelica tenuissima nakai extracts into mice, we detected significant level of il-6 and ifn-λ which has antiviral function such as type i ifns, at each time point starting from 6 to 36 hpt in the balf. and also, we detected the presence of iga in balf and sif in all the tested samples. however, minor levels of iga secretion were detected in all the samples from the pbs-treated mice (fig. 6b) . these results indicate that oral inoculation of angelica tenuissima nakai extracts stimulate mucosal immune cells of small intestine and ultimately induce the strong inhibition of influenza virus replication in bronchoalveolar through the mucosal immune response system. for the main component profile of the water extract of angelica tenuissima nakai, we used a hplc system. a reversed-phase hplc method was employed with a mobile phase of 1% formic acid (solvent a) and acetonitrile (solvent b) in the gradient mode as follows: 0-12 min, 0-20% b; 12-15 min, 20-50% b; 15-20 min, 50-58% b; 20-25 min, 58-100% b; and 25-30 min, 100-0% b. the flow rate was kept constant at 1.0 ml/min for the total run time, and 11 fractions were purified successfully. we then evaluated the antiviral effect of the identified fractions (fr-1 to fr-11) in raw264.7 cells. viral replication was monitored with pr8-gfp in response to pre-treatment of various fractions. an effective dose of 5.0 μg/ml was chosen on the basis of our preliminary experiments on the efficacy of purified fractions (data not shown). interestingly, treatment with fr-5 markedly inhibited the virus replication (fig. 7c) . of 11 fractions, fr-5 was detected at a wavelength and retention time of 325 nm and 16.33 min, and the concentration of fr-5 in 6.0 mg/ml (w/v) of angelica tenuissima nakai extract was found to be 5.48 μg/ml ( fig. 7a and b) . ultimately, fr-5 was confirmed the ferulic acid by lc-mass. the ferulic acidtreated cells displayed reduced gfp expression and reduced viral titers compared with the untreated cells. ferulic acid treatment reduced the viral titers by nearly 5.2-fold against pr8-gfp at 24 hpi (fig. 7c) . furthermore, treatment with ferulic acid (5.0 μg/ml) markedly increased cytokine secretion in the raw264.7 cells (fig. 7d) . therefore, our results strongly suggest that ferulic acid, a major constituent of angelica tenuissima nakai, might be able to induce the antiviral state in cells and subsequent inhibition of virus replication. historically, natural products and their derivatives have been invaluable sources of therapeutic agents. recent technological advances, coupled with unrealized expectations of lead-generation strategies, have led to renewed interest in natural products in drug discovery (koehn and carter, 2005) . plants have a long evolutionary history of developing resistance against viruses and have increasingly drawn attention as potential sources of antiviral drugs. for instance, many plant extracts and compounds of plant origin have been shown to have activity against influenza viruses (park, 2003; koehn and carter, 2005) . at present, plant and herb resources are unlimited, have provided mankind with remedies for many infectious diseases and continue to play a major role in primary health care as therapeutic remedies in developing countries. here, we evaluated the antiviral activity of angelica tenuissima nakai in vitro and in vivo. despite the known biological properties of the plant, no study has been conducted on the antiviral activity and the underlying mechanism of action. first, we found that the total aqueous extract of angelica tenuissima nakai displayed a broad spectrum of antiviral properties in vitro. this herb has been used for a variety of purposes for a long time, and no adverse effects of its use have been reported. notably, angelica tenuissima nakai did not show any significant cytotoxic effect on the tested cell lines. its cell cytotoxic concentration (cc 50 ) was several magnitudes higher than the effective concentration (ec 50 ) of the tested viruses, and the sis of the herb for various viruses indicate a higher safety margin of the extract for therapeutic or prophylactic purposes (table 1 ). in examining the in vitro antiviral activity, we determined that angelica tenuissima nakai inhibited the replication of influenza (fig. 2c) , vsv (figs. 1a and 2a) , hsv (fig. 1b) , coxsackie (fig. 1c) and ev-71 (fig. 1d ) viruses in immune (raw264.7) and epithelial (hek293t and hela) cells. upon viral infection, host cells initially recognize an infection and quickly evoke antiviral innate immune responses, including secretion of type i ifns and pro-inflammatory cytokines (takeuchi and akira, 2007) . secreted ifns and cytokines induce an antiviral state, which is important to protect the host cells against invading viruses (tenoever et al., 2007) . induction of an antiviral state at an early stage of virus infection is critical to control the spread and pathogenesis of viruses (boasso, 2013) by specific agents could be effective approaches for limiting viral infection (jackson, 2012; perlman, 2012) . likewise, we hypothesized that angelica tenuissima nakai induces an antiviral state via the induction of type i interferons and proinflammatory cytokines, and we determined the induction of antiviral, ifn-stimulated genes (fig. 4 ) and the secretion of ifn-β and il-6 ( fig. 3) by angelica tenuissima nakai in vitro. to elucidate the features in antiviral signaling, we also evaluated the effect of angelica tenuissima nakai on the phosphorylation of irf-3, p65, tbk1, stat1, erk, and p38, which are key signaling molecules in the type i ifn and nf-κb signaling pathways. upon stimulation of the pattern recognition receptors (prrs) or unknown receptors of the host cell by foreign materials containing pathogens or diverse ligands, downstream signal transduction is activated, including the activation of adaptor signal molecules or transcriptional factors, and can initiate the induction of type i ifns and pro-inflammatory cytokines to up-regulate the antiviral status of the host cell (mogensen, 2009) . in this study, we found that angelica tenuissima nakai extracts treatment can induce the phosphorylation of irf-3, stat1, and tbk1 in a time-dependent manner, providing evidence of downstream signal transduction in the type i ifn signaling pathway (fig. 3c) . additionally, the activation of nf-κb (p65, p38), which leads to a strong secretion of pro-inflammatory cytokines, was also observed (fig. 3c ). this phosphorylation could lead to the rapid production of type i ifns and various inflammatory cytokines that play a crucial role in stimulating the antiviral state and the subsequent clearance of viruses (price et al., 2000) . oral administration of angelica tenuissima nakai extracts not only increased the survival rate of mice subjected to lethal challenges with divergent influenza a subtypes, including h1n1, h5n2, h7n3, and h9n2, but also led to rapid weight recovery (fig. 5 ). mice treated with the abstract initially displayed little weight reduction; ultimately, the majority did not lose more than 25% of their body weight. by contrast, all of the mice in the control groups displayed weight loss of more than 25% within 7 dpi and were humanely killed. influenza virus causes a rapid reduction in the body weight of infected mice; hence, 25% body weight loss is considered the humane end point for sacrificing influenza virus-infected mice . in addition, lung virus titration (fig. 5e ) correlated with survival, and the extract-treated mice displayed reduced lung viral titers, suggesting that angelica tenuissima nakai is sufficiently potent to inhibit viral replication and promote the survival of mice against lethal infections of diverse influenza a viruses. to support the antiviral effects of angelica tenuissima nakai in vivo, we observed the immune status in balf of angelica tenuissima nakai inoculated balb/c mice. as shown in fig. 6 , after inoculation with angelica tenuissima nakai extracts into mice, we could detect significant level of il-6 and ifn-λ in balf. previous literature has indicated that elevated levels of serum il-6 correlate with induction of the antiviral state and therefore play an important role in the inhibition of virus replication (spellberg and edwards, 2001; melchjorsen et al., 2003) . and ifn-λ which is an exciting new chapter in the field of ifn research, is also able to activate the same intracellular signaling pathway and many of the same biological activities as those displayed by type i ifns, including antiviral activity, in a wide variety of target cells (lazear et al., 2015) . moreover, we also found significant level of secreted iga (siga) in balf and sif, after inoculation with angelica tenuissima nakai extracts. secretory iga (siga) is the most abundant immunoglobulin in body secretions, including saliva, tears, colostrum and gastrointestinal secretions, and is the main effector of the mucosal immune system, providing an important first line of defense against most pathogens that invade the body at the mucosal surfaces (woof and mestecky, 2005; mantis et al., 2011) . thus, significant level of il-6, ifn-λ or siga which induced in balf by angelica tenuissima nakai extracts, maybe correlate with the higher survival rates observed in the balb/c mice against divergent influenza a infection. macrophages have been widely used in drug development because of their significant role in the immune system, especially in antiviral responses (wilden et al., 2011; kim et al., 2013) . here, we used murine macrophages (raw264.7) to evaluate the antiviral effect of angelica tenuissima nakai in vitro. in addition, we used epithelial cells such as hek293t and hela, which are very susceptible to viral replication. it was recently reported that hek293t (graham et al., 2014) and hela (kolli et al., 2013) cells have fewer prrs than tolllike receptors (tlrs). this finding means that an aqueous extract of angelica tenuissima nakai may contain active components that can bind or penetrate the cell membrane. those active components may stimulate the cell surface prrs, cytoplasmic prrs, or both, and ultimately induce antiviral immune responses in immune or epithelial cells. surely, we tested angelica tenuissima nakai for endotoxin contamination using a limulus amebocyte lysate (lal) assay and found that it was not contaminated with endotoxin (data not shown). there have been phytochemical reports on various types of compounds from angelica tenuissima nakai, including phthalides, coumarins, terpenoids, and phenylpropanoids (islam et al., 2009; nam et al., 2014) . some of these compounds were found to have diverse biological activities, such as antioxidative (ka et al., 2005) , anticancer (park et al., 2011) , angiotensin converting enzyme (ace) inhibitory (liang et al., 2011) and antifungal effects . to confirm these reports, we conducted hplc analysis to identify the active compounds present in the total aqueous fraction. we observed 11 major peaks (fig. 7a) , and 11 fractions (fr-1 to fr-11) were successfully purified. of the 11 fractions, fr-5 was identified as having significant antiviral properties, like those displayed by angelica tenuissima nakai; fr-5 was later confirmed to be ferulic acid (fig. 7a) . here, we newly defined the antiviral effects of ferulic acid via type i ifn stimulation. thus, the antiviral and immunomodulatory effects of an aqueous extract of angelica tenuissima nakai might be due to a cumulative effect of ferulic acid including with other fractions or other unknown active compounds present in the extract. the relationship between the mechanisms of the antiviral effects and active compounds, including ferulic acid, should be studied further. interferon-mediated antiviral activities of angelica tenuissima nakai 69 in summary, we demonstrated that the aqueous extract of angelica tenuissima nakai can be a major alternative antiviral agent. the aqueous extract of angelica tenuissima nakai inhibit the diverse viral infection through the induction of type i ifn signaling and pro-inflammatory cytokines, leading to an antiviral state in epithelial and immune cells. the aqueous extract of angelica tenuissima nakai can reduce influenza-induced mortality by disrupting viral replication or preventing viral infection by creating an antiviral state in in vivo (lungs). given the antiviral activities, angelica tenuissima nakai extracts or its components, including ferulic acid may be used as a preventive or therapeutic agent to limit viral replication which have been caused a serious damage to human or livestocks. furthermore, angelica tenuissima nakai may also be used as a target for drug design to prevent the viral infection through the innate immune responses. prasanna weeratunga, md bashir uddin designed and executed all cell biological experiments; myun-soo kim, byeong-hoon lee, ji-eun yoon performed all virus infection experiments, analyzed the data; jin yeul ma, hongik kim analyzed the data. jong-soo lee designed the overall study and wrote the paper. the animal study was conducted under appropriate conditions with the approval of the institutional animal care and use committee of bioleaders corporation, daejeon, korea. protocol number: bsl-abls-13-008. none of the authors have any financial or personal relationships with other people or organizations that could inappropriately influence or bias this study. type i interferon at the interface of antiviral immunity and immune regulation: the curious case of hiv-1 animal models for influenza virus pathogenesis and transmission phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus phytochemical screening and antimicrobial activity of the plant extracts of mimosapudica l. against selected microbes treatment with a toll-like receptor inhibitory gpg oligonucleotide delays and attenuates lupus nephritis in nzb/w mice characterization of the anti-influenza activity of the chinese herbal plant paeonia lactiflora a validated lc method for 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macrophages middle east respiratory syndrome coronavirus (mers-cov). world health organization expression of rig-i, irf-3, ifn-ß, and irf-7 determines resistance or susceptibility of cells to infection by newcatle disease virus important role of interferon regulatory factor (irf)-3 in the interferon response of mouse macrophages upon infection by newcastle disease virus mucosal immunoglobulins in vitro and in vivo antifungal activities of decursin and decursinol angelate isolated from angelica gigas against magnaporthe oryzae, the causal agent of rice blast an m2e-based multiple antigenic peptide vaccine protects mice from lethal challenge with divergent h5n1 influenza viruses the authors thank dr. j. u. jung of the university of southern california, usa for providing green fluorescence key: cord-001365-6u80p5sj authors: weger-lucarelli, james; chu, haiyan; aliota, matthew t.; partidos, charalambos d.; osorio, jorge e. title: a novel mva vectored chikungunya virus vaccine elicits protective immunity in mice date: 2014-07-24 journal: plos negl trop dis doi: 10.1371/journal.pntd.0002970 sha: doc_id: 1365 cord_uid: 6u80p5sj background: chikungunya virus (chikv) is a re-emerging arbovirus associated with febrile illness often accompanied by rash and arthralgia that may persist for several years. outbreaks are associated with high morbidity and create a public health challenge for countries affected. recent outbreaks have occurred in both europe and the americas, suggesting chikv may continue to spread. despite the sustained threat of the virus, there is no approved vaccine or antiviral therapy against chikv. therefore, it is critical to develop a vaccine that is both well tolerated and highly protective. methodology/principal findings: in this study, we describe the construction and characterization of a modified vaccinia virus ankara (mva) virus expressing chikv e3 and e2 proteins (mva-chik) that protected several mouse models from challenge with chikv. in particular, balb/c mice were completely protected against viremia upon challenge with chikv after two doses of mva-chik. additionally, a129 mice (deficient in ifnα/β) were protected from viremia, footpad swelling, and mortality. while high anti-virus antibodies were elicited, low or undetectable levels of neutralizing antibodies were produced in both mouse models. however, passive transfer of mva-chik immune serum to naïve mice did not protect against mortality, suggesting that antibodies may not be the main effectors of protection afforded by mva-chik. furthermore, depletion of cd4(+), but not cd8(+) t-cells from vaccinated mice resulted in 100% mortality, implicating the indispensable role of cd4(+) t-cells in the protection afforded by mva-chik. conclusions/significance: the results presented herein demonstrate the potential of mva to effectively express chikv e3-e2 proteins and generate protective immune responses. our findings challenge the assumption that only neutralizing antibodies are effective in providing protection against chikv, and provides a framework for the development of novel, more effective vaccine strategies to combat chikv. chikungunya virus (chikv; togaviridae, alphavirus), the etiologic agent of chikungunya fever is an emerging pathogen that has recently caused several severe outbreaks throughout africa and southeast asia [1] [2] [3] . large outbreaks have occurred on la réunion island (an overseas department of france), mauritius, sri lanka, and india, among others [4] . in addition, autochthonous transmission has been seen in europe, with outbreaks occurring in both italy and mainland france [5] , suggesting temperate climates can support virus transmission. furthermore, as of january 2014, the european centre for disease control has confirmed 70 cases of chikv on the caribbean islands of st. martin, martinique, guadeloupe, and saint barthelemy, with many more suspected, indicating spread to continental america is possible. chikv is transmitted to humans by aedes aegypti and aedes albopictus mosquitoes, the latter of which has been an important vector in many of the recent outbreaks due to mutations in the envelope genes of the virus that allow for more efficient transmission [6] [7] [8] [9] . chikv causes a dengue-like illness associated with fever, rash and joint pain and was first described in modern day tanzania in 1952 [10] . the term chikungunya is derived from the makonde word meaning ''that which bends up'' and describes the posture of an infected individual [11] . recently, the u.s. army developed a live-attenuated vaccine candidate, called chik 181/clone 25 or 181/25, but it caused transient arthralgia in a small number of volunteers during phase ii clinical trials [12, 13] . experimental subunit [14] , recombinant viruses [15] [16] [17] , and vlp [18] based vaccines have also been described which are currently at various stages of preclinical or clinical development, however there currently are no licensed vaccines or antiviral treatments available for chikv. an alternative approach for developing chikv vaccines is the use of viral vectors. a complex adenovirus expressing the complete chikv structural poly-protein has been described and was shown to be immunogenic and protective in mice [19] . however, safety concerns regarding adenovirus based-vectors may limit this approach [20] . in contrast, modified vaccinia virus ankara (mva) has been tested in over 120,000 humans and was proven to be highly safe and effective in protecting against smallpox [21, 22] . mva was attenuated by over 500 passages of vaccinia virus (vacv) in chicken embryo fibroblasts (cefs), which resulted in large deletions of its genome that restricted its host-range [23, 24] . during passaging, mva lost the ability to productively infect mammalian cells, leading to abortive replication [25] . vacv, which is far more reactogenic than mva [26, 27] , has been used previously against other alphaviruses, namely sindbis and venezuelan equine encephalitis (veev) viruses, providing robust immunity against the latter, suggesting poxviruses as suitable vectors against alphaviruses [28] [29] [30] [31] [32] [33] . despite the presence of high levels of neutralizing antibodies elicited by most of the vacv vectored veev vaccine candidates, they were ineffective in providing protection against airborne infection, suggesting they were unable to elicit a sufficient t cell mediated immune response, which has been shown to be critical for protection against lethal veev encephalitis [34, 35] . mva, like its parent virus vacv, has been extensively tested as a vaccine vector, expressing viral, bacterial or parasite antigens and has been shown to induce both humoral and cell-mediated protective immune responses [25, [36] [37] [38] [39] [40] [41] [42] . furthermore, mva can be delivered effectively by different routes and has much greater stability than most other live virus based vaccine approaches [43] . additionally, because mva undergoes only abortive replication in mammalian cells, vector stability is not a problem as only one infection cycle occurs [25] .therefore, an mva vectored chikv vaccine would be an attractive option for resource-limited countries, where the majority of chikv infections occur. additionally, volz and sutter (2013) have targeted mva as an ideal vector for safe next generation vaccines, with chikv being mentioned specifically as an attractive candidate pathogen [44] . furthermore, garcia-arriaza et al. (2014) successfully used mva for expression of the entire chikv structural protein as a vaccine candidate, indicating mva can provide effective immunity against this virus [45] . in this report, we describe the construction and immunological evaluation of an mva-based chikv candidate vaccine based on the e3-e2 proteins. the vaccine was tested in two mouse modelsone immunocompetent and the other lacking a/b interferon signaling (a129)-and was uniformly protective. mice deficient in a/b interferon signaling have been used for many viruses and have been shown repeatedly to be a good model for chikv, providing many similarities to human infection [46] [47] [48] . complete protection against mortality was provided when mva-chik was administered in a prime-boost regimen. in addition, it provided 80% protection against mortality in this highly immunocompromised mouse model after only 11 days post-vaccination. surprisingly, while playing a role, neutralizing antibodies did not appear to be necessary for protection against chikv, contrary to other recent reports [49, 50] . most importantly, depletion of cd4 + t cells in vaccinated mice resulted in loss of protection, with 100% succumbing to infection upon challenge with wild-type chikv, indicating an indispensable role of mva-chik immune cd4+ t cells in protection. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the iacuc protocol (protocol #v01380) was approved by the institutional animal care and use committees of the university of wisconsin. african green monkey kidney cells (vero, atcc #ccl-81) and baby hamster kidney cells (bhk-21, atcc # ccl-10) cells were maintained in dulbecco's modified eagle medium (dmem; gibco, carlsbad, ca) supplemented with 5% fetal bovine serum (fbs), 100 u/ml of penicillin, 100 mg/ml of streptomycin, and 2.5 mg/ml amphotericin b, and incubated at 37uc in 5% co 2 . chicken embryo fibroblasts (cefs) were obtained from charles river (charles river laboratories international, inc., wilmington, ma) and maintained in optimem (invitrogen, carlsbad, ca) supplemented with 10% fbs under the same conditions. chikungunya virus strain la reunion (chikv-lr; genbank: dq443544.2) was used for both the construction of recombinant poxviruses and challenge experiments. the virus was kindly provided by dr. scott weaver (university of texas medical branch, galveston, texas). the chik-ires vaccine candidate has been previously described [51] and was used in this study as a positive control. briefly, the vaccine was constructed by replacing the subgenomic promoter in a cdna chikv clone with an internal ribosome entry site from encephalomyocarditis virus (emcv-ires) and has shown to be effective and safe in mice [51] and non-human primates [52] . the mva virus used in these studies was obtained through bei resources (niaid, nih,ref # nr-727). a recombinant mva virus expressing gfp (herein called mva-gfp) was based on the wild-type mva and its construction has been previously described [37, 53] . for the production of recombinant mva vaccines, the chikv e2 and e3 genes (hereafter called p62) were pcr amplified using phusion high-fidelity dna polymerase (new england biolabs, ipswich, ma) and cloned into a specially modified poxvirus transfer vector, pi2-red [37] . e2 was chosen because it has been previously demonstrated that most neutralizing antibodies mapped to epitopes in this region [49, 54, 55] . e3 has not been strongly associated with protection; however, it was included to allow proper folding of the e2 protein [56] . the expression of p62 was controlled by a synthetic early/late vaccinia virus promoter and used the vaccinia virus transcription terminator, which have been previously described [57] . the vector used in this study contained flanking sequences that when transfected into mva-gfp infected cells can recombine into the deletion iii region of the viral chikungunya virus (chikv) has recently re-emerged from africa to cause disease outbreaks in asia, europe, and more recently the caribbean. the virus is transmitted by aedes mosquitoes and causes a disease that is characterized by high fever and incapacitating joint pain that can cause great personal and economic loss. at present, no approved vaccine or antivirals are approved against chikv. in this study, we developed a novel chikv vaccine that is vectored by modified vaccinia virus ankara (mva), an attenuated vaccine vector which has been shown to be safe in humans and induce a strong immune response. the vaccine expresses the e3 and e2 proteins of chikv, the latter of which is thought to be the main mediator of protection. the vaccine was effective in two mouse models and protected against all markers of disease tested despite the absence of high levels of neutralizing antibodies, the gold standard of protection. depletion of cd4+ t cells from vaccinated mice resulted in loss of protection, implicating these cells in the protection induced by the vaccine. genome [58] . this vector contains dsred under the control of a late p11 promoter to allow for visual based selection and permits an easy distinction between recombinant (red) and wild-type (green) viruses (fig. 1a) . recombinant mva-chik viruses were generated as described previously [59, 60] . briefly, cefs were seeded into six-well plates the day before transfection and then infected at a multiplicity of infection (moi) of 0.05 pfu/cell for 1.5-2 hours with mva-gfp. cells were then washed with 16 pbs and transfected with appropriate transfer vectors using fugene hd (roche diagnostics, indianapolis, in) following manufacturer's protocol. cells were monitored for the presence of red fluorescence 24 hours after transfection. at 48-72 h post-transfection, monolayers were harvested, centrifuged at 5006g for 5 min at 4uc and cells disrupted by freeze-thaw (3 times) followed by sonication (2 times for 15 s using a cup sonicator). the disrupted cell extracts containing recombinant viruses expressing dsred were plated onto fresh cef cells and overlaid with 0.8% agarose. after 48-72 h, recombinant virus-generated plaques were detected by observing fluorescence and picked into 300 ml media with a sterile filter pipette tip. the cell/virus samples were subjected to freeze thaw and sonication (as described above) and plated to continue passaging. plaques were passaged until no wild-type (gfp expressing) virus was observed at which point pcr was performed to confirm the presence of only recombinant virus [61] . high titer virus stocks were prepared from pcr positive cultures and recombinant mva-chik viruses were further characterized. pcr analysis was performed on final virus stocks to ensure genetic homogeneity and stability. dna was extracted using the quick-gdna miniprep kit from zymo research per manufacturer's instructions (zymo research, irvine, ca). pcr was then performed using phusion polymerase with deletion iii specific primers (forward 59-atgcggcacctctcttaa-39, reverse 59-tgggctccttataccaagca-39). western blot analyses were used to determine the in vitro expression patterns of mva-chik constructs. for this purpose, bhk-21 cells were seeded at 3.0610 5 cells/well into six-well plates and 24 hours (hr) later infected at an moi of 5 pfu/cell in optimem in the absence of fbs. at 24 hr after infection, cells were harvested and lysed using radio-immunoprecipitation assay buffer (ripa; 150 mm nacl, 1.0% np-40, 0.5% sodium deoxycholate, 0.1% sds, and 50 mm tris, ph 8.0) at 4uc for 30 minutes (min) under gentle agitation. lysates were then centrifuged for 20 min at 12,000 rpm and supernatant was collected for further analysis. supernatants were diluted into 26 laemmli buffer (bio-rad, richmond, ca), heated at 95uc for 5 min and 50 ml were loaded into a bio-rad 4-20% precast gel. gels were transferred to a nitrocellulose membrane using trans-blot turbo blotting system following manufacturer's instructions. polyclonal serum obtained from specific-pathogenfree (spf) rabbits inoculated with a vaccine strain of chikv was used to probe blots at a dilution of 1:5000 and developed using bcip/nbt alkaline phosphatase system (bio-rad). bhk-21 cells were used for immunocytochemistry (icc) experiments. one day prior to infection, 2610 5 cells were plated onto glass coverslips, which had been placed inside of 24 well tissue culture plates. the following day, the cells were infected at an moi of 10 pfu/cell. infection was allowed to progress for 24 hrs and then fixed using 2% paraformaldehyde (pfa) solution in phosphate buffer. for permeabilization, cells were treated with 0.5% triton x-100 for 10 min at 4uc, followed by 50 mm nh 4 cl in pbs for 10 min at room temperature (rt), and then blocked with pbs containing 10% bovine serum albumin (bsa) for 3 to 4 hr at rt. cells were then incubated overnight at 4uc with a 1:1000 dilution of anti-chikv polyclonal rabbit serum. cells were subsequently washed three times with pbs containing 0.05% tween and then incubated with a 1:2000 dilution of anti-rabbit igg alexafluor-488 for 45 min at rt. cells were mounted using vectashield mounting medium (vector laboratories). images were acquired using an evos microscope with attached camera. elisas were performed as described by brewoo et al., 2010 [37] . briefly, 96-well elisa plates were coated with purified chikv (0.5 mg in 100 ml carbonate buffer, ph 9.6 per well) at 4uc overnight. coated plates were washed twice with 0.05% tween-20 in pbs (washing buffer) and incubated with blocking buffer (1% bsa in pbs) at rt for 1 hr. serum samples then were serially diluted from 1:100-1:12,800 in elisa diluent (0.1% bsa in washing buffer) and added in triplicate to the prepared elisa plates and plates were incubated at rt for 1 hr. known negative (uninoculated) and positive serum samples from mice inoculated with chik-ires from previous studies were used as controls. after washing, 100 ml per well of a 1:10,000 dilution of horseradish peroxidase (hrp)-conjugated rabbit anti-mouse igm+g (abcam inc, cambridge, ma) was added to each well and incubated at rt for 1 hr. plates were washed, and 100 ml per well of tetra-methyl-benzidine (tmb) chromogen (sigma, st louis, mo) was added to each well and incubated in the dark for 5 min. the reaction was then stopped by adding 100 ml per well of 2 mm h 2 s0 4 . colorimetry was measured using an elx800 absorbance microplate reader (biotek, winooski, vt) at test wavelength of 450 nm and a reference wavelength of 630 nm. the highest dilution that was positive (exceeded the mean of known negative serum samples plus three standard deviations) was considered the endpoint, and its reciprocal value was recorded as the titer. groups of four-to six-week-old female balb/c mice (harlan sprague dawley, indianapolis, in) or six-to ten-week-old mixed the different areas where deletion occurred in the vaccinia virus genome to create mva are shown. mva-chik was created by transfecting the plasmid containing chikv p62 under control of a se/l promoter which also expressed dsred2 under control of a p11 promoter. this plasmid contains flanking regions to deletion iii (deliii) which is where the recombinant gene was inserted into the genome with a fluorescent marker (a). pcr analysis of the deliii region. viral dna was extracted from purified, final virus stocks of mva-chik, mva-gfp and mva-wt. pcr was performed using primers specific for the deliii flanking regions. a dna ladder is included for comparison of size (b). monolayers of cef cells were infected with recombinant mva-chik viruses at moi of 5 or 10 pfu/cell for western blot or immunostaining, respectively. after 24 h post infection, cells were harvested and subjected to sds-page followed by western blot analysis (c) or fixed with 2% pfa as described in the methods. both the cellular pellet and supernatant were analyzed for expression of chikv e3/e2. the order is the same for both and is as follows, wild-type chikv (1), mva-chik (2), mva-gfp (3), and mock infected cells (4) . following fixation, cells for immunostaining were either allowed to remain intact or permeabilized with triton x-100 (d). anti-chikv polyclonal rabbit was used for primary staining for both assays. green represents chikv e2 positive cells, red is dsred protein produced by the virus and blue is nuclear staining with hoechst. doi:10.1371/journal.pntd.0002970.g001 gender a/b interferon signaling deficient mice (a129) received either primary only or primary and booster immunizations (28 days apart) with each vaccine candidate via intradermal (i.d.) injection into the hind, left footpad. a dose of 1610 7 tcid 50 units in 50 ml was used for all mva vaccinations. the dose chosen is either lower than or consistent with many previous reports of mva vectored vaccines [62] [63] [64] . negative control groups were immunized with mva-gfp at the same dose. as a positive control, mice were vaccinated with experimental control vaccine virus chik-ires [17] at a dose of 10 4 tcid 50 units. at either 11 days post-prime or two weeks post-boost (where applicable), all animals were challenged with wild-type chikv-lr by i.d. inoculation of 10 4 (balb/c) or 10 2 (a129) tcid 50 units in 50 ml into the hind, left footpad. mice were bled prior to boost and prior to challenge following vaccination to monitor levels of neutralizing and anti-virus antibodies. passive transfer studies were performed using a129 mice as previously described [37] . briefly, serum was collected from vaccinated balb/c or a129 mice and equal volumes from each sample were used to create a pool of sera for each mouse strain separately. 100 ml (balb/c) or 200 ml (a129) of this inoculum were then injected into naive mice intraperitoneally (i.p.). serum from chik-ires vaccinated a129 mice (100 ml) was used as a positive control. 24 hours after passive transfer, mice were challenged in the same manner as described above. following challenge, mice were bled three days consecutively to monitor viremia via the maxillary vein. with the exception of the balb/c experiment, all animal experiments were repeated at least once, with similar results. for depletion studies, groups of six-to ten-week-old a129 mice the depletion efficiency in pbmcs on the day of infection was more than 99% for both t cell subsets as assessed by flow cytometry by staining with anti-mouse cd4 fitc (rm4-5), antimouse cd8a percp (53-6.7) mabs (from bd bioscience) (data not shown). mice were monitored for morbidity and mortality for two weeks. histopathology was performed on tissues collected from a129 mice 7 days post infection (d.p.i.). footpad and leg tissues were severed from euthanized mice, cut in half to expose the tissues, and fixed in 4% buffered pfa for two days. hind limbs were then decalcified in 4% pfa containing 14% edta for 3-4 weeks, changing decalcification solution weekly. tissues were paraffin embedded, sectioned and stained with hematoxylin and eosin (h&e). pictures were taken using a sony nex-5n camera attached to a nikon microscope. blood samples collected from mice following challenge were used to assess viremia. viremia was assessed via end point dilutions in 96 well plates seeded with vero cells (seeded the night before at 2610 4 cells/well) and was expressed as tissue culture infectious dose 50 (tcid 50 ) per ml. for prime only studies a serum sample was taken ten days post-vaccination to measure neutralizing antibodies. for studies with prime and boost serum samples were collected on days 28 and 42 post-primary vaccination. neutralization titers were determined using a tcid 50 based assay that was modified from grosfeld et al. [65] . briefly, sera were incubated at 56uc for 60 min to inactivate complement and then serially diluted two-fold starting at a dilution of 1 in 5. dilutions were performed in dmem as described above except containing only 2% fbs. subsequently, 100 tcid 50 units of chikv were added to each well in the same dilution media. the virus/serum mixture was then incubated at 37uc for 1 hr. following incubation, 100 ml of the mixture was added to 96 well plates seeded with vero cells and monitored for 3-4 days for presence of cytopathic effects (cpe). t cell responses were analyzed as previously described [50, 53] . briefly, a129 mice were euthanized 2 weeks post-boost. after red blood cell (rbc) lysis, single-splenocyte suspensions were resuspended in rpmi-1640 medium supplemented with 10% fbs 16 penicillin/streptomycin and 0.14 mm of b-mercaptoethanol. splenocytes were stimulated with three chikv peptide pools that cover the entire protein sequences of e3, e2 and nsp2, respectively. the peptide pools were used at a concentration of 1 mg/well separately in 200 ml total volume for 16 h in the presence of brefeldin a. peptide pools were a generous gift from dr. daniel streblow [66] and were synthesized by thermo fisher scientific. cells were stained intracellularly for ifn-c apc (xmg1.2), il-2 pe (jes6-5h4), tnf-a pe (mp6-xt22) and cd40l (mr1) after surface staining of cd4 fitc (rm4-5) or anti-mouse cd8a percp (53-6.7). the samples were acquired on a bd facscalibur and analyzed with flowjo v7.6.5 (tree star). graphpad prism 6 software (la jolla, ca) was used for all statistical analyses. statistical analysis of viremia levels were performed using an unpaired t-test with welch's correction for unequal variance. survival analysis was performed to assess vaccine effectiveness against; reported p-values are from the mantel-cox test. genetic homogeneity of final virus constructs was analyzed by pcr. purified viral dna was used as a template for amplification by primers specific for the flanking regions of deletion iii. the presence of e3/e2 was confirmed in mva-chik by a band at the expected size of roughly 3 kb (fig. 1b) which indicated the maintenance of the insertion throughout plaque selection. mva-gfp (,1500 bp) and wild-type mva (657 bp) were included as controls. in addition, dna sequencing confirmed that no genetic alterations were present in the final construct (data not shown). the expression of p62 antigen by the mva-chik recombinant viruses and wild-type chikv was monitored by immunoblot analysis. cell pellet lysates and supernatants from infected cefs were tested for protein expression (fig. 1c) . the p62 (62 kd) protein was detected at 24 hr p.i. in both wild-type chikv and mva-chik infected cells, but not in mva-gfp or mock infected cells. interestingly, a cleaved e2 protein (,50 kd) was detected in wild-type infected cells, but not in the mva-chik lysates, suggesting the lack of furin cleavage and release of the e3 peptide from the p62 precursor protein. expression of e2 was observed in the supernatant of chikv infected cells, but there prior to boost (where applicable) and challenge, mice were bled and serum was monitored for both total ig(g+m) and neutralizing activity by tcid 50 was no detection of e2 expression in mva-chik, suggesting that the protein was not being secreted. to determine the cellular localization of chikv proteins, mva-chik infected cells were fixed and then probed for production of chikv e3/e2 proteins using polyclonal serum. in order to determine if the e2 protein was reaching the surface of the cells, a single cell set was permeabilized while the other set remained intact. permeabilized cells stained much brighter than those that were not (fig. 1d) . this indicated that chikv e3/e2 proteins were being maintained inside the cell and not reaching the cell surface as is observed with wild-type chikv infection. to evaluate the protective efficacy of the mva-chik vaccine, an active immunization study was conducted in a129 mice. this mouse strain is more sensitive to chikv infection and experience high viremia, footpad swelling and succumb to infection [51] compared to immunocompetent strains of mice. mice were vaccinated as described previously and then challenged with wt-chikv. mva-chik or mva-gfp vaccinated mice had background levels of neutralizing antibodies, whereas all chik-ires vaccinated mice seroconverted (fig. 2a) . serum collected from these mice post-prime and post-boost was tested for total anti-virus ig(g+m) antibodies by elisa. a clear booster effect was seen in mva-chik vaccinated mice, as the differences between post-prime and post-boost samples were highly significant (p,0.001) (fig. 2b) . this difference was not as profound with chik-ires vaccinated mice as they had high levels of anti-virus antibodies even prior to boost. mva-chik and chik-ires groups were protected 100% from lethal challenge after prime and boost (fig. 2c ). in addition, mva-chik mice were completely protected against both viremia and footpad swelling (figs. 2d-e) . next, we compared by histopathology the footpads of mva-chik and mva-gfp vaccinated a129 mice. vaccinated mice were challenged in the same way described above and subsequently were euthanized seven d.p.i. to assess local tissue damage. following infection, footpads from mva-chik vaccinated mice displayed mild inflammation with limited muscle damage (fig. 3a) . in contrast, footpads from mva-gfp mice exhibited severe necrotic muscle degeneration with edema, consistent with previous reports from unvaccinated mice (fig. 3b) [47, 67] . the immunocompetent balb/c mouse model was selected to evaluate the immunogenicity of mva-chik as it has been used previously with mva and chikv [37, 53, 68] . following vaccination, low levels of neutralizing antibodies were detected in mice immunized with mva-chik (fig. 4a) . immunized mice were then challenged with wild-type chikv to determine protection against viremia. viremia was not detected in any of the mva-chik vaccinated mice (n = 6) (fig. 4b) . in contrast, all mva-gfp immunized mice had significant levels of viremia (p = 0.027). to determine whether antibodies present in balb/c (avg. neut. titer of 9.2) or a129 (undetectable neut. titer) vaccinated mice were sufficient for protection against chikv, pooled immune serum was passively transferred into naïve a129 mice. as a positive control, an additional group of mice was treated in the same manner with a pool of serum (avg. neut. titer of 64) from a129 mice vaccinated with chik-ires candidate vaccine. all of the mice treated with either anti-mva-chik or mva-gfp immune serum had high levels of viremia and succumbed to infection (fig. 4c-d) . viremia in mice passively transferred serum from mva-chik vaccinated a129 was slightly reduced on day 2 post-infection, however this was not significant (p = 0.11). in contrast, chik-ires immune serum provided full protection against viremia and mortality (figs. 4c-d) . to determine the role of t cells in protection we first investigated whether mva-chik could elicit a t cell response in a129 mice by measuring cytokine production. upon in vitro restimulation with a chikv peptide pool that covers the protein sequence of e2, mva-chik immune cd4 + t cells produced the cytokine ifnc and upregulated co-stimulatory molecule cd40l at significantly higher levels as compared to mva-gfp vector control (p = 0.02 and 0.01, respectively) ( fig. 5a-b) . no response was observed when mva-chik splenocytes were stimulated with peptide pools against either e3 or nsp2 (viral protein negative control) (fig. 5c-d) , suggesting the immune response is specific to e2. in addition, there were no significant differences in other cytokines monitored, including tnfa, il-2, il-4, il-6, il-10, and il-17a (data not shown). furthermore, none of the cytokines tested showed a difference between the two groups in immune cd8+ t cells (data not shown). similar results were obtained when mva-chik immune splenocytes were stimulated with live or inactivated whole virus preparations (data not shown). to test whether mva-chik-specific immune cd4 + or cd8 + t cells were indeed protective, we depleted cd4 + or cd8 + t cells from vaccinated mice prior to challenge with wt-chikv. mice depleted of cd8 + t cells or control non-depleted mice had no footpad swelling and survived the challenge (fig. 6a-b) . in contrast, cd4 + depleted mice along with mva-gfp controls succumbed to infection (fig. 6b) . mice depleted of cd4 + t cells also had high levels of viremia and footpad swelling, similar to the mva-gfp controls (fig. 6a-c) . interestingly, cd8 + t cell depleted vaccinated mice remained healthy without footpad swelling throughout the duration of the study despite developing a low level of viremia three d.p.i. the recent re-emergence of chikv has resulted in several explosive outbreaks and underscores the need for an effective vaccine. several groups have recently employed varying strategies to construct effective vaccines against chikv. live-attenuated, inactivated, adenovirus vectored, dna, and vlp based strategies all have been tested and have been shown to be effective in providing protection against chikv [12, 13, 68, 69] . despite promising results, these vaccine candidates all have some drawbacks. safety, stability, and production of a broad long-lived immune response are necessary for any effective vaccine. safety concerns remain for both live-attenuated and adenovirus based approaches. inactivated and vlp based approaches require adjuvants and normally elicit weak t cell responses. in contrast, mva has been shown to be safe in thousands of human patients micro-neutralization assay (a) and elisa (b), respectively. prime only mva-gfp data is not shown because it does not differ with the prime & boost groups. mice were monitored for 14 days following challenge for survival (c) and footpad swelling (d). the first three days following challenge viremia levels were measured via tcid 50 (e). the dotted line indicates the limit of detection of the assay. doi:10.1371/journal.pntd.0002970.g002 and also in every animal tested [21, 22, 58] . furthermore, mice deficient in a/b/c interferon signaling experienced no clinical symptoms following vaccination with mva-chik (data not shown) and we have previously shown other mva constructs to be safe in highly immunodeficient scid mice [37] . in addition, the stability of poxviruses and mva's ability to be lyophilized are optimal for use in developing countries where the vaccine is needed most and cold-chains cannot be reliably maintained. mva also has been shown to stimulate a broad immune response, including the induction of cellular immunity [62, 70] . here, we describe the construction, expression, and preclinical efficacy and safety of a novel mva-chik vaccine candidate. balb/c mice, which are immunocompetent, were 100% protected from viremia upon challenge. a129 mice, which are deficient in a/b interferon signaling, and therefore highly susceptible to chikv infection, were also completely protected against challenge with wtchikv after prime and boost vaccinations. in addition, 80% of mice were protected from lethal challenge following prime only vaccination with mva-chik. despite the robust protection observed, mice did not produce a strong neutralizing antibody response following vaccination. although vaccinated a129 mice developed undetectable levels of neutralizing antibodies, they did produce a significant amount of anti-virus antibodies, which for other alphaviruses have been shown to be protective [71] [72] [73] . a129 mice passively transferred mva-chik immune serum from either balb/c or a129 mice were not protected against mortality, or footpad swelling following challenge, despite a slight reduction in viremia 2 days postinfection in mice transferred a129 mva-chik immune serum (p = 0.11). this suggests antibodies induced by vaccination were not sufficient to provide protection in this manner. this could be due to the fact that passively administered immune serum is significantly diluted in the circulation of naïve mice. further evidence that antibodies are not sufficient for protection alone was provided by depletion studies conducted in mva-chik vaccinated mice. when immune cd4 + t-cells were depleted, despite the presence of full levels of circulating anti-virus antibodies, mice were 100% susceptible to lethal infection. if anti-virus antibodies were essential for protection a reduction in mortality or viremia would be expected in the cd4+ depleted mice, which was not observed. these data suggest that mva-chik is providing protection by a novel mechanism that is not based solely on vaccine induced serum circulating antibodies. while antibodies do not protect via passive transfer or during depletion, it remains unclear what role they may be playing during infection of an mva-chik immunized mouse. immunocytochemistry and western blot analysis suggested chikv p62 protein was being maintained inside of the cell and that furin cleavage was not occurring to separate e3 from e2, respectively. p62 was chosen due the presence of immunodominant epitopes and the fact that previous reports have shown that it could be an effective immunogen against other alphaviruses [71, 74] . in addition, it has been previously observed that when expressed by a plasmid, the presence of e1 is not a requirement to allow e2 to be placed on the outside of the plasma membrane [75, 76] . barth et al. (1997) also demonstrated that in the context of an e1 deleted semliki forest virus, p62 is not transported efficiently to the plasma membrane while e2 alone is [77] . therefore, absence of e2 on the surface of infected cells is not completely unexpected and may have proven to be serendipitous in allowing the production of a more robust cd4 + immune response. this could provide an explanation for the lack of a strong humoral immune response, as antibodies would have limited access to this intracellular protein. in addition, an improperly folded protein would be expected to produce many virus binding antibodies but few that are neutralizing, which our data suggest. a recent report by metz et al. (2013) , suggested that e2 produced in combination with e1 in a baculovirus system is more immunogenic (i.e., produces higher neutralizing antibodies) than either protein alone [78] . this might suggest that e1 and e2 are more efficient together to obtain a robust neutralizing antibody response and might explain why mva-chik produces few neutralizing antibodies. analysis of ex vivo cellular responses indicated that mva-chik induced antigen-specific cd4 + t cells but not cd8 + t cells, suggesting that immune cd4 + t cells are the main effector cells. this is supported by the cellular depletion studies that showed the protective role of cd4+ t-cells. t-cell mediated protection has previously been demonstrated with another alphavirus, veev [79] . yun et al. (2009) were able to adoptively transfer cd4 + t-cells and obtain protection after multiple immunizations, where passive transfer of hyper-immune serum was not protective. interestingly, they did not observe a difference in viral titers in the brain from surviving mice, while mva-chik vaccination completely protected against viremia in our studies. recent studies based on another vaccine candidate, chik-ires, have shown that a correlate of protection based on antibodies can be established for chikv [50] . herein, we present data that suggest that this is specific to the vaccine being used and that there may be more than one way to provide protection against chikv. mva-chik selectively expresses e2-e3 proteins. it could be argued that the processing of this virus is substantially different than the live attenuated chik-ires vaccine or wildtype chikv. as a result, during infection a different set of epitopes are generated that may well be protective via alternative mechanisms. in another report, using mice deficient in either bcells or cd4 + t cells, data were presented that suggested that antibodies are essential in controlling chikv infection and that cd4 + t cells may exacerbate disease [49, 80] . hawman et al. (2013) found similarly, that mice deficient in both b and t-cells had less severe tissue pathology early after chikv infection when compared to wild-type controls [81] . however, in the same report it was demonstrated that virus was inefficiently cleared in the absence of lymphocytes, suggesting a possible dual role for these cells during early chikv infection where some tissue damage is allowed in order to control viral dissemination. while we observe very little evidence of tissue damage in vaccinated mice, it is possible that mva-chik induced specific cd4 + t-cells which are able to effectively control viral replication while minimizing collateral tissue injury. it remains possible that cd4 t-cells are required for recall responses to produce antibodies following challenge, although this is unlikely as mice would be expected to have early viremia, which we did not observe in vaccinated mice (fig. 2e) . currently, we are conducting studies to determine the role of cd4 + t cells in protection. our data are also contrary to the previous report by teo et al. (2013) , where no footpad swelling was observed upon antibody depletion of cd4 + t cells during chikv infection [80] . however, their studies were performed in naïve mice with a functional interferon system, in contrast, our studies used vaccinated mice without proper ifn a/b signaling, and thus might account for the differences observed. additionally, recent studies in humans show that there is a strong cd4+ ifnc positive t-cell response directed against the e2 protein, suggesting it might be playing a role in the protective immune response against the virus [82] . therefore, the data presented here may be relevant to human infection, and suggest that antibodies may not be the only mediator of protection against alphaviruses. a recently published report by garcia-arriaza et al. (2014) also has used mva as a vector as a chikv vaccine candidate [83] . their vaccine, which expressed the entire structural protein is effective at inducing a high level neutralizing antibody response accompanied by a strong cd8+ t-cell specific cell mediated response, in direct contrast to the results reported here. they report that the e2 protein can be found in both the plasma membrane and the cytoplasm, in contrast to our results which clearly showed e2 is not on the surface of the cell (figure 1 ). this could explain why the immune responses are so drastically different between the two vaccine candidates. while depletion studies indicated cd4+ t cells are indispensable for protection for mva-chik in this report, the mechanism of protection is unclear in their report, although high levels of neutralizing antibodies prior to challenge, balb/c mice were bled and serum was monitored for neutralizing activity by tcid 50 microneutralization assay (a). mice were bled two days following challenge and viremia levels were measured via tcid 50 (b). for passive immunization, prime and boost vaccinated balb/c and a129 mice were bled and serum taken from each mouse was pooled with equal volumes into separate pools from each strain. pools of serum (100 ml for balb/c and 200 ml for a129) were then injected i.p. into a129 mice which were challenged 24 hrs later with 10 2 tcid 50 units of wild-type chikv. viremia was measured 2 d.p.i (c) and survival was monitored for two weeks post-challenge (d). the dotted line indicates the limit of detection of the assay. doi:10.1371/journal.pntd.0002970.g004 figure 5 . mva-chik elicited a chik-e2-specific cd4 + t cell response in a129 mice. two weeks following boost, splenocytes from mva-gfp or mva-chik immunized mice were stimulated with chikv nsp2, e2 or e3 peptide pools (1 mg/well). t-cells were then stained for intracellular ifnc (a and c) or cd40l (b and d). representative dot plots (for only one mouse per group) are shown for both cytokines with mva-chik and mva-gfp stimulated with e2 peptide pools or background media control (a and b). for stimulation with e2, e3 and nsp2 peptide pools data are presented as the mean+sd of the percentages cytokine-positive cells among gated cd4 + t cells (3 mice/group) with background subtracted (c and d). stimulation was significantly higher for ifnc and cd40l in the mva-chik immune group, as compared to mva-gfp control (p = 0.02 and p = 0.01, respectively). no cytokine production in cd8 + t cells was observed (data not shown). doi:10.1371/journal.pntd.0002970.g005 induced by their vaccine are likely protective [50] . additionally, the vaccine constructed by garcia-arriaza et al. (2014) uses the entire structural protein consisting of capsid-e3-e2-6k-e1. in contrast, our vaccine contains only the e3-e2 portion. recent reports have shown that the capsid protein contains immunodominant epitopes for both b and t cells [55, 82] , and was therefore excluded from our vaccine construct in favor of e2 with its accessory protein e3. furthermore, we used a footpad injection which more closely mimics intradermal vaccination a human might be expected to receive. garcia-arriaza et al. (2014) used intraperitoneal vaccinations, which are not given to humans and therefore could potentially induce a vastly different immune response delivered by another route. in conclusion, these studies demonstrate the potential of mva to effectively express chikv e3-e2 proteins and generate protective immune responses despite the presence of low or absent neutralizing antibody responses. the mva construct expressing chikv p62 protein was effective in several mouse models and provided protection against two critical markers of disease, viremia and joint swelling. the vaccine was protective despite low levels of neutralizing antibodies which are normally considered to be the golden standard for protection against alphaviruses. therefore the results reported here challenge the assumption that only these antibodies are effective in providing protection against chikv or other alphaviruses. future studies combining a t-cell targeted vaccine, like the one presented here, along with a more 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infection is controlled by the adaptive immune response identical strength of the t cell responses against e2, nsp1 and capsid chikv proteins in recovered and chronic patients after the epidemics of 2005-2006 in la reunion island a novel poxvirus-based vaccine (mva-chikv) is highly immunogenic and protects mice against chikungunya infection we are grateful to andrea trujillo and attapon kamlangdee for their technical assistance and help with data analysis. key: cord-319517-denczc6t authors: salipalli, sandeep; singh, prafull kumar; borlak, jürgen title: recent advances in live cell imaging of hepatoma cells date: 2014-07-08 journal: bmc cell biol doi: 10.1186/1471-2121-15-26 sha: doc_id: 319517 cord_uid: denczc6t live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. with the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. the success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. this review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example. microscopy has contributed immensely to our understanding of cellular structure and morphology. however, traditional microscopic tools provide only limited information in terms of dynamical processes occurring in a living cell which are of great interest for biomedical researchers. advances in optical methods, in vitro culture systems and molecular biology led to the advent of live cell imaging techniques. this non-invasive technique provides better insight into the biological role of target molecules by allowing researchers to investigate the dynamic processes occurring in living cells in real time. the technique has many potential applications in various fields of biomedical science including developmental biology, cell biology and tumor biology and provides opportunity to study the dynamic behaviour of living cells in context to gene expression, protein-protein interaction, co-localization, cell division, chromosomal dynamics and intracellular transport of bio-molecules. the success of live cell imaging relies on various factors including the specific imaging system, climate controlling devices for cultured cells under investigation, construction of recombinant plasmid dna, transfer and expression of candidate genes and/or fluorescent proteins in mammalian cells. these factors greatly influence the fluorescent/bioluminescent signals obtained from the cultured cells. the gene transfer methods should not only be efficient in delivery and in ensuring stable expression but at the same time should exert minimum toxic effects to the cultured cells. furthermore, the chosen fluorescent or bioluminescent markers should be minimally phototoxic to the cells at their highest expression levels. amongst the bioluminescent markers, atp dependent and independent luciferases from various sources have been extensively used in imaging experiments [1, 2] . the use of bioluminescent markers is not only limited to in vitro assays or live cell imaging but is also applied to in vivo molecular imaging experiments. various lines of luciferase expressing transgenic mice and cells have so far been developed and are frequently employed in biomedical research, and a major breakthrough in the field of fluorescent protein imaging was the discovery of green fluorescent protein (gfp) by osamu shimomura who received the nobel prize in chemistry in 2008 together with martin chalfie und roger tsien [3, 4] . after the advent of gfp the technique of live cell imaging has taken a leap in understanding the detailed and complex cellular dynamics. apart from gfp and its variants, many other fluorescent proteins have been isolated from a variety of sources and are successfully used in imaging experiments of various cell types and their organelles. in this regard, live cell imaging has been employed to study functional genetics of liver specific diseases including steatosis, which results from accumulation of lipid droplets in hepatocytes [5] . efficient gene delivery in mammalian cells is another aspect of our review with appropriate choices of cell type specific promoters and their use for targeted gene delivery to hepatoma lines such as hepg2 and hep3b. nonetheless, the concept of gene transfer through plasmids started in bacteria via both, physical and chemical methods. similar approaches have been used in hepatoma cells and other higher eukaryotes and mammalian cells and include lipofection, deae-dextran, calcium-phosphate, viral vectors, peptides and electroporation [6] . lipofection has been used to achieve transient as well as steady transfection in hepatoma cells resulting in an improved and stable expression of transgenes even after several passages [7] . to develop protocols for cell type specific reporter activity, we discuss the use of alternate promoters and vectors for stable expression in actively dividing cells. bioluminescence is the phenomenon of the production of light by a chemical reaction within a living organism. it was first discovered in firefly (lampyridae species) and since then has been used for various screening and staining activities with an advantage of observing the cells under a compound microscope. firefly luciferase (fluc) emits luminescence (up to 560 nm) without the requirement of any external light excitation and uses atp for the conversion of its substrate luciferin to oxyluciferin in a luciferase enzyme catalyzed oxidation reaction. initially, fluc was used only in luminometery based reporter assays using cellular lysates. later luciferase expressing cells and mouse lines were developed for noninvasive imaging of rodents. injection of the luciferin substrate in mice produces luminescent signals that can be easily detected by in vivo imaging modalities. apart from beetle, luciferase has been isolated from members of the coelenterazine species, i.e. gaussia, renilla, pleuromamm and oplophorus. the luciferase from renilla sp. (rluc) uses a different substrate "coelenterazine" and produces a higher and stable luminescent signal as compared to the fluc [1] . rluc has an added natural advantage of being an atp independent enzyme, and thus requires less energy to produce luminescence. however, a major limitation of fluc and rluc is their short life span and therefore these luminescent proteins cannot be used for long duration imaging assays. this led to the development of a more photostable and robust luciferase in the form of an enhanced beetle luciferase (eluc) which again uses luciferin as the substrate and is an atp dependent enzyme [8] . a comparison between eluc and fluc showed phenomenal differences in the intensity and photostability of the two luciferases. however, the km values (michaelis constant) of the enzymes in regard to atp consumption were found to be almost equal, and it was concluded that the atp use is not the reason for a better performance of eluc. moreover, eluc was tested in cell lines such as nih/3t3 using various promoters, and it was shown that the luciferase primarily localizes in nucleus, cytosol and in peroxisomes. certain vectors and promoters also play an important role for their suitable expression namely pcmv vector used in mice cells along with the astrocyte specific promoter mper2 which outperformed fluc by a factor of 10-and 16-fold in cell extracts and live cells, respectively [8] . in contrast to bioluminescence where the enzyme catalyzed reaction initiates the excitation of luminescent molecule, fluorescence is triggered when photons from an external source excites the light absorbing pigments. green fluorescent protein (gfp) was the first fluorescent protein to be discovered in jelly fish (aequorea victoria). wild type gfp (wtgfp) is a 26.9 kda protein with a major and a minor excitation peak (395 and 475 nm, respectively) and a single emission peak at 509 nm. the chromophore of this fp is formed by three amino acid residues consisting of ser65-tyr66-gly67 and held by a single αhelix surrounded by 11 β-barrel sheets to prevent its quenching by water [8] . several modifications have been made to wtgfp to enhance its fluorescence intensity as well as stability by minimizing photobleaching. in the year 1995, heim et al. [9] introduced a single point mutation at the s65t residue that resulted in enhanced fluorescence at the same emission spectra but with a shift in excitation peak from 395 nm to 488 nm. the mutation also significantly reduced the time required for formation of the fluorophore from 2 hours to 0.45 hours. several additional mutations in the protein helped the molecule to fold optimally at 37°c while the protein expression was improved by codon optimization of the wtgfp according to the host organism [10] . additional mutants were generated replacing the serine65 with threonine, alanine, glycine, cysteine or leucine leading to a protein with a single absorbance peak at~489 nm [9] [10] [11] [12] . expression of gfp at high concentrations in the cells posed the problem of dimerization which was resolved by side directed mutations at a206k, l221k or f223r residues [13] . the enhanced gfp (egfp) was developed with the modification and mutations described above at various positions of wtgfp. proteins tagged with enhanced gfp can be visualized in cells with low light intensities causing less photobleaching to enable imaging and quantification of intracellular proteins and its pathways effectively [14] [15] [16] . to tackle the problem of mixed chromophores in live cell imaging experiments, several mutants of wtgfp including enhanced gfp (egfp) and emrald gfp were generated which retained its fluorescent properties but with non-overlapping spectral properties [17] . neutral chromophore gfp has also been generated with a shift in the excitation and emission wavelengths to uv and green light, respectively [12, 18] . the proteins were again mutated to create sapphire which displays increased emission at 37°c [10] . the new mutant of sapphire called turbo sapphire has better ph-stability with uv excitation and thus has a larger separation between the excitation and emission wavelengths. table 1 summarizes the development of different variants of fps and their physical properties. several fluorescent proteins from different species have been discovered with various excitations and emission spectra ranging from ultraviolet to far-red or infrared [10] . these have been used in several cells and organelles, and some of them are discussed in this review. development of different variants of gfp with different absorption and emission spectra provided the opportunity for simultaneous multi-color staining of the cells. early variants included the blue fluorescent protein (bfp), developed by introducing a point mutation (y66h) to shift the absorbance and emission spectra to 384 nm and 448 nm, respectively, and cyan fluorescent protein (cfp) which was discovered from mutants of a.victoria [17] . since the spectra of bfp and egfp are distinguishable, a combination of these two fps was first used for multicolor imaging in cells and fluorescent (förster) resonance energy transfer (fret) analysis [9, 31, 32] . however, a major drawback of bfp was its low intensity and faster photobleaching [33] . therefore, enhanced bfp (ebfp) was developed by using codon optimisation for human cells [34] . notably, azurite, an ebfp, was developed by the dauherty's group and was reported to be 40-fold more photostable and brighter than the conventional bfp. ebfp was generated by incorporation of two additional point mutations (f64l and s65t) in the bfp and was considered to be the brightest bfp for the time until further improvements were made [24] . subsequently, ebfp 1.2 was developed based on mutations at s30r, y39n, t65s, s72a, n105t, i171v, n198s and a206v in ebfp and was 4-fold brighter than ebfp [23] . further modifications of ebfp 1.2 led to the development of ebfp 1.5 which was more photostable than its predecessor ebfp 1.2 but with no increase in fluorescence. azurite, as previously mentioned, had increased photostability as compared to ebfp 1.2 and ebfp 1.5 due to the mutations at v150i and v224r, respectively. however, this caused a decrease in the fluorescence by 30% [24] . screening of a library of mutants generated by random mutagenesis identified ebfp2 with enhanced fluorescence and photostability when compared to ebfp and azurite. ebfp2 was reported to be 4fold brighter and 550-fold more photostable than ebfp and 1.4-fold brighter and 2.9-fold more photostable than azurite [23] . however, both ebfp2 and azurite tend to dimerize when expressed in cells at higher concentrations. this was overcome by replacing the alanine residue at position 206 by valine [35] . bfp was further modified to develop a stable red fluorescent protein (rfp). site directed and random mutagenesis of rfp generated mtagbfp with a shift in excitation spectrum and blue emission [36] . although mtagbfp is the brightest among all the bfps produced, it is 1.4-fold less photostable than ebfp2 [23] . therefore, mtagbfp was mutated at i174a to give rise to mtagbfp2, i.e. a 1.5 fold more photostable bfp with a similar spectral profile and maturation half time as that of the parent protein [37] . cfp and yellow fluorescent protein (yfp) were developed as a multi-color pair. cfp was also brighter and more photostable when compared to bfp [9] and was created by a point mutation (y66w) with a spectra intermediate between bfp and egfp. further improvements were also made to increase the stability and intensity of cfp [18, 32] . mutation at t203 position of wild type gfp with an aromatic amino acid resulted in shifting of excitation and emission over 20 nm into yellow wavelength. this led to the development of enhanced yellow fp (eyfp). although eyfp is a dimer and sensitive to chlorine, three variants of eyfp were developed namely citrine (monomer), venus -a fast maturing fp at 37°c and the yellow fluorescent protein for energy transfer (ypet)used in fret along with cyan fps [29, 30, 38] . red fluorescent protein (rfp) was first isolated from an anthozoan, diascoma, with excitation and emission of 558 and 583 nm, respectively [39] . the major advantage of rfps over gfps is their longer excitation wavelength causing less damage to the cells and much less auto fluorescence in cells at the red spectrum. also the maturation of fps obtained from anthozoan is more efficient as compared to that of jelly fish fps at 37°c. native rfp has the disadvantage of forming tetramers which at times tends to aggregate in the cells and also cause false oligomerization of target proteins to hinder their native function [40] . dsred, the common rfp available, has prolonged maturation time often taking days to turn red from a greenish complex; this feature has been used to observe aggregate formation in target cells [41] . monomeric rfps have also been developed from dsred through a series of mutations, and the resultant proteins exhibited different maturation rate as well as fluorescent properties, i.e. morange, mko, mstrawberry, mcherry, tag rfp, mplum, mkate and tdtomato [42] . due to its fast maturation rate, high photostability and wide ph tolerance mcherry is a widely used long wavelength fp. however, it tends to dimerize that limits its experimental use. later the protein was modified to prevent its dimerization in the cells. it is considered to be the brightest rfp with enhanced photostability, and it has only a 3 nm longer excitation and emission wavelengths compared to its predecessors [43] . similarly, tag rfp was developed from wtrfp isolated from sea anemone entacmaea quadricolo by introducing mutations at five different sites (r162e, q166d, s180n, f198v and f200y) [44] . as of today, live cell imaging has advanced mainly due to the advent of fps tagged biomolecules with specific functions in the cell. fps have successfully been cloned into bacteria to mammalian cell lines either alone as a marker or along with a gene of interest as a fusion protein [45, 46] . several organic dyes have been tagged to cell organelles to visualize the dynamics under a microscope, however, not all dyes are readily accepted by the cell organelles and in such conditions fp tagged proteins are used to target the organelle. multi-color live cell imaging is an effort to observe multiple organelles, their spatial organisation, interactions between organelles and their constituent components. therefore, the successful application of the technique demands fps and dyes with non-overlapping emission spectra [47] . in recent developments organelle staining dyes such as hoechst 33342, mitored, dioc 6 , syto 9 and rhodamine b were tested along with several other dyes for imaging of various organelles in live cells and for the study of host pathogen interactions [48] . in this regard, lipid droplet staining has been done since a long time but only with organic dyes such as oil red, and none of the fps mentioned earlier have been used for successful imaging of lipid droplets. the different fluorescent proteins widely used for staining of different organelles of a live cell are listed in table 2 . fluorogen activating proteins (fap) are proteins/peptides such as bovine serum albumin and igg that can bind and activate the fluorescence of target fluorogen molecules and may also alter their spectral properties. conjugation of 9-(2-carboxy-2-cyanoviny1)julolidine (ccvj) fluorogen to either bsa and/or igg not only improved its solubility but also the fluorescence intensity by 6.3-fold when compared to the unconjugated molecule [49] . the use of larger fps such as gfp in reporter assays hinders the function of the fusion proteins in the cells. therefore, small tag systems such as tetracysteine motif (ccxxcc) were genetically inserted into the target proteins that can bind with high specificity to biarsenical dyes, resorufin arsenical helix binder (reash) and fiash (fluorescein arsenical helix binder) in live cells and lead to an enhanced fluorescent signal [50] . of note, flash, a derivative of 4' , 5'-bis (acetoxymercuri) fluorescein, is a cell permeable non-toxic ligand that binds specifically to four cysteine residues (i, i + 1, i + 4 and i + 5) of the target proteins α helical structure to emit fluorescence [51] . out of the fourteen different flash ligands screened, flash-edt2 showed strong affinity for hexamer peptides and positive emission at 635 nm. fret studies in hela cells with fiash-edt2 genetically fused to the cooh-terminus of an enhanced cyan fluorescent protein (ecfp-fiash) evidenced a 3-fold increase in fluorescence at 635 nm when compared to the ecfp alone transfected cells [32, 51, 52] . however, emission of ecfp at 480 nm declined to about 30% in fiash-edt2 transfected cells due to fret interactions. reash-edt2, a resorufin based fluorescent complex with excitation and emission maxima of 593 nm and 608 nm, respectively, was successfully tested for fret studies with gfp and yfp probe pairs in hela cells [53] . similar protein-ligand complexes were synthesized and used namely crash-edt2, sflash-edt2, f2flash-edt2, choxash-edt2, splash-edt2-alexa594, cag flash-edt2 and ascy3-edt2. several modifications have been carried out to improve the quantum yield, tetracysteine binding affinity and to reduce the cytotoxicity of these complexes. specifically, malachite green (mg) and thiazole orange (to) derived intercalating faps were developed which bind to rna aptamer and dna, respectively, and lead to enhanced (2,360-and 550-fold, respectively) and bleaching stable fluorescent signal from the fluorogens [54] [55] [56] . several unique membrane permeant and impermeant faps were developed by screening a library of human single-chain antibodies (scfvs) using derivatives of thiazole orange and malachite green. the screened clones have comparatively smaller size (the smallest being 110 amino acids long that is almost half the size of gfp) and thousands-fold higher brightness as compared to the typical fps. moreover, different spectral variants of mg and to could be easily generated by different combination of the screened fluorogens and faps. the fluorescence and differential interference contrast imaging microscopy was performed in nih3t3 cells with the membrane impermeable (mg-11p) and membrane permeable (mg-ester) forms of mg fluorogens and fap hl4-mg scfv tagged to membrane protein plateletderived growth factor receptor (pdgfr). the mg-ester signal was detected from the plasma membrane while mg-11p emitting fluorescence inside the cells suggested a localization near endoplasmic reticulum [54] . figure 1 illustrates the uses of membrane permeable and impermeable mg fap fluorogens and their application with a fluorescent tagged protein to study trafficking of a transmembrane protein. additionally, these faps were also tagged along with gfp and other fluorescent proteins to study plasma membrane and transporter proteins such as the insulin regulated glucose transporter (glut4), β2 adrenergic receptor (β2ar) and cystic fibrosis transmembrane conductance regulator (cftr). these genes were designed such that the fap and gfp were present as intracellular and extracellular reporters and the fluorogens, being membrane permeable and impermeable, were used to observe the mobility and placement of the target proteins tested in nih3t3, c2c12 and hek293 cells [57] . faps have been tested and proved to be reliable fluorescent markers for detecting and imaging various activities in a cell like protein motility studies and even for transmembrane protein activity. faps provide wide scope in developing alternatives to traditional fluorescent proteins where size hinders the activity of the target proteins. bacterial transformations have led to the expression of various genes in the prokaryotic system with certain [45, 46] limitations as compared to the eukaryotic cells. however, the transfer of genes in the mammalian cells requires much more sensitive approaches, and several methods have been developed for effective transfer and stable expression of genes in these cells. with the advent of viral vectors much progress has been made in the field of gene transfer. the use of cell type specific promoters allows greater control over the expression of foreign genes. liver cell specific promoters such as glycerol-3-phosphate acyltransferase (gpat i and gpat ii) and phosphoglycerate kinase 1 (pgk-1), albumin, human alpha1-antitrypsin and hemopexin are shown to be active in hepatocytes of human and mice origin [7, 58, 59] . similarly, viral promoters and vectors have been tested in different cell lines for their specificity and stability of expression. pcns and pcns-d2 vectors were reported to express cloned genes in cell lines and can also act as a shuttle vector between bacteria and mammalian cell lines to facilitate an easy cloning process. this vector system has been tested for stable expression of luciferase in hepg2, hep3b, hela, snu638 and snu668 cell lines. the luciferase expression was higher in hepg2 and snu638 cells as compared to other cell lines. cytomegalovirus (cmv) and simian virus 40 are known to infect human and other primate cells and have been shown to induce a stable expression of transgene in various cell lines [60] . during the study of cmv promoter systems, a 5' untranslated region (5' utr) was identified which was shown to boost the expression of viral proteins facilitating the rapid propagation of viral particles in the host cell. incorporation of utr regions in the promoter of viral vectors boosted the expression of genes of interest in host cells such as cho or hepg2. notably, the use of two copies of the utr regions further improved the expression of target genes in actively dividing cells [61] . later, the utr regions were found to be the internal ribosome entry sequence (ires) being responsible for uninterrupted expression of genes. ires were first discovered in rna of poliovirus in 1988 by pelletier and sonenberg [61] and facilitate the viral replication machinery by providing means for viral rna to bind with the 40s subunit of the host ribosomal complex thus eliminating the need for eukaryotic translation factors [62, 63] . since then, several viral vectors have been developed including the recently explored corona virus of the sars virus family. this rna based vector provides a stable and prolonged expression for more than 30 passages in different cell types [62] . insertion of ires site in expression vectors also offers the possibility of separate expression of the cloned genes under a single promoter ( figure 2 ) [61] . table 3 summarizes the list of various promoters and plasmids that have been used extensively in mammalian cell lines. among all promoters used cmv and sv 40 are most efficient in a wide range of cell lines. apart from these cell specific promoters such as pgk1 and gpat enhance expression levels in hepatoma cell lines. a variety of methods have been developed for a robust and efficient gene transfer without causing toxicity to the host cell. earlier physical methods such as electroporation and microinjections [64, 65] were primarily used for gene transfer studies but have been sidelined gradually due to the obvious disadvantages of being time consuming and the associated technical difficulties. more advanced methods have come into place for gene transfer according to the need for expression as a plasmid entity or as an integrated system in genome which also can be used for gene knockouts. non-pathogenic viral vectors are commonly used for gene transfer studies. baculovirus of the autographa californica multicapsid nucleopolyhedrovirus (acmnpv) and bombyx mori nucleopolyhedrovirus (bmnpv) genus provide a safe and efficient means of gene delivery to target cells [66] . baculoviral vectors have been modified to infect mammalian cell lines either as the whole baculovirus or as a helper cell mediated gene delivery in cells. adenovirus terminal repeats were used for the replication of the target gene, and the whole plasmid was packed into the baculoviral vector bac-b4 for transfer in cell lines such as 293b5b1. the transfected cells showed not only stable expression but also a 100-fold increase in the vector titer during subsequent passages [67] . of note, dna fragments of approximately 38 kb were efficiently transferred into different cell lines using such a vector. direct incubation of oligodendrite and chicken muscle cells with baculoviral vector showed a transfection efficiency of 60% [68] . further increase in transfection and expression efficiency was achieved by additional modifications in baculoviral vectors such as an introduction of the metallothione promoter (bac-me). the bac-me vector showed a transfection efficiency of 90% with low leaky expression and reduced cytotoxicity in hela cells. the expression efficiency of these vectors was further enhanced by treating the transfected cells with znso 4 [69] . although the baculoviral vectors were successfully used for gene transfer experiments in several mammalian cell lines they were replaced by other viral vectors and methods due to the associated cytotoxicity specifically at higher cell to virus ratio. hence, non-viral vectors were developed for transfection of viral sensitive mammalian cell lines [70] . bacterial based transfection has also been employed by cloning sequences belonging to the pathogenic bacteria e.coli. the modified genes from pathogenic bacteria like listeria monocytogenes have also been used where the bacteria with its intracytoplasmic capabilities propagate into the host. likewise, e.coli was modified so that selflysis genes become activated as soon as the bacteria enter the host cell to release the vector into host cytoplasm [70] . other bacterial methods involve t4ss, a bartonella sp., for the introduction of single strand dna into the host cell. thus, after entry the bacteria releases ssdna into cytosol and along with the enzyme relaxase or integrase, which is bound to target dna, starts to replicate to finally produce plasmid dna as a whole by joining the loose ends present [70] . this mode of transfer is considered to be hazard free and is employed for a variety of cell lines. further methods of nonviral based gene transfer have been developed for higher efficiency and less toxicity to the cells. as mentioned earlier for efficient transfection the gene has to be integrated into the genome (constitutively) or is continuously expressed in the presence of a specific inducer reagent for the chosen promoter (inducible). owing to variable results the need remains to develop efficient methods as has been attempted in the form of a dual plasmid approach using the mice myogenic c2c12 cell line. according to this method t-rex plasmid system is first transfected into the cell line using a lipid based method with an efficiency of 70% and is activated by tetracycline. thereafter, a modified t-rex plasmid became available. after 18 days of initial transfection a second plasmid can be introduced with 40% efficiency that permits the study of complex biochemical reactions involving different factors [71] . in table 4 the various transfection efficiencies of different methods are summarized. bactofection seems to be an effective mode of transfection in cells without the risk of cytotoxicity as compared to some viral methods but much improvement has to be made for application of bactofection for a wide variety of cell lines. lipofection involves formation of a positively charged lipid-nucleic acid complex by suspending negatively charged dna/rna molecules with cationic lipids for a short duration of time. the positive charge on the complex facilitates attachment to the cell membrane and entry via endocytosis [70] . a comparative study concluded that lipofection is the safest method to transfer puast sv 40 expressed genes in yeast [60] genes into in mammalian cells amongst the various transfer methods [70] . different commercially available kits with a broad range of efficiency on different cell lines are available. the transfection efficiency of methods varies depending upon the cell type, cell number and ratio of dna and reagent used. using lipofection and as determined with a gfp marker an efficiency of about 60% was obtained in cho-k1 cell lines [72] . the major challenge faced by most of the non-viral delivery systems is the degradation of the transfected nucleic acid in the endosomes and/or lysosomes as they are mainly internalized by endocytosis. lysosomotropic agents like chloroquine and polyvinylpyrolidone (pvp) have been used effectively to increase the transfection efficiency of different delivery methods by reducing the lysosomic degradation of the transfected nucleic acid [73, [79] [80] [81] [82] [83] . however, the cytotoxicity of these agents raises concerns against their use. chloroquine is known to increase the intracellular ph leading to cytotoxicity and degradation of transfected dna at higher concentrations and at prolonged exposure times (>4 hours). amongst the two variants of pvp, pvp10 was reported to be cytotoxic at all concentrations while pvp40 showed negligible cell toxicity and even improved the transfection efficiency. pvp40 has been used for transfection studies in macrophages and hepatocytes [84] . sucrose is also known to cause intracellular swelling of vesicles in endosome and lysosomes due to osmotic pressure [85, 86] . sucrose in conjunction with lipid-dna complex or lipofection reagents was shown to increase their transfection efficiency in fibroblast cell. furthermore, no toxicity has been reported using sucrose as lysosomotropic agent and a concentration range of 5 to 500 mm was safe in various cell lines such as cho, cos7 and hek 293 t cells. beneficial effects of these lysosomotropic agents were compared against lipofectamine 2000 in cos7, cho and hek 293 cell lines. essentially, the transfection efficiency of lipofectamine was increased by 6-and 3-fold when used in conjunction with pv40 and sucrose, respectively. chloroquine, although being toxic showed an increase of 3to 6-fold when used at a concentration range of 0.01 to 0.1 mm as compared to lipofectamine alone [87] . poly(ethyleneimine) is another cationic polymer transfection reagent frequently used in a variety of cell lines. however high cytotoxicity is the major drawback associated with pei and therefore several changes in its molecular structure have been done to reduce its cytotoxicity and this includes pegylation and the introduction of carbohydrate, lipid and peptide moieties [75, [88] [89] [90] . a recent study showed the improved transfection efficiency and low cytotoxicity of an anionic glycopolymer derivative of pei in hepg2 and hek 293 t cells [91] . modified lipids were developed to enhance the transfection efficiency of vectors. specifically, cationic oligopeptide lipids are charged lipids with a linker molecule to provide cations and are said to be more efficient in the presence of the serum containing medium [74] . different ratios of nucleic acid and lipids were tested ranging from 1:1 to 9:1 and the ratio of 3:1 was found to be optimum in terms of cell viability and transfection efficiency [74] . other synthetic vectors have been tested for gene transfer in various cell lines. polyspermine imidazole-4, 5amide complex (psia) was employed with hepg2 as well as cos 7 cell lines for gene targeted therapy. psia transfection efficiency is of only 20-30% in hepg2. apart from the risk of psia degradation that hinders complex formation with plasmid dna the process of psia synthesis requires strict environmental (ph and temperature) conditions before dna can be introduced [92] . to improve transfection efficiency in various hepatoma cell lines like huh-7, hepg2 and hep3b different strategies were employed including lipofection, electroporation and microinjection. microinjection (100 μg/ml) and cationic lipids 47-60% 45-55% 65% 60% [73, 74] non-viral ---6 0 % [ 71, 75] e.coli (modified salmonells/listeris sp.) t4ss (bartonella sp.) 60-70% 50% >70% >60% 50-60% [76] [77] [78] lipofection (1 μg) methods were used for transfection of plasmid encoding fluorescent protein (pegfp and peyfp) tagged to human and mouse adrp genes in huh-7 cells as to study the pathways of lipid droplet metabolism. a transfection efficiency of 66% was achieved, and none of the methods affected the expression or the interaction of proteins in cells proving their reliability and safety. however, the transfection efficiency of these methods varied inversely with the size of the plasmid [93] . in this regard, adenoviral vectors have been employed in hepg2 cells to study the function of genes such as the cytochrome p450 monooxygenases in drug induced hepatotoxicity. notably, cyp1a2, cyp2d6, cyp2c9, cyp2c19 and cyp3a4 were transfected in hepg2 cells using recombinant adenoviral vector with a transfection efficiency of approximately 70% [94, 95] . in most cases transfection or transduction of primary cells proof to be difficult for several reasons like the stable state of non-dividing or quiescent cells. however, some viral vector-based methods have been used successfully such as the murine retroviral vector that can transfect primary hepatocytes stimulated by mitogens (epidermal or hepatocyte or keratinocyte growth factors). moreover, the associated high costs and varying efficiency of the growth factors make these transfection methods unfavorable [96] [97] [98] [99] . lentiviral vectors are capable of transfecting quiescent cells [100] [101] [102] and have been successfully used for gene transfer experiments in various hepatoma cell lines [76, 103, 104] . although the discussed methods and markers are compatible with various if not all cell lines, the cell lines chosen have to be noted to select the right kind of vector and promoter system. an overview of the various transfection methods in different cell lines is given in figure 2 . research in recent years significantly advanced an understanding of the molecular causes of fatty liver disease, and live cell imaging of hepatoma cells proved to be extremely valuable for studying lipid droplet formation. lipids are primarily stored in adipocytes as lipid droplets; however, under stressed conditions hepatocytes can also produce lipid droplets leading to pathological conditions like fatty liver disease [105, 106] . lipid droplet staining requires cell permeable dyes/fluorochromes which can bind specifically to the components of the lipid monolayer of droplets. the most commonly used dyes for lipid staining are oil red o, nile red and bod-ipy with an excitation-emission spectrum in the range of 400-500 nm and 500-600 nm, respectively. figure 3 depicts oil red o staining of lipid droplets in cultured human hepatocytes treated with palmitic acid (pa) and oleic acid (oa) and the cardiovascular drug amiodarone which is known to cause steatosis and steatohepatitis in patients. fps and dyes with non-overlapping fluorescence spectrum are required for differential staining of multiple biological molecules involved in lipid droplet metabolism. to address this issue, a long wavelength fp and a short range dye monodansyl pentane (mdh) was developed for lipid droplet staining with excitation and emission in the range of 405 and 480 nm [106] . such an approach allowed the study of perlipin 2 interactions on the lipid droplet as defined by the mdh stain. notably, in the early works of niemann et al. (2001) the use of mdh as a mean to study lipid droplets was reported [107] . mdh was also found to be photostable when compared to nile red and bodipy with a light emission of more than 15 minutes wherein the latter dyes were stable up to 10 minutes only [106] . mdh has a potential use for lipid droplet staining to facilitate an understanding of the formation of lipid droplets and their interaction with various proteins within hepatocytes. the lipid droplet monolayer has many associated proteins namely adipocyte differentiation-related proteins (adrp also known as perilipin 2/plin2) and perilipins (plins) which interact with other organelles in the cell. simultaneous detection of lipid droplets and adrp was achieved by transfecting huh-7 cells with adrp-gfp encoding plasmid and staining of lipid droplets with oil red o. although this method was useful in observing the cytoplasmic interaction of adrp/plin2 with lipid droplets the precise localization of gfp-adrp/plin2 at the surface of lipid droplet was distinctly observed only after incubating the cells with bodipy558/568 dodecanoic acid, a fatty acid sequestered in lipid droplets as evidenced by imaging by confocal microscopy in live and fixed cells conditions [93] . while this study was one of the first reports on lipid-protein interactions investigated by live cell imaging the use of bodipy hindered clear imaging due to background signals. to overcome these obstacles fret and frap methods were used as a means to study these complexes. moreover, mdh stain provides an efficient stain for lipid droplets which allows proteins to interact with the droplets to be tagged with longer wavelength fluorescent proteins and also mdh provides a more stable and robust stain for lipid droplets as compared to the traditionally used oil red o and nile red stains. fret is an approach to study protein-protein interaction and co-localization of organelles on the principle of energy transfer between two chromophores situated in close proximity (6-10 nm). the pair of fluorescent probes used has an overlapping spectral profile, and the emission wavelength of the donor probe (gfp) provides the required energy for the excitation of the acceptor probe (cfp). acceptor bleaching can be used to demonstrate whether the organelles are within the fret-distance (i.e. 6-10 nm), thus demonstrating true association on a molecular scale. fret was used to observe motility of certain proteins as well as organelles [108] . notably, laser scanning confocal microscopy based fret assays have been employed to study the co-localization of lipid droplets and mitochondria in porcine oocytes using the mitotracker green (mtg) and nile red (nr) fluorochromes. fret acceptor bleaching methods have also been used to examine mitochondria-lipid droplet co-localization [109] . to study protein-lipid interactions on the surface of the lipid droplets, perilipin 2 was tagged with cfp and followed by cyan fluorescence. since it forms a strong fret interaction with the lipid stain 22-(n-(7-nitrobenz-2-oxa-1, 3-diazo) aminostearic acid (nbd), the colocalized protein-lipid gave a signal of yellow-to-orange. the mouse perilipin 2 gene was cloned into the mammalian expression vector pecfpn1 and was stably expressed in mouse l fibroblast cells. subsequently, nbd was used to stain phosphatidyl choline to form nbd-pc complex, and a confocal microscopy based fret assay was employed to determine the interaction between lipids and proteins in the cell. similarly, co-localization of perilipin 2 and sphingomyelin (sm) near plasma membrane was demonstrated using nbd-sm and plin2-cfp vector constructs [110] . although cfp and yfp are most widely used probes yfp has the disadvantage of having low quantum yield and poor signal to noise ratio hampering the process of individual cfp intensity measurement. to counter this problem, two ecfp molecules were fused; however, the result was a further decrease in the intensity due to multiple excited states causing interconversion of excited states by homotransfer. notably, multiple excited states are due to two different crystal structure conformations of ecfp [111] . this problem was resolved by site directed mutagenesis of the his148 residue to aspartate, and the fluorescence intensity of the mutant was further increased by a factor of 2.5 as compared to ecfp. further mutations at s72a and y145a residues boosted the extinction coefficient of the monomer but caused a decrease in the quantum yield. the modified ecfp is known as cerulean and is proved to be a better fret donor than the conventional ecfp [111] . figure 4 depicts a basic understanding on how fret analysis can be done using ecfp and eyfp particularly for lipid droplet studies. fluorescent recovery after photobleaching (frap) is used to study single membrane bound structures, single cells and protein-lipid interactions. frap uses the concept of activating a fluorescent protein observing its intensity and subsequent bleaching by excess light to finally measure the bleached region for binding activity to the surface of the secondary structure or membrane. cfp and yfp have been commonly used to study the interactions of perilipin 1 (plin1) and perilipin 5 (plin5) with adipocyte triglyceride lipase (atgl) and its corresponding activator α-β-hydrolase domain-containing 5 (abhd5). on the basis of frap studies in cos 7 cells, it was concluded that plin5 interacts with atgl but not plin1 [27] . adrp/plin2 is shown to be located at the surface of cytoplasmic lipid droplets [112] , and localization studies for dna fragments encoding human and mouse adrp were ligated in-frame to the 3' end of egfp, thereby generating gfp-hadrp (expressed by plasmid pla4) and gfp-madrp fusion products. to examine whether the gfp-adrp products for both species retained the capacity to associate with these structures, constructs expressing the fusion proteins were transfected in huh-7 cells. after stimulation with fatty acid, the formation of lipid droplets in huh-7 cells can be identified by staining with lipophilic dyes and by antisera raised against adrp/ plin2. the signal from gfp-madrp was detected from the surface of spherical intracellular structures while egfp was seen throughout the huh-7 cells. the localization of endogenous hadrp and the gfp-madrp products coincided, indicating that gfp-madrp was located at the surface of lipid droplets therefore demonstrating that it is possible to detect tagged and untagged adrp on the same lipid droplet. to verify the localization of gfp-madrp, cells expressing the fusion protein were incubated with bodipy 558/568 dodecanoic acid, a fluorescent fatty acid analogue that is sequestered in lipid droplets. the dye was selected because of its spectral properties which allow differentiation from egfp and a better cell permeability as compared to oil red o. results in both live cells and paraformaldehyde fixed cells showed localization of gfp-madrp at the surface of lipid droplets stained with the bodipy dye. attempts to fuse egfp to the c terminus of adrp/plin2 yielded few cells that produced fluorescence of the chimeric protein but in this case the signal did not derive from the surface of lipid droplet. consequently, egfp fused to the n terminus of human and mouse forms of adrp/plin2 was used in further studies [93] . it is well recognized that caveolins are found on lipid droplets, but the functional significance of this association is poorly understood. adenovirus mediated transfer of cav1-gfp in nih3t3 cells demonstrated that caveolin-1-coated lipid droplets can grow larger than caveolin-1 devoid lipid bodies suggesting the importance of caveolins in determining the size of lipid droplets. this study provided the first detailed characterization of the impact of caveolins on molecular composition and the size of lipid droplets [113] . the most frequently employed tools (elisa, western blot, fret, frap co-immunoprecipitation) to study protein expression or protein-protein interactions rely on the use of antibodies tagged with either fluorophores or enzymes. the sensitivity of these techniques differs significantly, and the detection of proteins at low expression levels still remains challenging. the proximity ligation assay is a relatively inexpensive quantitative technique with high sensitivity. the technique is developed on the principles of antibody targeted assay, split reporter assay and polymerase based amplification of oligonucleotides. in this assay, the target proteins are recognized by specific antibodies (two or more) conjugated with short oligonucleotide strand. in case these probes are in near figure 4 fret studies to determine interaction between plin5 and the atgl protein on lipid droplet in adipocytes [39] . plin5 (1) and atgl (2) proteins were tagged with fret probes cfp (3) and yfp (4), respectively. the emission spectra of cfp act as excitation wavelength for the yfp. detection of yellow fluorescence after activation of cfp (436 nm) suggest possible interaction of the two tagged proteins. proximity, the oligonucleotides can be ligated by addition of complementary oligonucleotide and ligase enzyme and subsequently amplified using pcr or rolling circle amplification. this leads to the formation of more than 1000 copies of the complement of 100 bp in one hour using the phi29 polymerase [114] . the incorporation of multiple fluorescently labeled oligonucleotides in the polymerase catalyzed reaction causes amplification of the signal resulting in high sensitivity. apart from protein expression and proteinprotein interaction, this highly versatile technique can also be adapted to detect post-translational modifications, colocalization and interaction of proteins with other biomolecules ( figure 5 ). pla was recently used to demonstrate that the myc inhibitor 10058-f4 could prevent mycn/max interaction in situ and caused accumulation of lipid droplets in tumor cells [115] . the availability of a wide range of fluorescent proteins and organelle specific stains provides unprecedented opportunities for microscopic studies of diverse cellular processes including protein-protein interactions. however, for the visualization of certain cell components in live cells such as lipid droplets options are currently limited to organic dyes, i.e. bodipy, nile red and oil red o. the emission spectrum of these dyes overlaps with that of gfp and rfp; thus, it is not feasible to use them in combination for multi-color imaging. there is unmet need for the development of a new lipophilic dye which can be spectrally resolved from commonly used fluorophores. monodansylpentane, a cadaverin family dye with blue emission spectra, is the appropriate substitute for the red dyes for lipid staining. furthermore, this can be used along with far wavelength fluorescent proteins to obtain better insight into the lipid metabolism. discovery of novel proteins involved in the lipid droplet metabolism and their fusion with fluorescent proteins that can be spectrally resolved will help researchers to delineate the process of biogenesis of lipid droplets under live cell conditions. the advancements in culture systems to ensure long term metabolic activity of hepatocytes, the development of suitable transfection methods and reporter assays will be instrumental for the success of live cell imaging experiments. (2) formation of covalently joined circular oligonucleotide by ligase reaction: if the two probes are in close proximity, addition of two linear oligonucleotide leads to the formation of a covalently joined circular oligonucleotide molecule with the help of a ligase enzyme. (3) amplification via rolling circle mechanism: one of the probes linked to antibody act as a primer and addition of dna polymerase yields long single stranded concatemeric dna molecule composed of complements of the circular dna strands formed in the ligase reaction. (4) fluorophore labeling for detection: the amplified product is easily detected by addition of complementary oligonucleotides labeled with fluorophore. decision to publish, or preparation of the manuscript. we acknowledge support by deutsche forschungsgemeinschaft; the charge for this publication was covered by the dfg sponsorship "open access publication". optical imaging of renilla luciferase reporter gene expression in living mice enhanced beetle luciferase for high-resolution bioluminescence imaging the discovery of aequorin and green fluorescent protein discovery of green fluorescent protein association between nonalcoholic fatty liver disease and risk for hepatocellular cancer, based on systematic review construction of multi-purpose vectors, pcns and pcns-d2, are suitable for collection and functional study of large-scale cdnas identification and functional characterization of human glycerol-3-phosphate acyltransferase 1 gene promoters crystal structure of the aequorea victoria 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analysis expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases lipid droplet analysis in caveolin-deficient adipocytes: alterations in surface phospholipid composition and maturation defects proximity ligation assays: a recent addition to the proteomics toolbox myc inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells recent advances in live cell imaging of hepatoma cells we gratefully acknowledge support from the virtual liver network (grant 031 6154) of the german federal ministry of education and research (bmbf) to jb. the funders had no role in study design, data collection and analysis, the authors declare that they have no competing interests.authors' contributions jb initiated the review. all authors contributed to the writing and approved the final manuscript. key: cord-023026-2r84ndzv authors: nan title: posters date: 2013-06-14 journal: glia doi: 10.1002/glia.22530 sha: doc_id: 23026 cord_uid: 2r84ndzv nan university of colorado school of medicine, aurora, united states oligodendrocyte progenitor cells (opcs) migrate long distances before differentiating and myelinating axons. it is well established that both short and long-range soluble guidance factors, as well as contact with the surrounding extracellular matrix (ecm) and neighboring cells, can modulate opc migration and differentiation. however, the influence of cell-matrix interactions on opc responses to guidance molecules is less clear. previous in vitro studies of opc migration in response to semaphorins and netrin-1 yielded inconsistent results, but these studies were performed on a variety of ecm substrates, i.e., explants, collagen, poly-d-lysine. in the current study, we systematically studied opc migration in response to guidance factors on several different ecm substrates, to better understand the influence of ecm interactions on opc responses to chemotropic molecules. understanding the regulation of opc migration and differentiation has important implications for the treatment of demyelinating diseases such as multiple sclerosis (ms). for example, semaphorins 3a and 3f are expressed in active, but not chronic, ms lesions, and in demyelination/remyelination studies in animal models, semaphorins influence opc migration into lesions, affecting the rate at which remyelination occurs. in addition, aberrant expression of ecm ligands occurs in and around ms lesions. we performed live imaging experiments of opc migration on different ecm substrates in response to gradients of chemotropic molecules. these studies are combined with ihc, co-ip, western blot and rt-pcr analyses to determine the effects of ecm substrates on opc migration and signaling pathway responses to chemotropic molecules. our preliminarily results suggest that ecm substrate can regulate both the direction and rate of opc migration. semaphorin 3a is repulsive to opcs on laminin 1, while on fibronectin opc migration remains uniform in the presence of semaphorin 3a. since laminin 1 is the ligand for a6b1 integrin heterodimers, while opc migration on fibronectin is mediated by avb3 integrins, the repulsive effect of semaphorin 3a appears likely to function through b1 integrin. thus, signaling through the neuropilin-plexin receptor complex that underlies the repulsive effects of semaphorin 3a on opc migration may be modulated by interactions with b1 integrins. x. bai university of saarland, homburg, germany secondary injury processes after acute brain trauma involve activation of different cell types like astrocytes, oligodendrocytes and microglia cells. a complex and yet not completely understood sequence of cellular responses initiate functional recovery after the neurodegeneration process. by using double-transgenic mice gcpb (gfap-egfp 3 plp-dsred1) mice, we were able to identify a particular type of activated glia expressing astro-and oligodendroglial properties simultaneously (ao cells) that transiently appeared after three types of acute cortical injuries (stab wound injury, pial vessel disruption and middle cerebral artery occlusion). ao cells could be labeled by oligodendrocyte precursor cell (opc) markers olig2 and sox10, but not for markers of mature astrocytes or neurons. two-photon live imaging of gcpb mice revealed that ao cells originated from plp-dsred-positive oligodendrocyte lineage cells. electrophysiological inspection of ao cells in acutely isolated brain slices revealed the expression of delayed rectifying, voltage gated k 1 currents, a typical property of ng2 glia. therefore, we classified ao cells as opcs. to follow the fate of ao cells which disappeared about 15 days after the injury, we generated gfap-n-cre 3 plp-c-cre 3 rosa26eyfp triple transgenic (crec) mice. under non-injury conditions, few recombined cells were observed. however, numerous recombined cells appeared after 1 week of stab wound injury adjacent to the lesion site, and lasted over 8 weeks. we found that 55-70% of recombined cells were gfap-positive, 18-29% of them were pdgfra-positive and 19-30% were positive for the oligodendrocyte marker gstp. in line with that, about 46% of newly generated gfap-positive astrocytes were observed from ng2-creert2 x rosa26tdtomato mice after 1 week of stab wound injury. these results provide a strong evidence for opcs giving rise to astrocytes after acute cortical injuries. to further understand the molecular mechanism, we performed intra-cerebral injection of bone morphogenetic protein 4 (bmp4, known to promote astrocyte, but blocking oligodendrocyte differentiation) into gcpb and crec mice. bmp4, but not leukemia inhibitory factor (lif), significantly increased the number of ao cells as well as astrocytes in the respective mice. in conclusion, ng2/opcs display strong potential to differentiate into astrocytes after acute cortical injury. for instance, sox9 and sox10 have a crucial role in opc specification and differentiation, respectively. using microarray analysis of oligodendrocyte lineage cells, we previously identified sox17 as a major regulator of oligodendrocyte development (sohn et al., 2006) . in opc cultures, sox17 expression was maximal at developmental corresponding to cell cycle exit and onset of differentiation. these findings, suggest that sox17 has critical functions in the controlling of opc cell cycle exit and differentiation. in order to decipher the functional role of sox17 in oligodendrocyte development, we generated a tet-sox17:sox10 rtta double transgenic mouse line. this mouse strain allows a sox17 overexpression specifically in sox101 cells in a doxycycline dependant manner (tet-on system). in the cns of dox-treated transgenic animals, we showed that sox17 overexpression was specifically targeted in sox101 oligodendroglial cells. in the developing cns, sox17 overexpression was targeted in pdgfra1/nkx2.21 opcs and in olig21/cc11 differentiated oligodendrocytes. to assess the effect of sox17 gain-of-function on oligodendrocyte development and myelination, we analysed opc proliferation, apoptosis and differentiation, after doxycycline induction from e12.5 to p15. in the embryonic spinal cord, we showed that overexpression of sox17 did not modify cell proliferation or the number of pdgfra1 opcs. these data strongly suggest that sox17 is not involved in early steps of oligodendrocyte development. interestingly, we found that sox17 gain-of-function drastically reduces the density of cc11 mature oligodendrocytes at postnatal stages, correlating with a delay in myelination in transgenic mouse spinal cords. altogether, our data reveal critical functions of sox17 in oligodendrocyte lineage progression and myelination. supports: arsep and nmss (usa). m.f is a phd fellow of the french mesr (ed3c). m. rocha, p. fernandes, a.c. manhães, p.c. barradas, f. tenorio universidade do estado rio de janeiro, instituto de biologia, rio de janeiro, brazil nutrient restriction during the perinatal period exerts a profound influence on brain development. previous studies using an undernutrition protocol (0% protein diet) during the first half of the lactation period in rats showed that the offspring presented an altered feeding pattern, which reflected a metabolic imprinting effect on feeding behavior. there is a delay in the pattern of npy expression in the arcuateparaventricular (arc-pvn) pathway, reflecting an effect on the development of the hypothalamic circuitry, leading to metabolic imprinting. here we studied the effects of undernutrition on glial differentiation using the same model. we used rats from 5 to 30 postnatal (p) days of age whose dams were either fed a 0% protein diet (dg) or a normoprotein diet (cg) from p1 to p10. we assessed ki67, vimentin, gfap, ed-1 and cnpase distribution in hypothalamic nuclei using immunohistochemistry. our results showed impairment in cell proliferation, more evident in the pvn and in the lateral hypothalamus (lh), where dg animals showed a lower number of ki-67positive (ki671) cells at p5 when compared to cg. however, at p10 and p15, the number of ki671 cells was significantly increased in dg. from p20 onwards, staining intensity remained relatively stable and similar in all groups. dg animals showed a delay in astroglial differentiation, presenting a decrease in gfap expression and a peak of vimentin expression at p10 and p15 accompanied by an increase in vimentin/ki671 cells. an increase in the number of ed1 positive cells next to the third ventricle was also shown at the same ages in dg. cnpase staining was very faint in most nuclei and did not show any obvious difference in dg animals. our results suggest that glial differentiation is delayed in dg, which may contribute to the changes in hypothalamic circuitry that causes metabolic imprinting. recent studies have demonstrated that neural precursor cells (npcs) residing in the adult subventricular zone also exhibit the capacity to generate new oligodendrocytes following experimental demyelination. the relative capacities of these two cell types to contribute to oligodendrogenesis in this context remain largely unexplored. we have adopted transgenic approaches that enable either the specific labeling of npcs or oligodendrocytes and the interrogation of their subsequent fate within the corpus callosi of mice subjected to central demyelination induced by cuprizone. adult nestin-creer t2 : r26r-eyfp mice were administered tamoxifen (0.3g/kg/day) for 4 days by oral gavage, inducing the permanent expression of yellow fluorescent protein (yfp) in npcs and their progeny. mice were subsequently fed 0.2% cuprizone for 6 weeks followed by 6 weeks recovery on normal food to enable the analysis of remyelination. expression of yfp and other cellular markers was examined immunohistochemically to determine the fate and migratory potential of npcs in the remyelinating corpus callosum (cc). rostrocaudal analyses of cc in the cuprizonechallenged mice demonstrated that approximately 60% of yfp 1 npcs commit to an oligodendroglial fate. there was robust recruitment of npc-derived oligodendroglial cells, with a 14-fold increase in yfp 1 sox10 1 cells compared to unchallenged mice. most of these cells were mature oligodendrocytes (yfp 1 cc1 1 ) and their density in the remyelinating cc was highest adjacent to the dorsolateral corner of svz and decreased proportionally with distance in the mediolateral axis. on the other hand, fate mapping of opcs using pdgfracreer t2 : r26r-eyfp mice revealed that oligodendrocytes derived from parenchymal opcs were distributed in a converse manner to svz-derived oligodendrocytes. in addition, we found that, independent of the cellular source, many oligodendrocytes repopulated the corpus callosum as linear arrays of clonally derived cells, in a manner that recapitulated the postnatal development of these structures. these results demonstrate that lineage determination and maturation of oligodendroglia from npcs occurs efficiently in situ. we also reveal that remyelination occurs in a coordinated way, most likely secondary to interactions between a restricted pool of axons and a sentinel opc induced to proliferate in situ. play an important role in developmental oligodendrogenesis and myelination and mounting evidence from rodent models of demyelination suggest that they also promote remyelination. the therapeutic application of thyroid hormone to demyelinating disorders is limited by the potential for cardiac side effects mediated by thyroid hormone receptor (thr) a signaling. here we report that gc-1, a thyromimetic with selective thrb1 action promotes in vitro oligodendrogenesis from both rodent and human oligodendrocyte progenitor cells. in addition, we used pdgfar-creer;rosa26-eyfp double-transgenic mice to examine the effect of gc-1 on the fate of oligodendrocyte progenitor cells and find that treatment with gc-1 during developmental myelination promotes oligodendrogenesis in vivo. these results indicate that a b receptor selective thyromimetic can enhance ol differentiation in vitro and during developmental myelination and warrants further study in demyelinating models. the olfactory epithelial layer contains multipotent horizontal basal cells (hbcs) that differentiate into olfactory sensory neurons. we generated olfactory sphere (os) cells in cultures that were derived from adult rat olfactory mucosa. fluorescence-activated cell sorting and immunofluorescence analyses showed that os cells were derived from hbcs. os cells underwent neuronal and glial differentiation in vitro. to examine multipotent differentiation in vivo, os cells were transplanted into injured rat spinal cords. the transplanted cells integrated into host tissue and underwent glial differentiation. our data showed the stem cell properties of hbcs. oligodendrocyte progenitor cells (opcs) comprise the main cycling cell population of the cns parenchyma during the early postnatal period and at adult stages. however, the molecular mechanisms implicated in opc divisions are still by large obscure. with the aims to unveil i) whether opc divisions exploit the same molecular machinery of neuronal precursors, and ii) whether distinct opc subsets exist with different cell division machineries, we focused on a mutant mouse where citron-kinase, a crucial regulator of cytokinesis in neuronal precursors, is germinally ablated (cit-k ko). these mice have severe cns developmental abnormalities, reduction of selected neuronal populations due to apoptosis triggered by defective divisions, display ataxia, epileptic seizures and die by the third postnatal week. interestingly, they were reported to have myelin defects, suggesting that the cit-ko affects oligodendroglial cells. notably, already early after birth we found a 2-fold decrease in opc density in both the cortical grey (gm) and white matter (wm), compared to wild-type mice (wt). here, most opcs were hypertrophic and multinucleated, indicating a cytokinesis failure after nuclear division. at later ages, opcs progressively disappeared from the cortical gm, while in both wm and ventral areas of the telencephalon (i.e. striatum, hypothalamus), uni-and multi-nucleated opcs could be detected, although at lower densities compared to wt. these data suggests opc heterogeneity in both cit-k requirement for cytokinesis and susceptibility to death. however, even where normal uninucleated opcs persisted, we could not find pre-myelinating or myelinating cells, in line with additional differentiation defects. to discriminate the contribution of cell-autonomous vs. environmental factors in differentiation impairment, we performed homochronic grafts of wt fluorescently tagged (gfp1) cells into perinatal cit-ko mice. strikingly, gfp1 opcs invaded the whole cit-k ko brain and actively divided. however, they hardly ever differentiated into more mature cells at difference with cells grafted in the wt, indicating that altered environmental signals contribute to myelination defects in cit-k ko. in conclusion, our results show that cit-k is involved also in glial cell divisions and suggest distinct cit-k requirement and proneness to death of dorsal and ventral opcs. additionally, cit-k ablation results in pervasive nervous tissue alterations affecting opc maturation. further studies will clarify the molecular mechanisms underlying these alterations. knowledge of how these cells act in repair is lacking, partly due to a limited understanding of their behaviour in normal developmental processes. radial glia cells are a unique population of neural progenitors that have recently been suggested to function in neurogenesis in the cerebral cortex. however, many questions still remain regarding their specific role in the developing spinal cord. using an organotypic spinal cord slice culture model and two-photon microscopy we directly visualized radial glial cell behaviour in the developing spinal cord. 400 mm slices of rat embryonic spinal cord (e12-e16) were cultured in order to preserve the 3d cytoarchitecture of the cellular environment. using electroporation, slices were transfected with a brain-lipid binding protein (blbp) driven gfp expression plasmid, which specifically labels endogenous radial glial cells. slice cultures were then imaged using two-photon time-lapse microscopy, allowing for high spatially and temporally resolved 4d imaging. we were able to follow individual gfp expressing radial glial cells over time (24 hours to 6 days) and characterized precise morphological changes. at early developmental ages, gfp expressing cells initially exhibited a radial morphology with the soma located in the ventricular zone and processes extending out to the pial surface. over time, many cells developed a multipolar morphology and migrated away from the lumen, primarily using somal translocation. using immunohistochemistry we identified populations of transfected cells which also expressed gfap, representing the differentiation of blbp-expressing radial glia cells into astrocytes. we also found that radial glial cells differentiate into oligodendrocytes (olig2 and cnpase positive) but found little evidence for gfp co-localization with neuronal markers (neun, dcx, biiitubulin). using this model we can directly observe complex developmental behaviours of specific cell populations and elucidate the relevant genetic regulation and molecular mechanisms that govern spinal cord development. by understanding these intricate events, we will gain insight into how a simple tube of undifferentiated neuroepithelial cells generates different cell populations that will eventually develop into the adult spinal cord. methods: we used the new cre-expressing mouse strain cx 3 cr1 creer to express a diphtheria toxin receptor (dtr) specifically on microglia/macrophages. in these mice, administration of tamoxifen leads to cre-mediated excision of a loxp flanked transcriptional stop element in both macrophages and microglia, thus allowing for the expression of the dtr as well as an yfp reporter. due to their fast turnover macrophages are replaced by unaffected precursors whereas microglia persist in the modified stage. these mice were used for in vivo and ex vivo analysis of microglia by facs and histology. results: our optimized protocol with two tamoxifen injections at the age of 2 weeks resulted in 80-90% of reporter-positive microglia in adulthood, whereas all peripheral macrophages were reporter-negative. with additional three successive dt injections we could achieve a microglia ablation efficiency of 80%. interestingly, histological as well as facs analysis revealed 50% of microglia repopulation already 4 days after depletion. two weeks after depletion microglia numbers were even higher compared to unaffected controls. on day 6, we found a cd45.2 high ly6c 1 population, indicating replenishment by peripheral monocytes. in bm chimera experiments, where peripheral cells were labeled with a congenic marker, we obtained striking evidence showing that almost all of the repopulating cells are of peripheral origin. conclusions: after depletion microglia were rapidly repopulated, which emphasizes their critical role in brain homeostasis and function. even though ms has an onset in young patients, it persists throughout life and progresses along adulthood and aging. therefore, mscs from aged individuals should be tested for their pro-oligodendrogenic activity before moving into the clinical trials. this is insofar of clinical relevance, since the brain's capacity for self-repair and remyelination strongly declines with aging. thus, the aim of this study was to determine whether mscs from aged individuals maintain their known prooligodendrogenic activity. mscs were obtained from bone marrow of young (2 months) as well as old (17-20 months) rats and analyzed their stem cell properties as well as their pro-oligodendrogenic activity. as expected, mscs derived from aged bone marrow presented diminished stem cell properties and differentiation capacity. to test the msc prooligodendrogenic activity, npcs were incubated with msc conditioned medium. surprisingly, soluble factors derived from old rats mscs maintain their ability to induce an increase of mbp-expressing oligodendrocytes on npcs. moreover, aged mscs conserve their oligodendrogenic activity also on npcs derived from old donors (20 months). these results indicate that mscs keep their oligodendrogenic activity regardless of age and, therefore, this study provides a rationale for clinical trials of autologous msc transplantation in ms therapy. 6 munich cluster for systems neurology (synergy), munich, germany astrocytes fulfill essential functions under physiological conditions and are involved in various beneficial and adverse functions after brain injury (reviewed by sofroniew 2009). in order to improve functional recovery after injury, it is essential to delineate the beneficial from adverse effects. however, little is yet to know which extent distinct sets of astrocytes perform distinct functions or whether all astrocytes behave in a uniform manner. live imaging of astrocytes after stab wound injury in the adult cerebral cortex in vivo revealed a striking heterogeneity of astrocyte behaviour with distinct subsets extending long processes towards the site of injury, others proliferating and yet other subtypes undergoing only hypertrophy. most strikingly, the vast majority of astrocytes proliferating were located with their somata directly at blood vessels (capillaries or post-capillary vessels). this close proximity was confirmed at ultrastructural level and identified as juxtavascular positions, sometimes adjacent to pericytes located in the perivascular space. this novel population of reactive astrocytes at juxtavascular positions is of particular interest, as the capacity of astrocytes to reactivate proliferation ( cell stem cell, in press). therefore we examined whether this population of juxtavascular astrocytes would also preferentially proliferate in even more severe injury conditions, such as 1 hour of middle cerebral artery occlusion (mcao). the proliferation of juxtavasular astrocyte was analyzed immunohistochemically in fixed brain sections co-stained for s100b/gfap/ki67/cd31 at different time points after injury. at 3 days post injury, 18% of all astrocytes were actively dividing (ki671). strikingly the majority of these was again located at juxtavascular positions (68%), suggesting a general bias of astrocytes at this position to proliferate after injury, while astrocytes further distant from blood vessels virtually fail to divide. we shall further present data from conditional knock-out mice with reduced proliferative activity of juxtavascular astrocytes in order to elucidate the function of this highly specific astrocyte population and their proliferative reaction after injury. a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom the generation of oligodendrocytes (ols) from their precursors (opcs) occurs throughout adult life and is particularly important following demyelinating insults, such as occurs in multiple sclerosis (ms). glycogen synthase kinase 3-b (gsk3b) acts as a constitutively active negative regulator of multiple pathways that regulate proliferation, survival and differentiation of opcs. lithium is a potent inhibitor of gsk-3 and is used for the treatment of bipolar disorder, but in addition is neuroprotective in a wide range of diseases, including stroke, amyotrophic lateral sclerosis, and alzheimer's disease. lithium inhibits gsk-3 by binding directly to the enzyme's magnesium-sensitive site and indirectly by enhancing its phosphorylation. notably, by inhibiting gsk3b, lithium acts as a wnt mimetic to promote b-catenin-dependent transcriptional events, including enhancement of genes involved in apoptotic inhibition. here, we have examined the effects of lithium on oligodendrocytes in the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act (1986) . transgenic mice aged postnatal day (p) in the developing and adult cns multipotent neural stem cells reside in distinct niches. specific carbohydrates and glycoproteins are expressed in these niche microenvironments that are important regulators of cell fate determination and stem cell maintenance. in previous studies we described lewisx (lex) glycan as a specific marker of neural stem precursor cells (nspcs) in developing brain and showed it to be involved in nspcs migration and proliferation. using lex glycosylation as a biomarker we aimed to identify the lex carrier glycoproteins which could have a functional relevance for cns development. in addition to already known lex carriers we have found lrp1 as new major lex carrier and for the first time showed it's expression in nspcs and developing brain. lrp1 is essential for the normal neuronal function in adult cns, whereas the role of lrp1 in development is still unclear, mainly due to the lethality of the lrp1 knock-out in mice. in the current study we investigated the basic properties of lrp1 knock-out nspcs created by means of cre-loxp mediated recombination. the elimination of lrp1 in vitro was induced by the addition of cell permeable cre-recombinase to nspcs derived from embryonic brain of lrp1flox/flox mice. the functional status of lrp1-deficient cells was subsequently studied using proliferation, migration and differentiation assays. lrp1 knock-out cells maintained as neurospheres, retained the ability to migrate and differentiate. interestingly, lrp1 knock-out nspcs generated 3-times less opcs in comparison to lrp1wt/wt cells. this suggests that lrp1 is involved in the control of the oligodendroglial differentiation. astrocytes are multifaceted cells in the adult central nervous system and react to pathology of the adult brain with a finely graded continuum of morphological and behavioral changes. given that astrocytes in healthy adult brain rarely divide outside the stem cell niches, but activate both proliferation as well as a stem cell potential after injury (for review see robel et al., 2011) , it is important to understand their exact mode of cell division. indeed, a defining hallmark of a stem cell is the capacity to self-renew -often by asymmetric cell division. therefore we set out to image reactive astrocyte divisions in vitro by developing a primary culture system for reactive astrocytes obtained from the somatosensory cortex of adult mouse at 5 days after stab wound injury. continuous live imaging and single cell tracking (costa et al., 2008 (costa et al., , 2011 allowed us observing the mode of cell division mode as well the cell fate of their immediate progeny. results of our study reveal (i) that reactive astrocytes can undergo multiple rounds of cell division, (ii) the shh signaling has a profound influence on this behavior, (iii) reactive astrocytes divide with distinct modes of cell division, which can be influenced by the local microenvironment. taken together, this new culture system allows close examination of signals on the mode and multitude of reactive astrocyte cell divisions and provides a great tool to identify signals derived from the extracellular matrix, other cell types or as diffusible signals. interestingly, even without injury temporary depletion of proliferating opcs leads to behavioral deficits which will be described. to identify the molecular mechanisms by which ng2 1 cells may react and influence other cells after brain injury, we isolated sox10-gfp 1 cells by facs either from the intact or the lesioned cerebral cortex at three days after stab wound injury. comparative genome-wide expression profiling revealed exciting candidates for opc function in the physiological and pathological brain. oligodendrocytes are the myelin forming cells of the central nervous system (cns) that enable fast salutatory signalling transduction and mediate trophic support and protection of axons. oligodendrocytes originate from a population of precursors cells (oligodendrocyte precursor cells, opcs) that are formed at distinct developmental stages. the biological process leading to opc differentiation involves a cascade of molecular events that eventually constitute the complex and multifaceted cellular program that determines the generation of mature oligodendrocytes. to identify the earliest detectable transcriptional correlates of differentiation we used a microarray approach investigating changes in gene expression occurring in primary rat opcs within the first 12 hours after purification. amongst the most regulated factors that were detected was hairless (hr), a nuclear receptor co-repressor that has been associated with thyroid hormone signalling in the neonatal cns. validation of the microarray results by rt-qpcr, western blot and immunochemistry confirmed a rapid increase of hr expression during opc differentiation in media containing thyroid hormone. in the absence of thyroid hormone, hr expression was suppressed and associated with impaired opc differentiation. similarly, rnai mediated gene silencing of hr induced an impairment of opc differentiation demonstrating an important functional role of hr in the differentiation program. thyroid hormones regulate hr expression via the thyroid hormone receptors thra and thrb as rnai-mediated gene silencing of each of the receptors resulted in a decrease in hr expression. hr is known to negatively regulate rar-related orphan receptor alpha (rora) and vitamin d receptor (vdr). however, rnai-mediated gene silencing of rora and vdr did not change the course of opc differentiation. interestingly, an interaction between hr and histone de-acetylases (hdacs), of which hdac1 and 2 are known regulators of opc differentiation, has been noted in the past. using an in situ protein-protein interaction assay we were able to demonstrate a direct interaction of hr with hdac1 and hdac2 in differentiating opcs. moreover, chip qpcr analysis revealed hr-hdac1 recruitment to the opc differentiation inhibitor hes5. taken together these data show that thyroid hormone promotes opc differentiation via hr by modulating hdac1 and 2 function. as iodine deficiency results in hypothyriodism our results give an example of a developmental disease that is triggered by environmental factors (the absence of iodine) causing epigenetic dysregulation. c. stagni, a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom astrocytes respond to all forms of cns insults by reactive astrogliosis, which involves changes in their molecular expression and morphology, and in most severe cases glial scar formation. reactive astrogliosis is potentially neuroprotective, but can also act as chemical and physical barrier to axon growth and repair. however, mechanisms underlying this process are still poorly understood. the enzyme glycogen synthase kinase 3-b (gsk-3b) is a key regulator of many signalling pathways involved in cell proliferation, survival and differentiation, including wnt signalling. lithium is a potent inhibitor of gsk-3 and acts as a wnt mimetic to promote b-catenin-dependent transcriptional events. wnt signalling is altered following cns injury and in a range of neurodegenerative diseases, such as stroke, amyotrophic lateral sclerosis, and alzheimer's disease. notably, lithium is neuroprotective in these same diseases. here, we have examined the effects of wnt3a and lithium on astrocytes of the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act (1986). we used transgenic mice aged postnatal day (p) 35 inwhich gfap drives the expression of egfp. optic nerves (ons) were isolated with the retina intact and maintained in organotypic culture for 3 days in vitro (div), in control medium, or medium containing lithium chloride, or wnt3a. both agents resulted in a significant increase in the number of astrocytes, compared to controls (p < 0.05, unrelated t-tests), but lithium and wnts3a had markedly divergent effects on astrocyte morphology. in the presence of lithium, astrocytes became highly polarised, extending long thin processes that traversed the nerve perpendicularly to the long axis. in contrast, wnt3a treatment resulted in the generation of morphologically simple astrocytes, with short, fine branching processes that extended radially from a centrally located cell body. genome wide microarray analysis indicated wnt3a and lithium differentially altered chondroitin sulphate proteoglycans (cspgs), a family of extracellular matrix molecules highly expressed at cns injury sites. further experiments are required to define the molecular phenotypes of the novel astrocytes generated by these treatments, but the results provide evidence that wnt signalling regulates astrogenesis in situ and is therefore a potential molecular target to modulate astrogliosis. the contrasting effects of lithium suggest it is acting via other pathways, which require further study. studies of mouse schwann cell culture. the proliferation of scs is regulated by axonal signals and several growth factors. growth factors that are essential for rodent sc survival in culture include heregulin-b1 and forskolin. in the case of rat scs, these factors are also sufficient to allow a robust proliferation. in contrast, the proliferation of mousederived scs in culture requires the presence of other growth factors. we studied effect of several factors known to be expressed in scs after nerve injury on mouse sc proliferation. these included fibroblast growth factor type 2 (fgf), pituitary extract (pe), epidermal growth factor, platelet-derived growth factor bb, and transforming growth factor b1. we found that heregulin-b1 and forskolin are essential for mouse sc to survive in culture, fgf and pe were the best combination of growth factors promoting the mouse sc proliferation, and that neuronal axons provide effective mitogenic environment for mouse scs. the expression of the large myelin-associated glycoprotein in pns was studied in scs cultured alone and in cocultures of scs and drgns. when schwann cells are alone without axons, expression of the cytoplasmic domain of l-mag is seen in the nucleus of schwann cells. when schwann cells make contact with neuronal axons, the location of the expression shifts out of the nucleus to the perinuclear and cytoplasmic regions; the extracellular domain is not visible. until the schwann cells initiate the myelination programme. these results support the hypothesis that l-mag might have role in the regulation of the myelination programme in the pns. it has been postulated that trophic support provided by either stem cells or precursors is actuallyone of the most beneficial effects in the cell-based therapies. to address this issue, a comparison between the neuroprotective properties of opcs and the stem cells derived from warton jelly was carried out in the co-culture system with ischemically injured organotypic hippocampal slices. those two cell types were selected in respect of the advancement in their differentiation process. while the mesenchymal stem cells residing in warthon jelly are still pluripotent, the opcs are already the glia-committed precursors. to evaluate their neuroprotective properties, we set -up an ex vivo co-culture system of either stem cells or opcs isolated from neonatal rat brain with organotypic hippocampal slices subjected to a short oxygenglucose deprivation (ogd). the cell death, as well as the number of the newborn cells has been quantified in slices co-cultured for one week. a microarray analysis of a broad spectrum of trophic factors and cytokines expressed by opcs was performed for the purpose of finding the factor(s) contributing to the observed effect. three of them were selected for the subsequent blocking experiments with specificl antibodies to verify their influence on the injured tissue. the results obtained in the study prove that both cell types secrete soluble factors and are potent in exerting the neuroprotective effect in a paracrine-like manner, promoting cell survival and proliferation in damaged hippocampal slices. in conclusion, the observed advantageous qualities of stem cells and oligodendroglia-biased progenitors significantly contribute to anticipating a clinical improvement in cell replacement therapies. ng2 is a type i transmembrane glycoprotein and also known as chondroitin sulphate proteoglycan 4 (cspg4). in the central nervous system ng2-expressing cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes in the developing white matter. for temporally-controlled gene targeting of ng2 glia in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of cre recombinase (creert2) was inserted into the ng2 locus by homologous recombination. here, we investigated the differentiation potential of ng2 glia at different developmental stages of the forebrain. cre recombinase activity was induced at embryonic day 17.5, postnatal day 0 (p0), p3, p8, p30 and p60 by intraperitoneal tamoxifen injections into novel tgh(ng2-creert2) mice crossbred to rosa26-tdtomato and rosa26-eyfp reporter lines that helped to identify ng2 glia and its progeny. recombination and cell identification were determined 10 to 60 days after the first tamoxifen injection. induction of recombination at embryonic stages revealed that embryonic ng21 cells mainly generated more ng2 glia and oligodendrocytes, however, a significant number of astrocytes could be detected as well. recombined cells were predominantly restricted to the ventral brain. in contrast, after tamoxifen injections at p0 and p3 recombined cells were widely found in all brain regions, thereby suggesting the presence of a distinct subpopulation of ng21 cells after birth. interestingly, only very few recombined astrocytes could be found, which are probably derived from few remaining embryonic ng2 glia. starting from p8, ng2 glia stopped generating astrocytes, with the progeny largely restricted to the oligodendrocyte lineage. however, consistently, we found recombined neun1 cells with the morphology of neurons in the ventral cortex after administration of tamoxifen at p8. even more of such cells were present in the whole cortex when cre activity was induced in adult mice. the morphology, the presence of neun immunoreactivity and our electrophysiological characterization that demonstrated bona fide action potentials clearly identify these cells as functional neurons. our results suggest that ng21 cells display a broad differentiation potential that appears to be highly age-dependent during development. brdu-assay) and the number of surviving cells (by counting dapi-stained nuclei). after we had observed, that the choice of the basal medium (neurobasal, rpmi or dmem) exerts a pronounced effect on opc proliferation we here investigated the effect of cell density on opc proliferation. starting from a small seeding density (10.000 cells/cm 2 ) a tenfold increase in seeding density only resulted in a fourfold augmentation of the density of surviving cells after 7 days in culture. using plating densities of 5.000; 20.000 and 80.000 cells/cm 2 we observed, that an increase in seeding density led to a decrease in the proliferation rate, as tested with a brduassay and monitored after 3 days in culture. similarly, the percentage of a2b5-positive, immature, cells was lower, the higher the cell density. we then tested whether this finding is due to proliferation inhibiting factors secreted in cultures with high seeding densities and whether the remaining microglia population could be involved. to this aim we seeded opcs at low density (5.000 cells/cm 2 ) and cultivated them with the medium supernatant of cultures with high density (80.000 cells/ cm 2 ). furthermore we seeded opcs in low density (5.000 cells/cm 2 ) and co-cultivated them with an added density of microglia as counted in cultures of high density (80.000 cells/cm 2 ). the resulting cell counts after 3 days showed, that the percentage of brdu positive cells after cultivation with the supernatant was significantly reduced compared with control cultures, although the number of a2b5-positive cells was not influenced by this treatment. the addition of microglia to the cultures lead to a strong reduction of surviving cells, even though the percentage of brdu and a2b5 positive cells were not altered. so apoptosis, necrosis or phagocytosis might be responsible for the reduction of surviving cells. our findings suggest that the reduction of surviving opcs in high density cultures is due to secreted factors inhibiting opc proliferation and that microglia could be involved in this effect. in neuropathological studies, it is important to detect microglial cells; however, a specific microglia marker has yet to be determined. [methods] in this study, we examined and compared the usefulness of various microglia markers, including human glucose transporter-5 (glut-5), mhc class ii, cd68, and iba-1. [results] all of the microglia markers consistently stained for microglia in paraffin sections fixed with formalin. cd68 staining of microglia in the gray matter was not as well defined as the other microglia markers. moreover, cd68 did not clearly stain the microglial processes. with regard to mhc class ii, it was difficult to observe the microglial processes in the white matter. in contrast, both glut-5 and iba-1 clearly stained for microglia in the gray and white matter, and provided detailed staining of microglial processes. although it has previously been shown that perivascular cells are negative for glut-5, we found perivascular macrophages to be glut-5 positive. [conclusions] nevertheless, glut-5 and iba-1 may be considered as markers suitable for routine histopathological staining procedures. neurons and glia of the enteric nervous system (ens) originate from a pool of sox101 progenitors. in addition to being a scaffold for enteric neurons, enteric glial cells (egcs) have been suggested to function in mucosal integrity, neuroprotection, adult neurogenesis, neuro-immune interactions and synaptic transmission. currently, diverse glial populations are known to reside within the ens. however, whether, specific functions are carried out by specialized glial subtypes, with characteristic morphologies and molecular markers and within specific locations in the gut remains elusive. to address this question we used a repertoire of genetic tools, immunostaining and imaging techniques. for high single-cell resolution study of egcs, we mosaic analysis with double markers (madm) in conjunction with sox10-cre. we analyzed adult gut tissue to characterize the different glial populations based on morphology, position in the ens and their relationship with the intestinal epithelial and vascular system. moreover, we used reporter mice (sox10-icreer t2 ; r26r-yfp and gfap-icreer t2 ; r26r-yfp) to quantify the expression of three glial markers -gfap, s100b and sox10 in myenteric plexus (mp) preparations of adult mice. our investigation on madm-mp preparations, revealed three subtypes of enteric glia in the mp of adult mice. type i and type ii glia, present in the primary plexus of mouse ens have been previously defined. in addition, we characterized a third subtype in the extraganglionic space of the mp of adult mice, at present termed as type iii-extraganglionic glia. furthermore, our analyses using 3d-imaging, on madm-gut sections allowed us to characterize the relationship of enteric glia located within the mucosa, with the intestinal epithelium and vascular system of the villi. our marker expression analysis showed that while the majority of glial cells co-expressed gfap and s100b, a large fraction of glia within the myenteric ganglia was positive for either s100b ($30%) or gfap ($10%). we also found that s100b 1 /gfapglia were abundant outside the ganglia. most glial cells co-expressed sox10 with gfap and s100b yet a significant proportion ($10%) of mainly extra-ganglionic glia (type iii) expressed only sox10. surprisingly, $5% of the cells that were s100band sox10displayed high gfap expression. use of gfap-icreer t2 ; r26r-yfp mice showed that about $30% of yfp-labelled cells did not express gfap, implying dynamic regulation of gfap expression. overall, our high resolution studies reveal extensive heterogeneity of enteric glial cells in terms of morphology, position and expression of molecular markers suggesting differential functional roles. our future experiments will address whether different physiological roles of egcs can be assigned to specific subtypes. university of washington, seattle, united states m€ uller glia are the major type of glia in the retina, spanning its entire thickness. it is known that bmp signaling promotes astroglial differentiation in cortex, but its role in m€ uller glial development in the retina has not been studied. the purpose of this study was to investigate the role of bmp-smad1/5/8 signaling in m€ uller glial development. c57bl/6 mouse retinas were collected at various ages between postnatal day 0 (p0) and p21 in order to analyze the expression and activation of smad1/5/8. smad1/5/8 was transiently but robustly activated in the inner nuclear layer, including developing m€ uller glia between p5-p8. the timing of this transient activation of smad1/5/8 corresponds with the timing of m€ uller glial differentiation and maturation. to test the hypothesis that activation of bmp-smad1/5/8 signaling is essential for m€ uller glial development, retinas were explanted at p3 or p6 and treated with bmp4 or a bmp inhibitor dorsomorphin (dm) for 5 days in order to activate or inhibit smad1/5/8, respectively. when p3 and p6 retinal explants were treated with dm for 5 days in vitro, there was a significant reduction in m€ uller glial markers (cralbp, gs, sox9, and sox2). smad1/5/8 activation and cralbp expression were also significantly attenuated in vivo 1 day after intraocular injection of a bmp inhibitor dm at p5. our data suggest that activation of bmp-smad1/5/8 signaling is necessary for the development of m€ uller glia. this work was supported by 1r01ey021482 to tar. huazhong university of science and technology, wuhan, china rho-associated kinase (rock) has been identified as an important regulator of proliferation and cell cycle progression in a number of cell types. although its effects on astrocyte proliferation have not been well characterized, rock has been reported to play important roles in gap junction formation, morphology, and migration of astrocytes. in the present study, our aim was to investigate the effect of rock inhibition by [(1)-(r)trans-4-(1-aminoethyl)-n-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] (y27632) on proliferation and dna synthesis in cultured astrocytes from rat spinal cord and the possible mechanism involved. western blots showed that treatment of astrocytes with y27632 increased their expression of cyclin d1, cdk4, and cyclin e, thereby causing cell cycle progression. furthermore, y27632-induced astrocyte proliferation was mediated through the extracellular-signalregulated kinase signaling cascade. these results indicate the importance of rock in astrocyte proliferation. here we used cell transplantation experiments to determine to which extent this differential behavior is regulated by environmental signals differing between the wm and gm, or intrinsic fate determinants restricted to one or the other cell population. these experiments indicate that the wm promotes differentiation of opcs to mature oligodendrocytes, as the rate of differentiation of gm cells increased when exposed to this environment. interestingly, cells derived from the wm retained their higher differentiation rate also in a less supportive environment like the gm. these results reveal not only important environmental differences for opc maturation, but also support the concept of intrinsic heterogeneity among adult opcs, persisting even when exposed to more inhibitory environments. thus, this work provides the basis to identify molecular pathways regulated by distinct niche/environmental signals and involved in the heterogeneity of adult opcs. multiple sclerosis (ms) is a chronic inflammatory and neurodegenerative demyelinating disease of the central nervous system (cns) characterized by inflammation, which leads to formation of demyelinating areas due to loss of oligodendrocytes, astrogliosis and, finally, axonal degeneration. different studies have suggested that 3 0 -5 0 -cyclic adenosine monophosphate (camp) levels might play an important role in neuroprotection and neuroinflammatory response so the modulation of this nucleotide intracellular levels might control the neuroinflammatory pathological process and, consequently, to delay the progression of ms. intracellular camp levels depend on its synthesis by adenylyl cyclases, and on its degradation by cyclic nucleotide 3 0 ,5 0 -phosphodiesterases (pdes). specific inhibitors for the different isoforms of pdes family, particularly camp-specific pde, emerge as putative treatments of this kind of disease. pde7 has emerged as a new therapeutic target, not only for a variety of immunological and immunodeficiency conditions to alleviate chronic inflammation, but also for several neurodegenerative disorders, including ms, in which both immune system and cns are implicated. in the present work, we have detected the expression of pde7 in oligodendrocyte precursor cells (opcs) isolated from cerebral cortex of p0 and p15 mice and from adult human samples derived from neurosurgery of epilepsy. opcs are abundant in the mice and human cns during development but they also represent 5-7% of the total number of cells in the adult cns, where they serve as a source of new oligodendrocytes to replace those which die. however, in ms these endogenous opcs are not able to effectively replace dead oligodendrocytes. in vitro, we have demonstrated that two newly-developed pde7 selective inhibitors (tc 3.6 and vp 1.15) favour opc survival and differentiation towards mature oligodendrocytes, being both more effective than the commercial pde7 inhibitor brl-50481. erk intracellular pathway has been identified as a key in these cellular processes related with the camp/pka/creb pathway. moreover, we have observed that both specific inhibitors (vp1.15 and tc3.6) increase the differentiation of human opcs. these findings, combined with the already known anti-inflammatory effect carried out by these pde7 inhibitors, point them as potential therapeutic agents to treat ms. the knowledge of factors that affect the biology of murine opcs and even more, human opcs, are critical for possible remyelination in this kind of pathologies. their role in cns remyelination has not been fully investigated. previous studies using genetic fate mapping techniques revealed their recruitment into injured spinal cord and differentiation into astrocytes and oligodendrocytes. in this study, we used aninducible cre-floxed-stop-rosa26-yfp mediated lineage tracing strategy to characterize the fate of foxj1 expressing ependymal cells during remyelination of toxin-induced demyelination. we found that in unlesioned spinal cord, foxj1 labelled ependymal cells. however, it also labeled a population of cells in the spinal roots with immnochemical features of fibroblasts. following lysolecithin-induced demyelination in the dorsal funiculus, there were few yfp expressing cells in the lesion area at 5 and 14 days post lesion (dpl) indicating that the ependymal cells were not recruited in demyelinated area. however, in ventral white matter lesions, which were adjacent to damaged nerve roots, many cells were detected expressing yfp. the distribution of yfp positive cells overlapped that of remyelinating schwann cells and a number of yfp expressing cells colocalised with mature schwann cell marker periaxin. these data revealed a foxj1 expressing population in peripheral nerve which migrates into demyelinated lesions andcontributes to cns remyelinaiton. the generation of myelinating cells from multipotential neural stem cells in the cns requires the initiation of specific gene expression programs in oligodendrocytes (ols). we reasoned that micrornas (mirnas) could play an important role in this process by regulating genes crucial for ol development. here we identified mir-7a as one of the highly enriched mirnas in oligodendrocyte precursor cells (opcs), overexpression of which in either neural progenitor cells (npcs) or embryonic mouse cortex promoted the generation of ol lineage cells. blocking the function of mir-7a in differentiating npcs led to a reduction in ol number and an expansion of neuronal populations simultaneously. we also found that overexpression of this mirna in purified opc cultures promoted cell proliferation and inhibited further maturation. in addition, mir-7a might exert the effects just mentioned partially by directly repressing proneuronal differentiation factors including pax6 and neurod4, or prool genes involved in oligodendrocyte maturation. these results suggest that mirna pathway is essential in determining cell fate commitment for ols and thus providing a new strategy for modulating this process in ol loss diseases. a. guzman de la fuente 1 , j. huang 1 new sheaths can be regenerated following the recruitment of adult neural progenitor cells (opc) into areas of injury and their differentiation into myelinating oligodendrocytes. although this regenerative process (called remyelination) occurs efficiently in the early stages of multiple sclerosis, its efficiency gradually declines leaving axons demyelinated and vulnerable to degeneration. several signalling pathways are involved in regulating opc differentiation and subsequent myelination. understanding their mechanisms is necessary to design regenerative therapies to overcome remyelination failure. we have identified rxrc as a key positive regulator of opc differentiation and remyelination (huang et al., 2011). rxrc is a nuclear receptor forms a heterodimer with other nuclear receptors to activate its downstream signalling cascades. here we describe rxrc binding partners expressed in both opc and oligodendrocytes, and the binding of vdr, rarb and pparc to rxrc within oligodendrocyte lineage cells by coimmunoprecipitation. vdr-rxrc heterodimer is necessary for the oligodendrocyte differentiation. treatment of oligodendrocytes with vdr antagonist inhibits opc differentiation and abrogates the effect of 9-cis retinoic acid, an rxrc agonist, in opc differentiation. our data suggests a model in which rxrc is an essential anchor for different nuclear receptors, including vdr, with a shifting and complex rxrc signaling in oligodendrocyte lineage cells. cleavage of type i membrane proteins by a-and c-secretases to yield biologically active fragments is best exemplified for the evolutionarily conserved protein notch. here we show that the protein ng2, a type i membrane protein which has homologues in mouse (ng2) and drosophila (kontiki) and is expressed by multiple immature cell types including glia, is processed in a similar fashion to notch. in both mouse and drosophila this generates a major 290 kd shedded ectodomain, a c-terminal fragment (ctf) and a small intracellular domain (icd). the ng2 icd is present after overexpression in cells of biochemical isolates of nuclear fractions, and can be visualized in the nucleus by immunofluorescence of cultured murine glial cells. in glial nuclei of developing drosophila larva, a striking intranuclear staining of the kontiki icd is observed. levels of expression of ng2 mrna and protein in mouse glial cells are regulated by the neurotransmitter glutamate and overexpression of cleaved fragments modulates protein expression in murine glial cells. these results demonstrate that cleavage of ng2 takes place in mouse and drosophila glia and that this generates fragments with signaling properties. modulation of ng2 expression by glutamate provides a way for neuronal activity to regulate ng2 signalling. our goal is to understand the glial signaling that controls inflammatory responses in the central nervous system (cns). this glial response can play both a detrimental and beneficial role. toll like receptor (tlr) signaling is implicated in responses to pathogens or endogenous signals. tlr signaling mediates immune response by inducing cytokines, including type i interferons (ifn). ifn-beta, a member of the type i ifn family, is a first-line therapeutic for multiple sclerosis. type i ifns signal through a common receptor, ifnar. the aim of the present study was to investigate the in vivo response of microglia and astrocytes to cns administration of tlr ligand/agonist, and to examine whether this response involves type i ifn signaling. mice were administered tlr ligands/agonists by injection to the cisterna magna. we analyzed leukocyte entry to the cns by flow cytometry, and their localization by immunostaining. the induction of ifn-beta was examined by in vivo imaging of an ifn-beta reporter mouse. astrocytes and microglia were sorted by facs and gene expression was measured using quantitative real time-pcr. injection of ligands/agonists for tlr2, 3 and 4 resulted in infiltration of cd451 leukocytes after 18 hrs, most strongly by tlr2. immunostaining showed parenchymal localization of infiltrating cells in cerebellum. facs sorted astrocytes expressed equivalent levels of tlr3 mrna to microglia but lower levels of tlr2 or 4. astrocytes were induced by tlr3 signaling to express interferon regulatory factor 7, which regulates the induction of type i ifn. the induction of ifnbeta in cns in response to tlr3 signaling was verified in ifn-beta reporter mice. tlr2, 3 and 4 signaling led to increased levels of mrna for glial cxcl10. together these results suggest the involvement of type i ifn signaling. however, unlike cxcl10 gene expression, that was dependent on ifnar signaling, tlr2-induced leukocyte infiltration was not affected in ifnar deficient mice. these studies point to a role for tlr signaling in the innate glial response that regulates cns inflammation. microglial phagocytosis plays a key role in neuroprotective and neurodegenerative responses of the innate immune system in the brain. here we investigated the regulatory function of the signaling protein phosphoinositide 3-kinasec (pi3kc) in phagocytosis of bacteria and zymosan particles by mouse brain microglia in vitro and in vivo. using genetic and pharmacological approaches our data revealed pi3kc as an essential mediator of microglial phagocytosis. unexpectedly, microglia expressing lipid kinase deficient mutant pi3kc exhibited similar phagocytosis as wild-type cells. detailed analysis disclosed pi3kc dependent stimulation of camp phosphodiesterase as a crucial mediator of phagocytosis. both stimulation of protein kinase a and epac were shown to convey inhibitory effects of camp on phagocytosis. together our findings indicate pi3kc-dependent suppression of camp signaling as an indispensible regulatory element of microglial phagocytosis. v. kovaleva, e. berezhnaya, m. rudkovskii, a. uzdensky southern federal university, rostov-on-don, russian federation photodynamic therapy (pdt) is currently used in oncology, particularly, in treatment of brain tumors. the possible role of no-mediated signaling in photodynamic injury and protection of neurons and surrounding glial cells (gc) was studied. crayfish stretch receptor consisting of a single neuron enveloped by gc was photosensitized with alumophthalocyanine photosens (10 nm, 30 min incubation) and irradiated with laser diode (670 nm, 0.4 w/cm2). application of no generators notably 10 mm sodium nitroprusside and 10 mm nonoate reduced pdtinduced necrosis of gc and showed the same tendency in neuronal necrosis. nonoate significantly increased pdt-induced apoptosis of gc. inhibitor of neuronal no-synthase l-name (1mm) significantly increased the percentage of necrotic glial cells after pdt but did not influence neuronal necrosis. this confirmed the anti-necrotic effect of no in glial cells and involvement of neuronal no synthase in protection of glial cells. 1 mm l-name and 1 mm l-nna (another inhibitor of neuronal no-synthase) as well as 50 lm s-methhylisothioharnstoff sulfat (inhibitor of inducible no synthase) protected gc from pdtinduced apoptosis. therefore no may be involved in pdt-induced apoptosis of glial cells. inhibition of no-activated protein kinase g with 10 lm kt5823 decreased the percentage of necrotic glial cells but not neurons. therefore protein kinase g appeared to be involved in pdtinduced necrosis of gc, possibly independently on no. kt5823 also increased the level of apoptosis of gc indicating the anti-apoptotic role of protein kinase g. thus no is involved in regulation of pdt-induced necrosis of neurons and glial cells as well as in apoptosis of glial cells. g. tyzack, t. cymes, n. lau, a. lakatos university of cambridge, cambridge, united kingdom background and question. emerging evidence suggests that signalling by damaged neurones leads to reactive astrocyte response that may have a fundamental impact on synaptic reorganisation. soluble factors, such as cytokines, have been described to induce such astrocyte activation. however, rapid contact dependent signalling between damaged neurones and astrocytes is also a possibility. in light of recent findings of ephb1 upregulation on injured neurons closely surrounded by reactive astrocytes, we hypothesized that ephb1 can signal to ephrin-b1 expressing astrocytes. methods. to test this in simplified in vitro assays, purified astrocyte cultures were treated with clustered-ephb1-fc, and the characteristic features of glial activation, such as cytoskeletal protein expression, stat3 phosphorylation, and p-stat3 nuclear transfer were monitored. results. we have demonstrated that 1) astrocytes express ephrin-b1, 2) ephb1 triggers a two-fold increase in gfap and ezrin expression, 3) ephb1 induces both stat3 phosphorylation and nuclear transfer by a three-fold value, and 4) all effects were significantly diminished by knocking down ephrin-b1 in astrocytes. conclusions. our data show that ephb1 induces astrocyte activation by triggering ephrinb1-mediated reverse signalling via stat3 activation. this work therefore suggests a potential novel route in neuron-astrocyte communication after injury. a further characterisation of this signalling pathway may provide a better understanding of mechanisms underlying glial activation and its role in plasticity. n. kramann 1 , r. pf€ ortner 1 , u.-k. hanisch 1 , k. hagemeier 2 , t. kuhlmann 2 , w. br€ uck 1 , c. wegner 1 1 university medical center g€ ottingen, department of neuropathology, g€ ottingen, germany 2 university hospital m€ unster, institute of neuropathology, m€ unster, germany introduction: laquinimod (laq) is an oral well-tolerated molecule that has been shown to reduce brain atrophy, disability progression and relapse rate in patients with relapsing-remitting multiple sclerosis. recent experimental data indicate that laq minimizes demyelination, inflammation and axonal damage in mice with cuprizone challenge. aim: since astrocytic nfjb activation plays a crucial role in cuprizone-induced demyelination, we investigated the effects of laq on cns cells in vitro and in vivo. methods: in vitro experiments used primary cells to test effects of laq on oligodendroglial survival as well as on cytokine secretion and nfjb activation in astrocytes and microglia. primary mouse astrocytes and microglia were pre-treated with 0, 0.25 and 2.5 mm laq and stimulated by pro-inflammatory cytokines. a dual-luciferase reporter assay was used to measure nfjb activity. for in vivo experiments, 10-week-old male c57bl/6j mice were challenged with 0.25% cuprizone and treated daily with laq (25 mg/ kg) or water. to assess astrocytic and microglial nfkb activation in vivo, nuclear translocation of nfjb p65 was assessed in astrocytes and microglia by double immunofluorescence with antibodies against p65 and iba1 or gfap. results: in vitro, astrocytic but not microglial nfjb activation was markedly reduced by laq as evidenced by nfjb reporter assay. in astrocytes, pre-treatment with 0.25 and 2.5 mm laq significantly reduced the induced nfjb activity after tnfa stimulation compared to stimulated controls. pre-treatment with 2.5 mm laq also significantly reduced nfjb activation after stimulation with the combination of il-1b and ifnc compared to untreated stimulated controls. oligodendroglial viability and survival were not affected by laq treatment. to confirm the in vivo relevance of these findings, we also examined astrocytic and microglial p65 translocation in mice with and without laq treatment after cuprizone challenge. the proportion of astrocytes with nuclear p65 immunoreactivity was significantly reduced in laqtreated mice (14.0% 6 0.9%) compared to untreated controls (25.8% 6 1.1%). microglia did not display marked translocation of nfjb p65 after cuprizone challenge. conclusion: our data indicate that laq prevents cuprizoneinduced demyelination by attenuating astrocytic nfjb activation. in vitro, laq reduced the astrocytic nfjb activation by up to 46% as evidenced by nfjb reporter assay. similar quantitative findings were obtained in vivo when laq treatment also led to a 46% reduction of astrocytes with nfjb activation, as evidenced by nuclear translocation of p65. these findings suggest that targeting the astrocytic nfjb pathway might have therapeutic effects in demyelinating cns disorders. . we asked whether brain-derived neurotrophic factor (bdnf), known to be highly expressed in a circadian manner in scn, might be involved in the mechanisms governing these astroglial rearrangements. using an analog-sensitive kinase allele murine model (trkb f616a ) and semi-quantitative electron microscopy, we found that the pharmacological blockade of the tropomyosin-related kinase receptor type b (trkb), the high affinity receptor of bdnf, abolished the day/night changes in glial coverage of vip dendrites. the bdnf/trkb signaling pathway therefore exerts a permissive role on the ultrastructural rearrangements that occur in scn across the day/night cycle. in contrast, the extent of glial coverage of non-vip dendrites was not different at daytime and nighttime in trkb f616a mice submitted to trkb inactivation or not receiving any pharmacological treatment. considering the well-established role of bdnf in the clock's photic synchronization process, these dataprovide strong support to the view that the daily astroglial rearrangements in scn could be involved in the photic entrainment of circadian rhythms. above all, they highly suggest that they are necessary for the modulatory action of bdnf on the scn responses to light. features of this form of neurodegenerative ataxia, including a striking reduction in lifespan when polyq atrophins are expressed in the glia. a sensible hypothesis is that affected glia will act non-autonomously on neurons as in other neurodegenerative pathologies. this is an emerging field that yields new and important therapeutic possibilities. we now wish to elucidate the mechanisms through which degenerating glia impairs organism functions, and identify genes involved cell-autonomously and non-autonomously in generating this aspect of the pathology. first, we investigated the impact of glial polyq atrophin on lifespan. using different glia marker, we have demonstrated that the expression of polyq atrophin in glia, or specifically in astrocyte-like cortex glia, has a dramatic effect on drosophila lifespan while brood brain barrier glia do not have any phenotype. we will then investigate the effect of other subtypes of glia looking at lifespan and motility to identify the relevance of each subtype in drpla. second, to identify new regulators of gliopathology we will conduct unbiased genetic screens for mutations in neurons that modify the lifespan of drpla flies, expressing polyq atrophin within the glia. our studies will be paramount in deciphering the complex glia-neurons interactions in ataxias and may be beneficial for several ataxias. they will open up new avenues for therapies aimed at targeting glianeuron interactions during disease progression. in alzheimer's disease, microglial clearance of beta-amyloid (ab) is considered neuroprotective. whether ab uptake regulates microglial cell responsiveness, and whether macrophages participate, remains unclear. here we investigated whether in vivo phagocytosis of endogenously-produced ab disturbs the turnover of microglia and macrophages by changing rates of cellular proliferation and apoptosis. using app swe /ps1 de9 tg mice, we show that plaque-associated microglia and cd11b 1 cd45 high macrophages bind and ingest endogenously-produced ab in vivo. plaqueassociated microglia and cd45 1 leukocyte-like cells were also observed in brains from patients with alzheimer's disease. phagocytic microglia and macrophages had a reduced rate of proliferation compared to abcells. instead, high proportions of apoptotic annexin v 1 microglia and macrophages were found in aged app/ps1 tg mice. phagocytic microglia and macrophages had greater caspase activity and increased p53 expression after in vivo uptake of ab, than cells that did not phagocytose ab. in vitro, we found that ab uptake induced caspase activation in microglia, whereas blocking ab uptake triggered apoptosis-induced cell death in macrophages. our data demonstrate that ab uptake impairs microglial/ macrophage viability and responsiveness. amyloid clearance may fail as phagocytic microglia and macrophages undergo ab-induced apoptosis. f. michetti, s. ceccariglia, a. d'altocolle, f. pizzolante, m. barba, a. delf a, c. gangitano universit a cattolica del s. cuore, rome, italy trimethyltin (tmt) is a neurotoxicant producing neuronal degeneration in the mammalian central nervous system , especially in the hippocampus (for reviews, 1,2). magnetic resonance imaging (mri) investigation in tmt-treated rats has evidenced dilation of lateral ventricles, possibly correlated to alterations in blood brain barrier permeability. we have investigated in the hippocampus and cortex of rats the expression of aquaporin 4 (aqp4), a glial water channel protein which is regarded to play a role in brain oedematous conditions, to explore the molecular mechanisms involved in the phenomenon. tmt-treated aqp4 expression was tested both by real-time pcr and western blotting analysis in hippocampus and cortex homogenates. to confirm molecular results and visualize the aqp4 cell distribution, double-label immunofluorescence for aqp4 and gfap was performed. real-time pcr and western blotting data show a significant upregulation of aqp4 starting from 14 days of tmt treatment in the hippocampus and, at a lower degree, in the cortex. accordingly, the immunofluorescence shows an intense astrogliosis and aqp4 immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting from 14 days after intoxication. aqp4 immunolabelling was localized in astrocytic end-feet encircling the blood vessels, as expected. the study of the rhodamine b fluorescent tracer, intraperitoneally administered, also revealed an intense vascular reaction, characterized by hypertrophic vessels with abnormal course and dimensions in the brain of tmt-treated rats, indicating a vascular involvement in the tmt-induced neurodegenerative processes. aqp4 over-expression and the concurrent astrogliosis occurring in the brain of tmt-treated rats might be candidated to play a role in alterations of vascular permeability and brain oedema formation evidenced by mri studies. amyotrophic lateral sclerosis (als) is a neurodegenerative disease affecting predominantly motoneurons but with a particular pathological role of non-neuronal cells. previous research suggested that increased concentration of extracellular potassium could lead to motoneuron dysfunction. the na 1 /k 1 -atpase should regulate the homeostasis of potassium ions and thus help maintain regular neuronal activity. this homeostasis may be regulated by na 1 /k 1 -atpase in both neurons and astrocytes. in order to reveal the possible role of the na 1 /k 1 -atpase in als pathology, we explored the expression of the alpha1 catalytic subunit of na 1 /k 1 -atpase in neurons and astrocytes from different brain regions of the sod1 g93a rat model of als. labeling immunofluorescently the alpha1 isoform of the na 1 /k 1 -atpase revealed a decreased signal in the trigeminal nucleus and cerebellum of the als rat. double immunofluorecence with neun for neurons showed that the reduced alpha1 was on the neuronal surface in the trigeminal nucleus. in addition, the alpha1 ring-like staining of cerebellar granule neurons was lower in als as compared to the wild type rat. in comparison to neurons, astrocytes from the wild type rat were characterized with a lower expression of alpha1. in the examined brain regions we did not observe a prominent difference in alpha1 expression between astrocytes of wild type and als rats. based on the findings of this study the prominent reduction of na 1 /k 1 -atpase level observed in neurons and not in astrocytes could contribute to further understaning of impaired ion homeostasis affecting proper neuronal physiology in als. the role of glial na 1 /k 1 -atpase needs to be further studied with markers of other alpha subunit isoforms. although commonly used as nutritional supplements, excessive intake of bcaas might favour the establishment of neurotoxic conditions as indicated by the severe neurological symptoms characterising inherited disorders of bcaa catabolism such as maple syrup urine disease (msud). recent evidence indicates that bcaas induce excitotoxicity through mechanisms that require the presence of astrocytes. in the present study, we evaluated the effects of bcaas on microglia, the main immune cells of the brain. as an experimental model we used primary microglial cells harvested from mixed glial cultures that had been kept in normal or high bcaa medium (h-bcaa). we show that h-bcaa microglial cells exhibit a peculiar phenotype characterized by a partial skewing toward the m2 state, with enhanced il-10 expression and phagocytic activity but also increased free radical generation and decreased neuroprotective functions. we suggest that such an intermediate m1/m2 phenotype might result in a less efficient microglial response, which would promote the establishment of a low grade chronic inflammation and increase the likelihood of neurodegeneration. although based on in vitro evidence, our study adds on to an increasing literature indicating that the increasing use of dietary integrators might deserve consideration for the possible drawbacks. in addition to excitotoxicity, the altered immune profile of microglia might represent a further mechanism by which bcaas might turn into toxicants and facilitate neurodegeneration. the proteolipid protein gene (plp1), located on the x-chromosome, undergoes alternative splicing to produce the most abundant proteins of central nervous system myelin: plp and dm20. related to the extent of myelin defect, mutations in the plp1 gene in humans are associated with a spectrum of x-linked disorders, from the severe pelizaeus-merzbacher disease (pmd), to the mild form spastic paraplegia type 2 (spg2). phenotype-genotype correlation exists, large duplications of plp1 gene are predominantly found in pmd and null mutations in spg2. plp is almost 100% conserved across mammalian species, and the large spectrum of phenotype severity is also found plp transgenic mice. experiments on these models have contributed to our understanding in the pathophysiology of plp related disorders. in one hand, mice with extra copies of the plp1 gene (1plptg mice) have neurological symptoms and cns pathology similar to those found in pmd patients while mice with plp1 gene inactivation (plp null mice) share similarities with spg2 patients. then, these two mouse models represent very useful tools to evaluate the efficacy of a therapeutic strategy for the severe and the mild form of the plp related disorders on the neuropathological and behavioral aspects. olesoxime (tro19622), a cholesterol-like small molecule, has been shown to rescue motor neurons from cell death and to promote axonal regeneration both in vitro and in vivo. more recently, olesoxime has also been shown to promote central remyelination by accelerating oligodendrocyte maturation both in vitro and in vivo. altogether, these results strongly suggest that olesoxime may be a potential drug candidate for the treatment of white matter neurodegenerative disorders and notably to plp related disorders. here we treated 1plptg mice with olesoxime for 2 months starting at birth, as well as plp null mice from 3 to 12 months of age (presymptomatic treatment) or from 9 to 15 months of age (symptomatic treatment). behavioral and neuropathological analysis revealed that olesoxime failed to correct the development of symptoms in mice modeling the severe form of plp related disorders. on the contrary, treatment of plp null mice with olesoxime resulted in a 3-month delay in the onset of abnormal behaviors related to anxiety and in the slowing of central nerve conduction. these beneficial effects are observed only when the drug is administered before disease onset. treatment with olesoxime also reduces the effect of aging on rotarod performance decline in both wt and plp null old mice. altogether these results suggest that olesoxime could be of interest as a treatment to delay the onset of symptoms in plp related disorders provided that patients suffer from a mild form of the disease and are treated before the establishment of symptoms. objectives: glial cells play an important role in amyloid-beta (aß) clearance, however little is known about the cellular routes involved in internalization of different aß aggregation forms by adult human microglia and astrocytes. therefore, we evaluated the uptake mechanism of aß 1-42 by primary human adult microglia and astrocytes isolated from brain tissue of non-demented control and ad cases. methods: cells were preincubated for 1 hour with different inhibitors, before exposure to fluorescence labelled (fam) oligomeric and fibrillar forms of synthetic aß. the number of amyloid positive cells was quantified by flow cytometry. inhibitors of various pathways tested, include cytochalasin b (inhibits actin polymerization; general endocytosis inhibitor), nocadozole (inhibits tubulin depolymerization; general endocytosis inhibitor) and fucoidan (scavenger receptor inhibitor). results: cytochalasin b inhibited aß fibril uptake (90% reduction, p < 0.01) and to a lesser extent oligomer (50% reduction, p < 0.05) uptake in microglia, whereas no effect was seen on aß uptake by astrocytes. in contrast, nocodazole was found to inhibit aß fibril uptake (54% reduction, p < 0.05) and oligomer (77% reduction, p < 0.01) uptake in astrocytes, whereas no effect was seen on aß uptake by the microglia. interestingly, fucoidan prevented aß uptake in both astrocytes (oligomers:86% (p < 0.01); fibrils: 79% (p 5 0.01) and microglia (oligomers:67% (p < 0.001); fibrils: 79% (p < 0.05)). conclusions: these data indicate that both human astrocytes and microglia use scavenger receptors to internalize aß oligomers and fibrils. whether different types of scavenger receptors are involved in aß uptake by astrocytes and microglia will be further investigated. tel-aviv university, tel aviv, israel alzheimer's disease (ad) accounts for the majority of cases of dementia worldwide, affecting over 50% of the population over 85. the disease is characterized by a progressive memory loss, cognitive deterioration, behavioral disorders, deposit of beta-amyloid (ab) peptides, neurofibrillary tangles, reactive astrocytosis and activation of microglia cells. mutations in the genes that encode app (amyloid precursor protein) and components of the proteases that generate amyloid beta cause familial forms of ad. microglia, the resident immune cells of the central nervous system (cns) were suggested to play both beneficial and harmful roles in the course of the disease. therefore, modulating the microglial response is being considered as a promising approach for the treatment of this disease. we have recently shown using in vitro and in vivo systems that the ectoenzyme cd38, regulates microglial activation. in view of the significant role of activated microglia in ad and the important role played by cd38 in microglial activation, we hypothesized that cd38 deficiency would affect the course of the disease. in order to test this hypothesis, we implicated the app swe ps1de9 transgenic mouse model for ad, and compared the cognitive performance (as evident by behavioral studies) of aged app swe ps1de9 mice, to app swe ps1de9 cd38 -/mice. in addition, abload, accumulation of microglia and astrocytes was assessed in the brains of these mice. our preliminary results show that cd38 deficiency has a neuroprotective role in this ad mouse model as indicated by improvement in cognitive performance and dramatic reduction in ab load. oxidative stress induced by reactive oxygen species (ros) is associated with various pathological conditions, including aging, neurodegenerative disorders, traumatic and ischemic insults. the mechanism by which the gap junction protein connexin43 (cx43) contributes to oxidative stress-induced cell death or the "rescue" of the dying cells is still unclear. cx43 is highly expressed in astrocytes. we have previously shown that astrocytic cx43 is critical for neuroprotection during ischemic insults in vivo. to investigate the protective effect of cx43 in oxidative stress pathways, we induced oxidative stress in wild-type and cx43 knockout astrocytes using hydrogen peroxide (h 2 o 2 ). cx43 knockout astrocytes exhibited increased h 2 o 2 -induced cell death as measured by the loss of membrane integrity using the dye rhodamine b dextran, supporting a cell protective effect of cx43; this effect was not observed in wild-type astrocytes. to examine the involvement of cx43 in h 2 o 2 -induced cell death, we studied the expression and distribution of cx43 in response to h 2 o 2 in wild-type astrocytes. h 2 o 2 treatment altered the expression level and phosphorylation of cx43. this was accompanied with changes in cx43 distribution and gap junction plaque formation. hemichannel activity was measured using dye uptake. h 2 o 2 (0.7 mm) increased dye uptake, an effect that was blocked by gap26 (syntethic mimetic peptide, 200 mm) or carbenoxolone (100 mm), two connexin hemichannels blockers. however, h 2 o 2 treatment did not result in any measureable gap junction activity in cx43 knockout cells as measured by a scrape-loading dye transfer assay. these findings suggests that gap junction activity contributes to cx43-mediated protection in response to ros. one of the receptors found on microglia is the triggering receptor expressed on myeloid cells 2 (trem2), which signals intracellularly via an adapter molecule referred to as tyro protein tyrosine kinase-binding protein (tyrobp, known also as dap12). in humans, loss of function of either trem2 or tyrobp leads to nasu-hakola disease, which is characterized by neuroinflammation and bone cysts. by using a trem2 knock-out (ko) mouse line, we provided further insights on the trem2 function in neurodegenerative diseases. first, we observed mild signs of dopaminergic neurodegeneration and a slight increase in microglial iba1-immunoreactivity in different brain regions of 3 months trem2 ko mice compared with the wild type (wt) control animals. however, the transcription levels of inflammatory cytokines (tnfa, il1b) or inducible nitric oxide synthase (inos) were unaffected in mice of different ages (3, 9 or 12 months). to shorten the time required for neurodegeneration to occur, we have injected the mice intraperitoneally with lipopolysaccharides (lps) on four consecutive days and analyzed the dopaminergic neurodegeneration in the substantia nigra after a three weeks period. while the quantification of dopaminergic neurons in the substantia nigra showed a slight decrease in number for wt animals, a higher loss was recorded in the trem2 ko mice after systemic lps challenge. concomitantly, significant increase of microglia activation was detected in the lpstreated trem2 ko mice compared with lps-treated wt animals. our results are pointing towards a neuroprotective effect of the microglial trem2 receptor under chronic and systemic inflammatory conditions. increasing evidence suggest that brain energy metabolism is severely altered in multiple sclerosis patients, which may contribute to the process of neurodegeneration. imaging studies reveal a disturbed cerebral perfusion and glucose uptake and metabolism in ms patients. moreover mitochondrial dysfunction is recognized to play a crucial role in ms pathology. to date little is known about the distribution of glucose transporters (gluts), key molecules for the cellular uptake of glucose, in human brain and their role in the pathogenesis of ms. therefore, the aim of this study was to provide a complete overview of the distribution and expression levels of glucose transporters in control and ms brain. our data so far show that mrna levels of glut1, 3 and 4 as well as their regulators hypoxia inducible factor (hif)-1a, hif-2a and peroxisome proliferator-activated receptor gamma coactivator (pgc)-1a, are increased in normal appearing white matter (nawm) of ms patients compared to controls. immunohistochemical analysis revealed that brain endothelial cells not only express glut1 but also glut3 and glut4. glut 1, 3 and 4 are present in resting microglia and highly expressed in activated microglia and macrophages in active ms lesions. interestingly reactive astrocytes expressed increased levels of glut1 and glut4 compared to control, in addition glut3 positive astrocyes were observed in ms brain. axons expressed high levels of glut3 in active ms lesions, which was evidently reduced in chronic lesions. strikingly, all glucose transporters studied were down regulated in chronic ms lesions. together, these data emphasize the extent to which glucose metabolism appears to be affected in all stages of ms. a better understanding of these metabolic changes, especially in the nawm and chronic stage of the disease, is essential to develop strategies to improve neuronal survival and to gain more insight in the mechanisms underlying lesion formation. pericytes are vascular mural cells which are enriched within the capillary basement membranes of the brain. recent studies have provided evidence that pericytes regulate the blood-brain barrier (bbb) and modulate cerebral blood flow. neurovascular dysfunction and bbb damage are hallmarks of alzheimer's disease (ad), but the role of pericytes in ad pathogenesis is still unclear. we have examined post-mortem samples of middle frontal gyrus in collaboration with the university of cambridge after ethical approval by the local irb. ten cases with neither history of neurological disorder nor evidence of neuropathology were compared with 10 ad cases provided by the neurological foundation of the new zealand human brain bank. we performed immunostainings for laminin and plateletderived growth factor receptor (pdgfr)-b which mark vessels and pericytes, respectively. we also stained for ab 1-42 for evidence of amyloid pathology. quantification of pericyte and vessel density was performed using stereological methods, which are considered as gold standard in morphometric quantification. the spaceballs method was used to measure the density of capillaries. pericyte density was assessed using the optical fractionator. the age of the subjects did not differ between the control and ad groups (76,2 6 4,0 vs. 75,4 6 5,5 years). the cortex of ad cases contained more capillary vessels than controls (mean 6 sd: 308 6 40 vs. 365 6 41 mm/mm 3 ; p < 0,01; fig. 1a ). the capillary density increased in correlation to the total plaque load (r 5 0,58; p < 0,001). in contrast, we could not observe a significant difference in the number of pericytes per mm capillary length between control and ad cases (8,56 6 1,21 vs. 8,17 6 0,94 cells/mm; p 5 0,44; fig. 1b ). relative to tissue volume, the cortex of ad individuals showed a tendency to contain more pericytes than controls (2624 6 406 vs. 2985 6 588 cells/mm 3 ; p 5 0,13; fig. 1c ). this is the first study using state-of-the-art techniques to investigate the relationship between capillaries and pericytes in ad brains. impaired clearance of b amyloid across the pericyte-regulated bbb and deficient microvascular regulation of blood flow may contribute causally to ad pathogenesis. in consequence, loss of pericytes could contribute to disease progression. however, our results show that ad cortical pathology is characterized by an increase in the density of capillary vessels without deficit in pericyte coverage. thus, they do not substantiate the notion that pericyte loss is a significative feature of ad. m. noack, j. leyk, c. richter-landsberg carl von ossietzky universit€ at oldenburg, oldenburg, germany histone deacetylase 6 (hdac6), a member of the class ii hdacs, is a unique cytoplasmic a-tubulin deacetylase that interacts with the microtubule (mt)-associated protein tau. in healthy human brain six tau isoforms are expressed generated by alternative mrna splicing. these contain either three (3r-tau) or four (4r-tau) microtubule binding repeats regulating mt assembly and stability. tau hyperphosphorylation impairs its mt-binding activity. tau deposits in nerve cells and glia are the characteristic hallmark of a number of neurodegenerative diseases termed tauopathies. in progressive supranuclear palsy and corticobasal degeneration, pathogenic glial cell inclusions are observable in oligodendrocytes, the myelin forming cells of the cns. hdac6 plays an important role in the degradation and accumulation of misfolded proteins by promoting transport of aggregated proteins to the aggresome, localized at the microtubule-organizing center where protein aggregates are deposited and processed by autophagy. the present study was undertaken to elucidate the functional significance of hdac6 in oligodendrocytes and its role in pathological protein aggregate formation. towards this cultured rat brain oligodendrocytes and oln-t40 cells, an oligodendroglial cell line expressing the longest human tau isoform, were used. treatment of primary oligodendrocytes with tubastatin a (tst), a selective inhibitor of the tubulin deacetylation activity of hdac6, caused a significant increase in the level of acetylated tubulin, as determined by indirect immunofluorescence and immunoblot procedure. mts appeared more bundled and stabilized. inhibition of hdac6 attenuated the phosphorylation of tau at the phf-1-epitope (serine 396/ 404) and its enhancement at the 12e8-epitope (serine 262, located within the microtubule binding repeat region 1). furthermore, altered protein levels of tau isoforms containing either three or four mt binding repeats were observed. while 3r-isoforms were reduced, 4r-isoforms were increased in primary oligodendrocytes suggesting an impact on microtubule binding ability. hdac6 knockdown in oln-t40 cells revealed no alteration in tau phosphorylation at phf-1-epitope, but also augmented phosphorylation at the 12e8-epitope. cells were morphologically damaged and mt organization was disturbed. the data indicate that hdca6 modulates tau expression and phosphorylation, its inhibition leads to the accumulation of 4r-tau which is also prominent in tauopathies with oligodendroglial pathology. to test the hypothesis, that these cells are part of a possible compensatory mechanism to cope with the 6-ohda-induced loss of dopaminergic neurons, we studied the expression of tyrosine hydroxylase (th), the rate-limiting enzyme in the catecholamine synthesis pathway, in the cortex of 6-ohda-lesioned animals. performing immuncytochemistry at 4d after the lesion we demonstrated a 21-fold increase in the number of th-positive (th1somata in rat cortex following 6-ohda injection) compared to sham-lesioned animals. th1somata in the parietal cortex were restricted to the layers ii-v with most of the cells being bipolar. combining th immuncytochemistry with classical nissl stain yielded complete congruency. a small fraction of th1cells co-expressed calretinin thus pointing to an interneuron affiliation. virtually none of the th1cells expressed other markers of interneurons (calcium binding proteins, neuropeptides) or the glial markers gfap and nestin. in contrast, we found a co-localization of th with markers of glial progenitor cells (sox2 and s100b) and with psa-ncam, which has been shown to be expressed in immature, but not recently generated cortical neurons in cortical layer ii of the cat (varea et al.; front. neurosci. 5: 17 2011). taken together, these findings indicate that 6-ohda lesions might induce a glial de-differentiation followed by a fate re-direction towards neuronal committed cells. future studies have to be performed to confirm this and to find out if the th1cells are capable of dopamine synthesis. objective: using the mouse optic nerve (mon) wm model, we tested whether hydrogen in drinking water reduced functional wm ischemic injury, and if it reduced cellular lipid peroxidation and mitochondrial dysfunction including mitochondrial and nuclear dna oxidation. methods: functional integrity of mon was determined by quantitatively monitoring the area of mon compound action potential (cap) before, during and after a standardized 60 min period of oxygen and glucose deprivation (ogd), the ischemia equivalent ex vivo. nuclear 8-oxoguanine (nu8-oxog) was used as a marker of oxidative dna damage. results: a 60 min period of ogd caused prompt loss of the cap, followed by an average 20% recovery. in mice that had received hydrogen-containing drinking water for 7-10 days, the cap area did not disappear during ischemia and recovered to a significantly greater extent during reperfusion (normal oxygen and glucose levels). immunostaining of axonal neurofilament by smi-31 showed significant protection in mons from mice drinking hydrogen-water. accumulation of nu8-oxog was observed mainly in oligodendrocytes after ogd. the levels of 8-oxog after ogd were significantly reduced in optic nerves from hydrogen-water drinking mice. the 'protection' induced by 7-10 days of hydrogen-water drinking lasted several days. conclusions: our results show that several days of hydrogen exposure reduced the extent of cns wm irreversible injury associated with ogd. the importance of these observations is that oligodendrocytes may play an important role in the protective effect against ischemic injury. these observations raise intriguing therapeutic options. amyloid-b (ab) deposits in the brain extracellular space (ecs) and neurofibrillary tangles are typical features of alzheimer's disease (ad). both affections are expressed in triple transgenic (3xtg-ad) mice, making them a useful model for studying the pathophysiology of ad. in these mice, significant changes in astroglial morphology appear in the limbic brain structures [1, 2] , which may affect the diffusion of neuroactive substances through the ecs. using the real-time iontophoretic method, we determined the ecs volume fraction a (a 5 ecs volume / total tissue volume) and the geometrical factor tortuosity k (k 2 5 free / apparent diffusion coefficient) in the ca1 region of the hippocampus, dentate gyrus (dg) and prelimbic cortex (plc) in brain slices obtained from 10-month-old (10m) and 20month-old (20m) 3xtg-ad mice and age-matched wild-type (wt) controls. to study potential diffusion anisotropy, the measurements were performed along 3 orthogonal axes: medio-lateral, rostro-caudal and ventro-dorsal. astroglial morphology and the presence of ab were assessed using immunohistochemical staining for gfap and ab. we found no anisotropy in the ca1 region, dg, or plc, thus the data for each structure along the three orthogonal axes were pooled. the ecs diffusion parameters measured in the ca1 are shown in table 1. in the ca1 of 10m mice, there was no significant difference in the ecs diffusion parameters between wt and 3xtg-ad mice. in 20m mice, a decreased by 9% in wt but increased by 27% in 3xtg-ad mice in comparison with the values found in the younger animals. during aging, there was also a significant decrease in k in wt but not in the 3xtg-ad mice. a similar pattern of changes in the ecs diffusion parameters during aging was also observed in the dg and plc. unlike in the ca1, we found significant differences in young animals: a was lower in the dg and k was higher in the dg and plc of 3xtg-ad mice than in wt mice. in 20m 3xtg-ad mice, ab deposits accompanied with astroglial atrophy or hypertrophy were observed. we suggest that the amyloid load in 3xtg-ad mice and the subsequent prevailing astroglial atrophy affect the normal aging process by inducing an increase in the ecs volume during aging instead of the normal decrease. this leads to a larger ecs space in aged 3xtg-ad mice than in age-matched wt mice, which may alter the efficacy of extrasynaptic as well as synaptic transmission and contribute to the impaired cognitive functions in ad. the study was supported by the grants: gacr p304/11/0184, gacr p304/12/g069. significant differences between 10m and 20m mice of the same group are marked with *, differences between wt and 3xtg-ad mice of the same age are marked with 1. sequence or any treatment. however growing evidences are in favor of the involvement, besides neurons, of several partners such as glia and muscles. to better characterize the time course of pathological events in an animal model that recapitulates human als symptoms, we investigated functional and cellular characteristics of hsod1 g93a mice. we have evaluated locomotor function of hsod1 g93a mice through dynamic walking patterns and spontaneous motor activity analysis. we detected early functional deficits that redefine symptoms onset at 60 days of age, i.e. 20 days earlier than previously described. moreover, sequential combination of these approaches allows monitoring of motor activity up to disease end stage. to tentatively correlate early functional deficit with cellular alterations we have used flow cytometry and immunohistochemistry approaches to characterize neuromuscular junctions, astrocytes and microglia. we show that (1) decrease in neuromuscular junction's number correlates with motor impairment, (2) astrocytes number is not altered at pre-and early-symptomatic ages but intraspinal repartition is modified at symptoms onset, and (3) microglia modifications precede disease onset. at pre-symptomatic age, we show a decrease in microglia number whereas at onset of the disease two distinct microglia sub-populations emerge. we are now establishing the transcriptomic profile of microglia over the course of the disease. in conclusion, precise motor analysis updates the onset of the disease in hsod1 g93a mice and allows locomotor monitoring until the end stage of the disease. early functional deficits coincide with alterations of neuromuscular junctions. importantly, we identify different sets of changes in microglia before disease onset as well as at early-symptomatic stage. this finding not only brings a new sequence of cellular events in the natural history of the disease, but it may also provide clues in the search for biomarkers of the disease, and potential therapeutic targets. in order to gain more knowledge about the disease pathophysiology, two mouse models for vwm are developed in our lab. co-cultures between astrocytes (wt and vwm) and wt oligodendrocyte progenitor cells (opcs) are done to investigate the effect of astrocytes on opc maturation. furthermore, induced pluripotent stem cells (ipscs) from both vwm and wt mice are compared in their glial differentiation efficiency in vitro and in vivo. the astrocyte-opc co-cultures show that vwm astrocytes inhibit opc maturation. this suggest that astrocytes might have a crucial role in the development of the oligodendrocyte and myelin pathology in vwm. currently our research focuses on rescuing the maturation inhibition in vitro, which can aid the development of stem cell therapy for vwm. particularly in response to b-amyloid. using the appps1 alzheimer's disease mouse model, we observed the production of the common interleukin (il)212 and il-23 subunit p40 by microglia. genetic ablation of various il-12/il-23 signaling molecules, of which deficiency in p40 had the strongest effect, resulted in a drastic decrease in cerebral amyloid burden. although deletion of il-12/il-23 signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p40-specific antibody likewise resulted in reduction of cerebral b-amyloid in appps1 mice. furthermore, intracerebroventricular delivery of antibodies to p40 significantly reduced soluble ab species and reversed cognitive deficits in aged appps1 mice. our results suggest that inhibition of il-12/il-23 signaling reduces cerebral amyloidosis and cognitive dysfunction, and may pose a novel potential pharmacological target to combat alzheimer's disease. charit e -universit€ atsmedizin berlin, berlin, germany question: microglia are attracted to and surround b-amyloid deposits, the main hallmark of alzheimer's disease (ad), suggesting a role for these cells in disease pathogenesis. however, recent in vivo data using the cd11b-hsvtk system for microglial depletion suggests that resident microglia are not sufficiently capable of restricting and clearing bamyloid plaques. to underpin the cd11b-hsvtk system and shed more light into the biological mechanistics of microglial depletion mediated by ganciclovir (gcv), we aim to intravitally monitor the depletion process. methods: we are using cd11b-hsvtk transgenic mice crossed to fractalkine-gfp 1/mice harboring green fluorescent microglia. a chronic cranial window is implanted onto the head of the offsprings followed by depletion of microglia and subsequent in vivo two-photon (2p) imaging. as 2p imaging is able to visualize the upper 300 mm of the cortex, a new topical depletion approach of cd11b-hsvtk 1 microglia was established. here, gcv is applied directly onto the brain surface in the area of the cranial window through a catheter. results: manipulation of the brain skull by installation of the cranial window and placing a catheter for topic gcv application resulted in strong depletion of microglia up to 85% in fractalkine-gfp 1/mice crossed to cd11b-hsvtk mice. conclusions: taken together, we established a minimal invasive technique for visualization of the microglia depletion process in cd11b-hsvtk mice using intravital 2p microscopy. these tools will allow the underpinning of the cd11b-hsvtk system as well as the study of relevant biological questions involving resident and peripheral derived microglia in ad. (icrea) , barcelona, spain x-linked adrenoleukodystrophy (x-ald) is a metabolic genetic disorder of the central nervous system characterized by demyelination in brain and/or axonopathy in the spinal cords, adrenal insufficiency and accumulation of very long-chain fatty acids (vlcfa) in plasma and tissues. the disease is caused by inactivation of the abcd1 transporter with the consequence production of oxidative stress mediators and damage. here we aimed to determine: i) the existence of endoplasmic reticulum (er) stress and the ensuing unfolded-protein response (upr) in brains and fibroblasts from x-ald patients, and in the x-ald mouse model (the abcd1 null mouse); and ii) whether the early and rampant oxidative stress formerly identified accounts for the er stress. indeed, here we report signs of er stress and upr response in human and mouse tissues. a common feature is the activation of atf6 and lower amounts of ire1, two er transducers constituting the core signature of upr in x-ald. chaperone grp78 and grp94 expression, by contrast, differ between variants and stages of the disease. we highlight how chaperones are down-regulated in x-ald mice at 12 months of age, pointing to selective depletion of upr-related factors overtime. treatment of the mouse model with a combination of antioxidants reversed the initial activation of transducers and chaperones, indicating that oxidative stress caused er stress. altogether, we postulate that oxidative-stress elicited er stress occurs in x-ald, and that a defective upr may contribute to axonopathy progression. thus, potentiation of the upr may be a therapeutic avenue in x-ald. representing opc-like cells with the potential to differentiate upon growth factor withdrawal and addition of triiodothyronine (t3) into premyelinating oligodendrocytes. upon differentiation, significant fewer cg4_wt-a-syn cells express the myelin basic protein (mbp; see figure a) as a marker for terminal differentiation and additionally, mrna levels were remarkably reduced compared to control cells (see figure b ). as mrna levels of the myelin protein proteolipid protein 1 (plp1) as well as the major myelin-gene regulatory factor (mrf) are also reduced in differentiated cg4_wt-a-syn cells (see figure b) we hypothesized that the process of differentiation is delayed upon a-syn expression. using various differentiation-promoting agents targeting distinct maturation-associated pathways we aimed to dissect the a-synmediated effect on differentiation in more detail and evaluate potential disease-modifying approaches for this devastating neurodegenerative disorder. methods: two nutritionally distinct groups of male newborn ratswell-nourished (w; n 5 26) and malnourished (m; n 5 33) were treated from the 5th to the 24th day of postnatal life with daily intraperitoneal injections of saline (sal) or cona (sigma-aldrich) at doses of 1 mg/kg (group w-l1 and m-l1) or 10 mg/kg (groups w-l10 and m-l10). two groups of "naive" (non-injected) pups were used as additional controls. when the pups reached adult age (90-120 days), under ip anesthesia (1 g/kg urethane 1 40 mg/kg chloralose) they were submitted to the csd recording (ecog and slow potential change) in two points of the parietal cortical surface. csd was triggered every 20 minutes by applying 2% kcl for 1 min at a point in the frontal cortex. this study was approved by the ethics committee of our university, case no.: 23076.031272/2010-53. results: compared to w-sal controls (mean 6 sd velocity in mm/ min 5 3.41 6 0.10), m-sal rats presented significantly higher velocities (4.26 6 0.16; p < 0.05). in both nutritional conditions l1 and l10 treated groups presented significantly lower csd velocities (3.12 6 0.14 and 2.82 6 0.18 for the w-l1 and w-l10, respectively, and 3.74 6 0.13 and 3.25 6 0.16 for the m-l1 and m-l10). this effect was dose-dependent, and was greater in the m-condition. the na€ ıve-and salgroups had similar csd velocities. conclusions: the lectin cona administered early in life to rats decelerated csd in a dose-dependent manner at adulthood, suggesting a long-lasting effect. the largest relative reduction was observed in the m group, suggesting modulation of the effect by the nutritional status. the involvement of glial cells in this effect is discussed. the endocannabinoid system is of growing interest as a therapeutic target in neurological diseases. the two major endocannabinoids 2arachidonoylglycerol (2-ag) and n-arachidonoyl ethanolamine (anandamide, aea), are the endogenous ligands for the cannabinoid receptors 1 (cb 1 ) and 2 (cb 2 ). cb 1 is mainly expressed in the brain and associated with neuroprotective effects, whereas cb 2 is primarily found in hematopoietic cells and associated with anti-inflammatory effects. in neurodegenerative diseases, e. g. amyotrophic lateral sclerosis (als) and parkinson's disease (pd), an increased cb 2 expression on microglia has been reported and is therefore an interesting therapeutic target. furthermore, an increase of endocannabinoid levels occurs in the affected neuronal tissues. stimulating this disease-related increase in endocannabinoids might thus be of therapeutic interest. in our project, we evaluated the novel, highly selective inhibitor kml29 of an enzyme central to the degradation of the endocannabinoid 2-ag, the monoacylglycerol lipase [1] . an in vitro monoacylglycerol lipase inhibitor screening assay for the comparison of the utilized inhibitor with others was performed. additionally, behavioral experiments including body temperature measurement, pain threshold measurement and motor activity analysis in healthy control mice were performed. furthermore, endocannabinoid system baseline levels of healthy control mice vs. sod1 (superoxide dismutase 1) mice as an als mouse model were compared at the gene expression and protein level. in vitro inhibition of the monoacylglycerol lipase by the recently published compound kml29 resulted in lower ic 50 values and therefore indicated a higher potency of kml29 when compared to the other monoacylglycerol lipase inhibitors jzl184 and cpc12 applied in the assay. in vivo administration of the reported monoacylglycerol lipase inhibitor kml29 resulted in significant decrease in body temperature, increase in pain threshold in b6sjlf1/j mice as well as in impairments in the nocturnal motor activity of c57bl/6j mice when compared to vehicle-treated mice. in summary, our data show a comparatively high potency in vitro and significant dose-dependent effects on physiological parameters in vivo of the monoacylglycerol lipase inhibitor kml29 and therefore lay the foundation for a future therapeutic study targeting neuroinflammation in mouse models of als and pd. glaz/apod protects against oxidative stress and promotes axon regeneration after injury, but its mechanism of action is unknown. we hypothesize that glaz/apod modulates membrane dynamics and oxidation. to contrast this hypothesis we study glaz protective role on the polyq-based spinocerebellar ataxia type i (sca1) model in drosophila. human polyq-ataxin1 expression in fly photoreceptors triggers glaz up-regulation. overexpressing glaz in photoreceptors or in retinal support cells rescues neurodegeneration. when expressed in retinal support cells, the glaz rescue is dependent on lipocalin-receptor expression by photoreceptor neurons, and the glaz-gfp fusion protein co-localizes with photoreceptor markers, suggesting the existence of receptor-mediated endocytosis of glaz. upon neurodegeneration, oxidative stress and induction of autophagy coexist. to test whether the glaz beneficial effects are mediated by the modulation of autophagy we quantified atg8a expression and monitored the accumulation of ubiquitinated proteins and p62. overexpression of glaz reduces atg8a induction, but concurrently reduces the accumulation of ubiquitinated proteins and p62. upon autophagy stimulation by rapamycin treatment, the levels of sca1-dependent accumulation of ubiquitinated proteins and p62 are further reduced by glaz. in addition, glaz decreases the sca1-dependent induction of gsts1, a sca1 genetic modifier contributing to the clearance of lipid peroxides. our data support that glaz enters the degenerating neurons by a receptor-mediated mechanism and promotes the resolution of autophagy, increasing its flow and helping to clear polyq-induced protein aggregates. moreover, the beneficial effects of glaz are linked to lipid peroxide clearance, either directly or through receptor-mediated modulation of protective gene networks. micinn(bfu2011-23978); jcyl(va180a11-2); fund. rodr ıguez-pascual. we investigated the effect of three different intensities of running exercises on the survival of sod1 g93a mice. at the early-symptomatic stage (p60), males were isolated and randomly assigned to 5 conditions: 2 sedentary groups ("sedentary" and "sedentary treadmill" placed on the inert treadmill), and 3 different training intensity groups (5 cm/s, 10 cm/s and 21 cm/s; 15 min/day, 5 days/week). we first demonstrated that an appropriate "control" of the environment is of the utmost importance since comparison of the two sedentary groups evidenced an 11.6% increase in survival in the "sedentary treadmill" group. moreover, we showed by immunohistochemistry that this increased lifespan is accompanied with motoneurons survival and increased glial reactivity in the spinal cord. in a second step, we showed that when compared with the proper control, all three runningbased training did not modify lifespan of the animals, but resulted in glial and motoneuronal changes. conclusions/significance: we demonstrate that increase in survival induced by a slight daily modification of the environment is associated with motoneurons preservation and strong glial modifications in the lumbar spinal cord of sod1 g93a . using the appropriate control, we then demonstrate that all running intensities have no effect on the survival of als mice but induce cellular modifications. our results highlight the critical importance of the control of the environment in als studies and may explain discrepancy in the literature regarding the effect of exercise in als. alzheimer's disease is the most prevalent neurodegenerative disorder, characterized by occurrence of senile plaques, neurofibrillary tangles and aberrant function of classical neurotransmitters and peptide messengers, such as neuropeptides and growth factors. in the last years a role of astrocytes in mediating and amplifying the progression of neurodegenerative disorders has been proposed. moreover, a growing body of evidence has shown that astrocytes can release non-peptide and peptide transmitters to influence neuronal development, function and plasticity. here we analyzed the impact of the main component of senile plaques, the amyloid-b (ab), on two key components of peptide vesicles, carboxypeptidase e (cpe) and secretogranin iii (sgiii), in astrocytes in vitro and in vivo. we consistently located cpe and sgiii proteins in cultured and in situ astrocytes. traffic and secretion of astroglial cpe and sgiii were analyzed under basal and stimulated conditions in primary cultures. we found that exposure of astrocytes to ab1-42 markedly impairs expression, traffic and secretion of glial cpe and sgiii. protein levels were decreased in the media, but increased into the cells, by ab1-42 incubations. the effect of ab on cpe and sgiii in glial cells was also investigated in vivo using the amyloid-forming transgenic mice appswe/ps1de9. an aberrant accumulation of cpe and sgiii was found in numerous activated-astrocytes surrounding senile plaques through all the cns of aged transgenic mice. taken together, the present study shows that ab dramatically alters expression, traffic and secretion of cpe and sgiii. because cpe and sgiii are essential in the process and targeting of neuropeptides and growth factors, an involvement of the astroglial secretory pathway in the pathology of alzheimer's disease is suggested. glia cytokine that is produced subsequent to h-i, that collaborates with other cytokines to stimulate the production of astrocytes from subventricular zone glial progenitors. the specific goal of this study was to evaluate gliogenesis after h-i when the tgfb1 receptor alk5 is antagonized in the vanucci p6 h-i rat model. by q-pcr, the relative amount of tgfb1 mrna peaked 7 days after h-i. therefore, sb-505124, and alk5 antagonist, was given intraperitoneally (i.p.) 7 days after the injury to a group of rats while another group received pbs. animals were sacrificed at different time points and brain samples processed for western blot analysis. validating the importance of alk-5 signaling, sb505124 reduced the levels of phosphorylated smad 2/3 that increased after h-i. in another study, sb-50512 was administered i.p. from p10 to p15 and animals sacrificed at p20 for immunohistochemistry for gfap, iba-1, gstpi and mbp. gfap and iba-1 positive cells were dramatically increased in the injured striatum and corpus callosum after h-i and mbp staining was decreased. by contrast, both the extent of injury and the degree of reactive gliosis was decreased by sb-505124 treatment. altogether, our results indicate that sb-505124 inhibits alk5 signaling in the damaged brain. furthermore, sb-505124 administration decreases the extent of microgliosis and astrogliosis while preserving myelination in the damaged brain after neonatal h-i. supported by nih r01 hd052064 and a grant from the leducq foundation awarded to swl. [2] . despite efforts to improve air quality, dep persists as a problem and the share of diesel engined passenger cars in western europe is increasing steadily. honey bees are an important pollinator of food crops and wild flowering plants. they not only contribute to food security by providing pollination services of an enormous economic value but also play an important ecological role. over the last five years, bee keepers worldwide have reported a decline in honey bee populations. the reasons for this decline remain unknown, but it is thought to be multi-factorial [3] . honey bee colonies are confronted with a number of stressors, for example infection with the mite varroa destructor or exposure to pesticides. to forage successfully honey bees rely on their learning and memory abilities. our research aims to establish whether dep might be a factor contributing to declines in honey bee populations through impairment of healthy cns function. methods: we have investigated the impact of dep exposure on the learning abilities of the honey bee using the proboscis extension reflex and classical conditioning. western blotting and histology were used to determine regional expression levels of stress proteins in the brain in response to an acute diesel exhaust exposure. we are examining the morphological appearance of glial cells to investigate the impact of dep in the honey bee cns of dep exposed, and control, forager bees. results and conclusions: we hypothesize that dep acts as a stressor in the brain of the honey bee causing changes in glial cells that are detectable using morphological and molecular techniques -similar to microglial activation or immunological responses of astroglia in the mammalian brain. this work will further our understanding of how dep acts on the brain and how it might impact on the health of the animal. current results indicate that diesel exhaust pollution impacts on the neurobiology of the honey bee and this may contribute to decline by impairing cns functions. hepatic encephalopathy (he) is a serious neurological disorder caused by liver failure. the prime candidate responsible for he pathology is an increased ammonium concentration; and following acute liver failure, ammonium can reach levels of up to 5 mm in the brain. astrocytes are a major target of ammonium toxicity and earlier studies suggested that this toxicity includes a disturbance in intracellular calcium signaling. in the present study, we analyzed the effect of acute hyperammonia on the intracellular calcium concentration of astrocytes in tissue slices of mouse brain, using quantitative intracellular calcium imaging with the indicator dye fura-2. astrocytes were identified by staining with the vital dye sr101; cerebellar bergmann glial cells were identified based on their morphology. in all brain regions studied, the hippocampal ca1 area, somatosensoric cortex, and cerebellar cortex, bath perfusion with 5 mm ammonium for 30 min induced a small, but persistent elevation in intracellular calcium, averaging about 50 nm. in addition, a fast and transient increase by on average 100 nm that lasted about 10 minutes was seen in a small percentage of astrocytes in hippocampal and cortical slices at the onset of ammonium perfusion. this transient increase was never observed in cerebellar bergmann glial cells. both the transient, as well as the plateau phase of ammonium-induced calcium changes were unaltered in the presence of ttx, indicating that they are independent from action potential generation by neurons. furthermore, ammonium-induced calcium increases largely persisted upon removal of extracellular calcium, indicating that they are caused by release from intracellular stores. taken together, our experiments demonstrate that ammonium evokes complex changes in the intracellular calcium concentration of astrocytes in the intact tissue, which differ both between cells in one preparation as well as between different brain regions. because of the central role of astrocyte calcium in gliotransmission, the observed ammonium-induced calcium dysbalance might result in disturbed neuronglia interaction and contribute to the pathology of he. it has been shown in a number of animal models that microglia in the degenerating brain are primed to show exaggerated cytokine responses to subsequent stimulation with toll-like receptors agonists such as lps and poly i:c. it is not clear whether the degenerating brain shows similarly exaggerated responses to pro-inflammatory cytokines. in the current study we hypothesised that glial cells in the hippocampus of animals with chronic neurodegenerative disease (me7 prion disease) would display abnormal responses to central cytokine challenges. in normal animals it has previously been established that intracerebral cytokine challenges elicit specific pathways, i.e. il-1b a cxcl-1 a neutrophil recruitment or tnfa a ccl-2 a monocyte recruitment. unilateral 1ll doses of tnfa (300ng/ll), il-1b (10ng/ll) or saline were administered intrahippocampally via pulled glass microcapillary, in normal (nbh) and me7 mice. these animals were terminally perfused for formalin fixation and paraffin embedding at 2, 24 and 72 hours post challenge. at 2 hours post challenge me7 microglia produced il-1b following either il-1b or tnfa challenge while nbh microglia did not. furthermore, there was very robust nuclear localisation of the nfkb subunit p65 in the astrocyte population and this was associated with very marked astrocytic synthesis of the chemokines cxcl-1 and ccl-2 in response to both cytokine challenges in me7 animals. conversely, very limited expression of these chemokines was apparent in nbh animals similarly challenged. thus astrocytes are the primary chemokine synthesizing brain cell and in the prion-diseased brain are they primed to produce exaggerated chemokine responses to acute stimulation with pro-inflammatory cytokines. this abnormal pattern of chemokine expression is predicted to have consequences for leukocyte infiltration and the ramifications of an altered cellular infiltration pathway for the degenerating brain will be discussed. these findings suggested that the presence of mutated htt in astrocytes alters glial glutamate transport capacity early in the disease process and may contribute to excitotoxicity. to decipher whether astrocytic mutated htt expression was directly associated with msns dysfunction, we investigated the functional properties of individual msns in proximity of mutated htt expressing astrocytes in acute slices. results from whole-cell patch clamp technique showed that msns surrounded (0 -100 microns distance) by astrocytes expressing mutated htt vs. astrocytes expressing normal htt did not differ with regard to passive membrane properties (input resistance, resting membrane potential), action potential firing rates nor spontaneous excitatory postsynaptic current properties (averaged amplitude and frequency). thus, astrocytic expression of mutated htt does not lead to readily apparent functional consequences in msns in these conditions. our data show that 11 month old tg mice displayed a significant increase of ab-plaques in the brain, compared to wt mice. furthermore, we show that the a1c subunit colocalizes with 90% of the abpositive plaques. the cellular expression of a1c was predominantly found in activated gfap1 astroglia around plaques. qpcr profiles of the hippocampus revealed expression of the majority of calcium channel subunits, which was stable during aging. the auxiliary beta4 subunit was expressed in these mice but did not co-localize with a1c1 astroglia around plaques. in cultures of primary astrocytes, ab(42) slightly enhanced the a1c mrna expression after 3 days. we are currently underway to characterize this expression in more detail. in summary, our data provide evidence for a largely stable expression of most ltcc subunits in alzheimer tg mice during aging. finally, the expression of a1c but not beta4 is upregulated in activated astrocytes located around ab-plaques and ab(42) may directly induce astroglial ca v 1.2 calcium channels. this study was supported by the sonderforschungsbereich sfb f4405-b19 and f4406-b19 of the austrian science funds. increasing evidence indicate that both physical and cognitive stimulation induce plastic changes in the brain, such as increase in synaptic and spine densities or neurogenesis, and counteract b amyloid (ab) pathology recovering cognitive function. in the present study we, for the first time, demonstrate the effects of psychostimulation on glial fibrillary acid protein (gfap) architecture in the dentate gyrus (dg) of 3xtg-ad, a well-established mouse model of ad. starting from 3 months of age, 3xtg-ad and their corresponding controls (non-tg) were housed either in the presence of a running wheel (run) or in the enriched environment (enr) for 9 months. as reported previously (olabarria et al., 2010), in standard housing, 3xtg-ad animals showed marked atrophy of gfap-positive profiles, evidenced by significant reduction in gfap surface area, by 39%, and volume, by 42%, compared with non-tg mice. however, physical and cognitive stimulation affected astrocyte morphology by increasing their cytoskeleton surface area and volume, both in non-tg and in 3xtg-ad mice. the surface area of gfap-ir astrocytes increased by 145% and 154% in 3xtg-ad mice housed in run and enr, respectively, compared with transgenic mice kept in standard housing. in addition, the cell volume of gfap-ir astrocytes was higher by 169% and 199% in 3xtg-ad mice housed in run and enr, respectively. the hypertrophy was also evidenced by an increase in the surface area and the volume of astroglial somata and processes in both non-tg and 3xtg-ad mice. further correlation analysis between 3xtg-ad and their corresponding control mice housed under the same conditions, revealed that run and enr not only reversed the genotype-induced astrocytic atrophy, but even induced a hypertrophy in 3xtg-ad mice, with gfap positive profiles rising to the levels of non-tg mice. thus our study indicates that long-term exercise and enriched environment restore and improve astroglial plasticity in the context of adlike pathology, which may recover cognitive functions by compensating disease-associated degeneration of neuronal-glial network. methods: intraperitoneal administration of wa at a dosage of 4mg/ kg of body weight was initiated from postnatal day 40 (p40) till end stage in sod1g93a mice, from 9 months till end stage in sod1g37r mice and from 6 months of age in tdp43a315t mice. results: wa was able to improve the survival by more than a week in sod1g93a and by two weeks in sod1g37r familial mouse model. in addition wa administration also conferred significant neuroprotection in tdp43a315t transgenic mice model. beneficial effects of wa in sod1g93a mice model was accompanied by reduction in loss of motor neurons and also by reduction in level of misfolded sod1 protein in the spinal cord as detected by immunoprecipitation with antibody specific to misfolded sod1. wa was found to be an inducer of heat shock protein 27 (hsp-27), which could possibly explain reduced level of misfolded sod1 and increased neuroprotection in wa treated mice. moreover real-time imaging with the use of biophotonic sod1g93a transgenic mice carrying luc (luciferase) and gfp (green fluorescent protein) reporter genes under the control of the murine gap-43 promoter revealed that wa was able to reduce neuronal stress at post symptomatic stage. conclusion: taken together, our results suggest that wa may represent a promising therapeutic drug for treatment of als. the temporal lobe epilepsy (tle) is the most common form of epilepsy that originates from the hippocampus and then propagates to other limbic areas such as the amygdala and entorhinal cortex. the pathological feature associated with tle is hippocampal sclerosis which is characterized by atrophy, induration, gliosis and loss of neurons in ca1, ca3 and the dentate hilar regions. some animal models, albeit do not exactly match the complex etiologies identified in humans, are found to recapitulate most of the pathological features observed in tle. there is evidence that administration of kainic acid can cause seizures in the ca3 region of the hippocampus that can lead to loss of neurons and astrogliosis characteristic of tle, but the underlying mechanisms associated with the degeneration of neurons remain unclear. since lysosomal enzymes, cathepsins b and d, can have important roles in the loss of neurons in a variety of experimental conditions, we evaluated their potential roles along with the insulin-like growth factor-ii (igf-ii) receptor, which is involved in the intracellular transport of these enzymes, in the kainic acid treated rats. our results clearly showed that systemic administration of kainic acid evoked severe loss of neurons along with hypertrophy of astrocytes and microglia in the hippocampal region of the adult rat brain. the levels and expression of cathepsins b and d as well as igf-ii receptor increased progressively with time in the hippocampus of kainic acid treated rats compared to control rats. the activity of both cathepsins b and d was also found to be enhanced in the hippocampus of treated rats. our double labelling studies revealed that expression of both cathepsins were initially increased in the pyramidal neurons and then decreased with time. this was accompanied by the expression of immunoreactive igf-ii receptors as well as cathepsins b and d in a subset of gfap-labelled activated astrocytes and iba1-labelled microglia in kainic acid treated rats. these results, taken together, suggest that enhanced levels/expression and activity of lysosomal enzymes may have a role in the loss of neurons observed in kainic acid treated rats. in addition, increased ng2 glia immunoreactivity and myelin loss were found specifically in the gray matter of patients' motor cortex and spinal cord ventral horn. furthermore, oligodendroglial specific monocarboxylate transporter 1 expression is decreased not only in als mouse models but also in patients. given that oligodendrocytes not only facilitate saltatory conduction of action potentials by myelinating axons, but also support neuronal functions metabolically through monocarboxylate transporter 1 (mct1), the injury to oligodendroglia in als may have pathogenic consequences. methods. to further investigate whether oligodendrocytes play a role in als development, we selectively removed mutant human sod1 (g37r) from postnatal ng21 cells in pdgfar-creer;loxsod1(g37r) mice. results. the administration of 4-hydorxytamoxifen (4ht) caused significant decrease in the expression of mutant human sod1 transgene in ng2 glia as determined by quantitative pcr analysis. we found that excision of mutant hsod1 from ng2 glia dramatically delayed disease onset and early disease, and significantly prolonged animal survival. in addition, at disease onset stage, activated astroglial and microglial responses were delayed in the animals received 4ht treatment. moreover, the removal of mutant sod1 helped to preserve mct1 expression at disease onset. conclusions. these data indicate that expression of mutant sod1 in ng21 cells and their oligodendrocyte progeny has a deleterious effect on motor neuron survival, and suggest that a key negative consequence of mutant sod1 expression in oligodendrocytes is to diminish their capacity to provide metabolic support to neurons. recent studies suggest that innate immunity might be also involved in the pathogenesis of neurodegenerative diseases, cerebral ischemia and brain injury. although the recent works demonstrated that adaptive immune system is involved in the motor neuron disease process, the role of innate immune system in motor neuron disease was not fully investigated. to assess the contribution of innate immunity in the pathogenesis of amyotrophic lateral sclerosis (als), the gene expression profile of lumbar spinal cord from symptomatic mutant sod1 mice was obtained by microarray approach and subsequent pathway analysis indicated the involvement of innate immune pathway. next, to test the role of innate immunity in the pathogenesis of als, sod1 g93a als model mice were mated with myd88 and trif (tir domain-containing adaptor inducing ifnb) deficient mice. myd88 and trif are the essential adaptor proteins for toll-like receptor mediated signaling pathway. as compared with sod1 g93a mice, myd88/trif double-deficient and trif-deficient sod1 g93a mice exhibited the substantially shorter survival times with accelerated disease progression. the disease duration was shortened by 50% in trif-deficient sod1 g93a mice. in contrast, elimination of myd88 in sod1 g93a mice showed marginal effect in survival time. in addition, the expression levels of pro-inflammatory chemokines, ccl5 and cxcl-10 were significantly suppressed in the spinal cord of trifdeficient sod1 g93a mice, as compared with sod1 g93a mice. to determine the cell type in which trif-dependent pathway contributes to the production of these chemokines, we examined the expression of poster abstracts s75 glia chemokines in lps-stimulated primary microglia or astrocyte derived from trif-deficient or myd88-deficient mice. trif-dependent induction of these chemokines was observed only in lps-stimulated microglia. moreover, we found that infiltration of t-lymphocytes and other immune cells were significantly decreased in the spinal cord of symptomatic trif-deficient sod1 g93a mice. these results suggest that the basal level of trif-dependent innate immune activation of microglia is beneficial to slow disease progression of als models through the maintenance of pro-inflammatory chemokines and the infiltration of the immune cells to the spinal cords. the detailed analyses to clarify the role of these infiltrating immune cells are underway. alzheimer's disease (ad), the most common age-dependent neurodegenerative disorder, causes a chronically progressive decline in cognitive functions. there is growing evidence that glial changes are early involved in this pathology. pdapp mouse, a well-defined model of ad, accumulates toxic soluble and deposited ab, derived from proteolytic processing of the amyloid precursor protein (app) and develops adlike synaptic deficits and cognitive impairment. on the other hand, autophagy has been associated to the neurodegenerative process, in particular with the clearance of aggregation-prone proteins like ab. the amyloid plaques, mainly located in cortex and hippocampus, are closely surrounded by reactive and hypertrophic gfap1 astrocytes. the aim of this work was to evaluate the potential astrocyte autophagic activity during the progression of ad in pdapp mice from 5 to 20 months (m) of age. lc3ii/i ratio was studied by western blot. amyloid deposit load, stained with congo red, exhibited a continuing rise according to age in transgenic mice. the markers gfap and lc3 were analyzed by immunofluorescence and confocal microscopy on hippocampal sections showing an increasing colocalization that reached the top at 14 m, where 52.5 6 9.9% of plaque associated gfap cells were lc31. at 20 m, this proportion was lower. conversely, the subpopulation of astrocytes located far from ab deposits were gfap1/lc3-, besides a decreased cell volume compared with control mice astrocytes. additionally, the density of astroglial cells in the stratum radiatum diminished with aging (5 compared to 14 m, total gfap1 cells, p < 0.05) along with higher plaque load, in a more prominently manner than in control mice. our results clearly show that 1) astroglia is strongly affected during ad progression: two different morphologic subpopulations -close to or far from deposits-are distinguished in the hippocampus. aging correlates with a glial density reduction; 2) a gradual increase of autophagic activity in plaque associated-astrocytes is verified by the first time, suggesting a contribution to ab clearance until 14 m in pdapp mice, with a significant decay after this age. it also shows widespread neuron loss and gliosis in the brain. interestingly, in cstb-/-mice pronounced microglial activation has been detected in selected brain areas already in presymptomatic mice, preceding astrocytosis and neuronal death. in this study, we examine the microglial contribution to the neuronal dysfunction and death in epm1. we aim to characterize the functional properties of cstb-/-microglia and their effects on the survival of cstb-/-neurons. our results show that the cstb mrna level in cultured wild type mouse microglia analyzed by real time quantitative pcr is high in relation to primary astrocytes or neurons. a gene-expression profiling of microglia from cstb-/-mice and from wild type mice has been obtained by using the affymetrix mouse exon 1.0 st array and reveals a down-regulation of interferon-regulated pathways in cstb-/-microglia. the stimulation of primary wild type mouse microglia with the endotoxin lipopolysaccharide (lps) up-regulates cstb mrna expression measured by real time quantitative pcr. cstb-/-microglia show an altered inflammatory response to the stimulation with lps by increased secretion of chemokines demonstrated by cytokine array analyses. moreover, the release of nitric oxide from cstb-/-microglia measured by griess assay is elevated. our data indicate an altered response of primary cstb-/-microglia to inflammatory stimuli, which may contribute to neurodegeneration in epm1. the data provide a basis for further detailed studies on the pathophysiology and therapy of this devastating disease. activation of astrocytes and microglia surrounding amyloid extracellular plaques in the brain is a hallmark of alzheimer's disease (ad). increasing evidence suggests that chronic neuroinflammation and elevated levels of several cytokines play important roles in ad development. beside astro-and microgliosis, demyelination and oligodendrogenesis can be observed in human patients as well as in murine models of ad. oligodendrocyte progenitor cells, also termed ng2 cells because they express the neuron/glial antigen 2 (ng2) proteoglycan, comprise about 5% of total cell number of the adult brain and are the main proliferating cells present. ng2 cells receive gabaergic and glutamatergic synaptic input and can secrete pro-and antiinflammatory substances upon activation by microglia-derived cytokines. overall, ng2 cells are highly reactive cells and one of the first cells which respond to changes in the cns. however, their role during ad pathology is still uncompletely characterized. using immunohistochemistry, we have analyzed their expression pattern in a transgenic murine model of ad (coexpressing a mutated form of the amyloid precursor protein (app) and the presenilin1(ps1) gene). we focused on the spatial arrangement of ng2 cells in the hippocampus of transgenic mice at 3, 7 and 10 months of age and age-matched controls (n 5 4/ time point). our goal is to provide an anatomical and structural basis for elucidating the role of ng2 cells in ad. the first results indicate an uniform distribution of ng2 cells in the hippocampus of app/ps1 mice, with an accumulation at the sites of plaque deposits in close association with microglia. ng2 cells are normaly absent from the pyramidal and granular cell layers. however, when plaques deposits were found at these principal cell layers, ng2 cells could be seen surrounding them, suggesting an active migration. changes in ng2 cell phenotype were noted from 3 months of age on. like microglia, ng2 cells assume an activated morphology, as in a more "bushy" appearance. in numerous plaques, ng2 immunoreactivity was strongly increased, mostly in the intermingled processes that encircle the core of the plaques. whether these activated ng2 cells proliferate and differentiate into oligodendrocytes or communicate with activated microglia in the plaque microenvironment remains to be investigated. further, levels of the glutamatergic ampa receptor expression will be evaluated. ultimately we aim at understanding how ng2 cells and microglia interact in ad, which could be usefull in the development of novel therapies. accumulating evidence supports an increasing role of astrocytes in the initiation or progression of a variety of neuropathological conditions. in alzheimer's disease (ad), numerous data indicate that astrocyte properties are modified with potential deleterious effects on neurons. accordingly, we have described changes in the expression of astroglial connexins, the gap junction channel and hemichannel forming proteins, in the vicinity of amyloid-b (ab) plaques in brains from ad patients and murine models of ad. also ab peptide was shown to trigger astroglial cx43 hemichannel activation in culture and in acute slices leading to neuronal degeneration. hence, we have investigated hemichannel function of astroglial connexins in 8-9 months old app swe /ps1 de9 mice that exhibit ab plaques in the cortex and hippocampus. ethidium bromide (etbr) uptake performed in acute brain hemisphere slices was used as an index of hemichannel activation and was quantified in astrocytes identified by gfap immunostaining. in app swe /ps1 de9 mice, compared to wildtype mice of the same age, an increased uptake of etbr was detected in the overall population of astrocytes. this uptake was blocked by carbenoxolone and lanthanum ions, indicating connexin hemichannel involvement. such activation may be linked to the increase in resting astroglial [ca 21 ] i described in this mouse model. interestingly, etbr uptake was higher in reactive astrocytes contacting ab plaques than in non-reactive astrocytes located far from (! 50 mm) these deposits. we are currently investigating their respective pharmacological profiles. moreover, in app swe /ps1 de9 mice knock-out for cx30 generated in our facility, etbr uptake was comparable to that observed in app swe /ps1 de9 mice, suggesting that cx43 is the major contributor to the hemichannel activity observed. altogether, these results indicate that neuroglial interactions could be affected by astroglial hemichannel activation and could account for neuronal alterations or death observed in neurodegenerative diseases. since it is well established that amyloid plaques are associated with activated astrocytes we asked whether concentrations of the astrocyte-derived proteins glial fibrillary acidic protein (gfap) and s100b in human csf might serve as additional biomarkers. to functionally link the role of astrocytes to mechanisms involved in memory formation, we asked whether the amount of gfap-positive astrocytes correlates to the level of ltp in the hippocampus of aged ad transgenic mice. we used elisa kits for the quantitative analysis of gfap (ibl-international, hamburg, germany) and s100b (ibl). in a mixed disease cohort (n 5 52 diseased patients, n 5 16 controls), we correlated standard biomarkers (abeta and tau-protein) and gliaderived proteins. furthermore we compared mean levels between groups of ad patients (n 5 36) and healthy control patients (n 5 35) and correlated levels of astroglial proteins in the csf and cognitive performance (mmst values). in transgenic (app/ps1 transgenic mice), aged (10-15 months) mice (n 5 10), we performed ltp experiments at the schaffer collateral synapses in the ca1 region of the hippocampus. the number of gfap-positive astrocytes was quantified contralaterally. results: in the mixed cohort, we found a significant correlation of t-tau and gfap levels in csf (r 5 0.261, pbetween the s100b levels and the patients cognitive performance as measured by the mmst values (r 5 -0,42) as well as between the gfap levels and the mmst values (r 5 -0,38). when correlating the number of astrocytes in the ca1 region to the level of ltp, 30 min after induction, we found a significant correlation of the number of astrocytes per mm 3 to the level of ltp (r 5 0,648, p conclusions: the astrocyte-derived proteins gfap and s100b can be reliably detected in human csf. measurement of astroglial biomarkers might allow an improved pathobiological staging of ad, also since astrocytes appear to play a functional role in memory formation in the context of an ad-like pathology in mice. retinitis pigmentosa is a group of inherited disorders affecting photoreceptors or retinal pigment epithelium (rpe) that leads to progressive loss of vision. the pde6b rd1 mouse model is a very well-known animal model for retinal degeneration, in which rods carry a mutation in b subunit of rod cgmp-phosphodiesterease. glial cell line-derived neurotrophic factor (gdnf) has already been shown to rescue morphology as well as function of rod cells in pde6b rd1 mouse. this effect was indirect, through stimulation of m€ uller glial cells (rmg). to better understand the neuroprotective effect of gdnf, primary retinal rmg were stimulated in vitro with gdnf and the secreted proteome was analyzed by proteome profiler arrays. among others, cyr61/ccn1, a member of ccn family, was found strongly induced upon rmg stimulation with gdnf. when applied directly to medium, cyr61 significantly reduced photoreceptor death in organotypic ex vivo cultures of pde6b rd1 retinas. in order to identify the target cells of cyr61 we treated, arpe19 and mio-m1 cell lines with cyr61, and observed an increase in phosphorylation of akt and erk1/2 signaling molecules. these results suggest that stimulation of rpe and rmg cells may have a protective influence on photoreceptor survival in organotypic ex vivo conditions. we postulate cyr61 as a novel potential candidate for future therapeutic approaches in neurodegenerative retinal disorders. since astrocytes react to multiple physiological and pathological stimuli in the microenvironment of the brain, they could be mediators for gene x environment interactions in the pathogenesis of psychiatric disorders. altered gene/protein expression and regressive changes have been reported for astrocytes in post-mortem studies of psychiatric disorders, i.e. schizophrenia (scz), bipolar disorder (bp) and autism spectrum disorders (asd). by contrast, no genetic link to astrocytes has emerged from the extensive genomic analysis of psychiatric disorders. genetic findings mostly relate to neurons for disorders rooted in neurodevelopment (asd, scz). it is timely to perform a formal analysis of genomic information emerging for psychiatric disorders for a contribution of genes expressed by astrocytes. methods: we generated a meta-list of genes highly expressed in rodent astrocytes using published gene lists and literature mining. datasets were prepared for candidate genes and genes from gwas in attention deficit/hyperactivity disorder (adhd), asd, bp and scz, by using literature and databases (szgene, sfari base, dbgap). datasets for copy number variations (cnvs) associated with neurodevelopmental delay were also assembled. then the overlap between the meta-list of genes highly expressed in astrocytes and gene datasets for psychiatric disease was determined. overlapping genes were pooled and subjected to david bioinformatics analysis. results: the meta-list of genes highly expressed in astrocytes contained n 5 2,680 genes (13.4% of the genome). datasets for risk genes in psychiatric disorders showed random overlap with the meta-list (adhd 18%; asd 13%; bp 12%; scz 13%). cnvs related to neurodevelopment were not enriched for astrocytic genes (8%). when the overlapping genes were pooled from all disorders, a small set of shared astrocytic genes emerged (n 5 23). cntnap2, nrxn1 and sdc2 were linked by david analysis (p 5 0.013). conclusions: this correlative analysis makes it unlikely that genes highly expressed in astrocytes make a major contribution to the genetic risk in four major psychiatric disorders. no genetic link was found between astrocytes and neurodevelopmental delay. changes in gene/ protein expression and pathology in astrocytes in the adult brain in scz and bp may be explained by chronic neuronal dysfunction, chronic medication or acute changes. the neuronal ceroid lipofuscinoses (ncls, batten disease) are a group of autosomal recessively inherited lysosomal storage disorders affecting children and young adults, each of which is caused by a mutation in a different gene. a common feature across all ncls is the early, localised glial activation that occurs long before the onset of neuron loss. this glial activation appears to be an accurate predictor of which neuron populations are vulnerable and will subsequently die. both astrocytes and microglia express the ncl gene products and it is likely that normal glial cell function may be compromised. given the close functional relationship between neurons and glial cells it is possible that glial activation and/or dysfunction may affect neuronal health. we have begun to explore the biology of astrocytes and microglia in the juvenile form of ncl using primary cultures from cln3 -/mice. defects in both astrocytes and microglia, including altered protein secretion profiles and impaired morphological and proliferative responses were apparent. co-culturing with mutant microglia and astrocytes influenced the survival and morphology of wild type neurons, with more profound effects upon cln3 -/neurons. intriguingly, these effects were largely reversed by substituting wild type glia, which rescue mutant neurons. a pilot study has provided similar evidence for glial dysfunction in infantile ncl, including and altered protein secretion and response to stimulation. these changes appear to be different from those observed in juvenile ncl and we are in the process of exploring whether the glial cell phenotypes discovered so far, and their impact upon neurons are specific to the different forms of the disease. the accumulation of aggregated proteins in nerve cells and glia underlie the pathogenesis of many neurodegenerative diseases. glial pathology is characteristically observed in tauopathies, such as corticobasal degeneration and progressive supranuclear palsy, and alexander disease, a primary disease of astrocytes caused by mutations in the gfap (glial fibrillary acidic protein) gene. irreversibly damaged proteins can be degraded by the proteasomal system. for proteasomal degradation they are covalently linked to ubiquitin in a three-step enzymatic pathway (e1, ubiquitin-activating enzyme; e2, ubiquitin-conjugating enzyme; and e3 ubiquitin ligases). the polyubiquitin chain needs to be removed before the translocation of the substrates to the lumen of the proteasome. this is achieved by deubiquitinating enzymes (dubs), which oppose the functions of e3-ligases and assist in targeting substrates to specific pathways. to assess whether impairment of dubs may contribute to age-related neurodegenerative diseases and the regulation of cell death and survival, we have subjected primary cultures of rat brain astrocytes to pr-619, a dub inhibitor with broad specificity. immunoblot analysis demonstrates that after a 18h treatment, pr-619 caused the induction of heat shock proteins, hsp70 and hsp32, and an increase in p62, which is considered a cargo receptor for ubiquitinated proteins and a common constituent of ubiquitinated protein inclusions. as analyzed by indirect immunofluorescence, protein aggregates positively stained by antibodies against ubiquitin and p62 assembled in the perinuclear region at the microtubule organizing center (mtoc). these aggregates were surrounded by a cage-like network of gfap intermediate filaments, thus resembling aggresomes. using mitotracker and lysotracker staining procedures, the fluorescent images demonstrate that after dub inhibition lysosomes and mitochondria are recruited to the mtoc. this process was dependent on an intact microtubule network, since it was interrupted after treatment with nocodazole, a microtubule destabilization drug. furthermore, pr-619 caused mitochondrial fragmentation which may be a stress response to enable the removal of damaged mitochondria by mitophagy. in conclusion, dub inhibition impairs the cellular architecture, leads to protein aggregate formation and mitochondrial alterations in astrocytes. our data sustain the hypothesis that an imbalance in dub activities may contribute to the pathogenesis of neurodegenerative diseases. shown that the collapsin response mediator protein 2 (crmp-2), which play a significant physiological role in neuronal cell bodies and axons within the cns, is phosphorylated during the neurodegenerative phase of the inflammatory disease. methods: we investigated the limitation of axonal degeneration by transducing retinal ganglion neurons with a phosphorylation mutant of crmp-2 utilising an intraocular adeno-associated virus 2 (aav2) delivery system in eae-induced mice. the other group of mice injected with aav2 consisting of the green flourescent protein reporter only (aav2-gfp) with eae was used as a control (n 5 8 per each group). results: we showed substantial preservation of axons of the optic nerve in the eae-induced mice injected with the aav2 carrying the crmp-2 phospho-mutant compared ($10-fold difference) with those mice injected with aav2-gfp. the aav2-gfp transduced axons showed significant degeneration during the peak stage of eae. conclusion: our data suggest that phosphorylation of crmp-2 may be a central mechanism that governs axonal degeneration during inflammatory demyelination of the cns (as occurs in ms) and inhibition of phosphorylation of crmp-2 may be of therapeutic potential for the progressive phase of the disease in ms patients. y. shinozaki, s. koizumi univ. of yamanashi, yamanashi, japan atp, a major gliotransmitter integrating neuron-glia networks, is known to be released or leaked from injured cells. in the cns, atp dramatically changes glial phenotypesand could affect pathogenesis of brain disorders. however, whether such glial responses facilitate or rather inhibit the diseases is still under debate. to address this issue,we studied the neuroprotective role of atp/purinergic signaling using in vivo stab injury model on mouse cerebral cortex. without injury or the contralateral side of the injured brain exhibited no fluoro-jade(fj)-positive neuronal death, cd45 1 infiltrating leukocytes or gfap 1 reactive astrocytes. three days after injury, fj signals, cd45 1 cells and reactive astrocytes were increased in the ipsilateral side of the brain. the cd45 1 cells were enclosed by reactive astrocytes-formed glial scar. administration of an atp-degrading enzyme apyrase, a broad p2 receptor antagonist ppads, and a p2y 1 receptor antagonist mrs2179 significantly reduced the stabincreased fj signals. immunohistochemical analysis revealed p2y 1 receptor was expressed in gfap 1 astrocytes. p2y 1 receptor knockout (p2y 1 ko) mice also exhibited reduced fj signals and infiltration of cd45 1 cells. they exhibited higher level of gfap expression, more remarkable hypertrophy of astrocytes and tighter glial scar formation than wild type mice did. the accelerated glial scar formation reduced cd45 1 cell number and inhibition of astrocytes by fluorocitrate increased cd45 1 cell number. although glial scar formation was accelerated, no enhanced proliferation was observed. we then analyzed the mechanism of enhanced glial scar formation and neuroprotection using in vitro scratch-wound model. in cultured astrocytes, the scratch induced a transient increase in extracellular atp, which was followed by decrease in extracellular atp mainly due to an increase in ectoatpase activity. suppression of purinergic signaling by either apyrase, ppads or mrs2179 enhanced scratchevoked astrocytic migration rather than proliferation. neither activation nor inhibition of p2y 1 receptors in cultured cortical neurons affected the scratch-induced damages. our data suggest that the decrease in atp/ p2y 1 receptor-mediated signal should be a trigger that transforms astrocytes into migratory phenotype, which forms tighter glial scar structure thereby suppressing neuronal death. background: oligodendrocyte damage and loss are key features of multiple sclerosis (ms) pathology and oligodendrocytes appear to be particularly vulnerable to ros. in vitro studies showed that ros induce cell death and prevent the differentiation of oligodendrocyte precursor cells (opcs) into mature myelin-producing oligodendrocytes. hence, a potential therapeutic strategy to protect these cells from ros-mediated damage is urgently needed. here we investigated the efficacy of several compounds that are able to boost antioxidant enzyme production, including monomethyl fumarate (mmf), tert-butylhydroquinone (tbhq), sulforaphane (sfn) and protandim. these compounds are thought to exert their protective function via activation of the nuclear-factor-e2-related factor-2 (nrf2) transcriptional pathway, which is involved in the production of antioxidant enzymes necessary for oxidative stress defense. methods: primary rat oligodendrocytes were treated with different concentrations of mmf, tbhq, sfn and protandim. expression of antioxidant enzymes were analyzed by pcr and western blot analyses. to study the beneficial effects of the different nrf2 activators, oligodendrocytes were first incubated with nrf2 activators and subsequently exposed to various concentrations of hydrogen peroxide. oligodendrocyte cell survival was measured by a live/dead cell viability assay. results: sfn, mmf and protandim are well-tolerated and induce nrf2driven antioxidant enzyme production in oligodendrocytes. protandim was the most potent compound with regard to antioxidant enzyme induction and protected oligodendrocytes against ros-induced cytotoxicity. conclusions: our findings indicate that several nrf2 activators are able to induce antioxidant enzyme production in oligodendrocytes. interestingly, protandim, a dietary supplement consisting of herbal ingredients, was the most potent protector of primary rat oligodendrocytes. in future experiments we will determine whether protandim can also promote the differentiation of opcs under oxidative stress and test the clinical efficacy of protandim in an experimental animal model for ms. the arising of the "tripartite synapse" concept and the discovery of the astrocytic excitability have been highlighting astrocytes as active elements concerning information flow in the brain. however, the impact of such remarkable features in complex brain function is still under-explored mostly due to the difficulty of studying neuron-astrocyte interactions in vivo. the aim of this work was to use an in vivo model of astrocytic dysfunction and investigate the impact of this treatment in complex cognitive functions. the rationale consisted in studying behavioral performance that relies on the prefrontal cortex, such as behavioral flexibility and working memory in an animal model of astrocytic dysfunction. for that purpose, we used a pharmacological model in which wistar-han rats were subjected to bilateral intracranial injections of aminoadipate in the prelimbic portion of the medial prefrontal cortex to cause astrocyte depletion specifically in this region, mimicking pathological states such as depression, in which marked decreases of gfappositive cells are observed. this animal model was tested for its cognitive abilities by the attentional set-shifting task (asst) and water maze-based tests. a clear impairment of cognitive function was observed in the animals treated with aminoadipate. a detailed morphological and histological analysis showed that along with the astrocytic lesion, neurons were also affected at the site of lesion. this seems to contribute to the cognitive decline observed in this model and may explain similar observations under pathological states. a. sternotte, e. hermans universit e catholique de louvain, brussels, belgium amyotrophic lateral sclerosis (als) is an adult disease characterized by a selective loss of motor neurons, resulting in progressive paralysis and death within 3-5 years. in several inherited cases, the disease is caused by point mutations in the gene encoding for superoxide dismutase 1 (sod1) which promotes its aggregation, leading to biochemical damages, including mitochondrial dysfunction. alteration of mitochondrial membrane potential and/or respiratory chain leads to atp depletion and these metabolic dysfunctions could contribute to motor neuron death in patients and animal models of als. in motor-neurons, the combination of altered axonal transport with mitochondrial dysfunction likely leads to considerable decrease in atp levels at distant neuromuscular junctions. commonly know as the fuel gauge of mammalian cells, amp-activated protein kinase (ampk) is a key enzyme in the control of cellular atp and energy homeostasis. in this study, the implication of ampk in the progression of als was specifically investigated by measuring the expression and activity of this enzyme in the spinal cord of mice overexpressing the mutated human sod1 (hsod1 g93a ), a commonly used experimental model of als. no consistent modification of the enzyme was detected in spinal cord samples throughout the progression of als. indeed, such experiment did not discriminate between cell types present in the tissue. hence, a more detailed characterization of ampk in cultured astrocytes derived from als animals revealed a robust induction of the enzyme activity, which correlates with decreased atp levels in these cells. in a second part of our study, we have investigated the consequences of ablating ampk in the mouse model of als by breeding ampk knock out mice with hsod1 g93a mice. while we did not detect any difference in the lifespan of ampk(-/-)/hsod1 g93a mice as compared to hsod1 g93a mice, we observed substantial alterations in the progression of the disease in selected behavioural tests. in particular, analysis of the gait (catwalk) which enables early detection of muscle weakness revealed that the onset was delayed by up to 30 days while the progression of the disease after onset was accelerated. further studies will be conducted to establish the importance of atp-in the disease, and to validate this enzyme as a putative pharmacological target in als and in other neurodegenerative diseases involving mitochondrial dysfunction. institute of neuroscience, sibs, cas, shanghai, china ng2 glia is the fourth type of neuroglia in vertebrate nervous system, which also known as oligodendrocyte progenitor cell (opc) in a developmental point of view. in adult brain, ng2 glia has a small cell body with highly branched extensions and represents a new type of glial cell differing from other glial cell types such as astrocyte, microglia and mature oligodendrocytes. published studies by others have shown that ng2 glia is able to promptly respond to brain injury and activated in several neurodegenerative disease animal models including 6-ohdainduced rat parkinson's disease model. however, the pattern and function of these activated ng2 glia remain largely unknown. here, we performed a time-course study of ng2 glia activation in mptp mouse model of parkinson's disease using immunohistochemistry. it was found that ng2 glia was activated as early as 24 hours after the last mptp injection in the substantia nigra (sn) of mptp-treated mice. this temporal character is very similar to those of microglia, indicating ng2 glia respond to mptp challenges very quickly. the activation of ng2 glia became stronger over time and reached the peak at about 5 days after the final mptp injection. then, the extent of ng2 glia activation declined gradually and diminished 10 days after mptp injection. interestingly, we found that prominent ng2 glia activation could only be observed in the substantia nigra. the dorsolateral striatum which is the projection target of nigral dopaminergic neurons, however, was devoid of ng2 glia activation in all the time-points examined. in summary, we characterized the ng2 glia activation in mptp mouse model of parkinson's disease. these data suggest that activated ng2 glia may play a role in the degenerative process of dopaminergic neurons in pd. hallmarks of cns inflammation, including microglial and astrocyte activation, are a feature of post-mortem tissue from amyotrophic lateral sclerosis (als) patients and in transgenic mice overexpressing mutant superoxide dismutase-1 (sod1 g93a ). administration of glucocorticoids does not significantly alter disease progression, but this may reflect poor cns delivery. here, we sought to discover whether cns-targeted liposomally-packaged glucocorticoid would inhibit the cns inflammatory response and reduce motor neuron loss. sod1 g93a mice were treated with saline, free methylprednisolone (mp, 4mg/kg/week) or glutathione pegylated liposomal mp (2b3-201, 4mg/kg/week) and compared to saline treated wild-type animals. animals were treated weekly with intravenous injections for 9 weeks from 60 days of age. animal weights and motor behaviour were monitored during this period. at the end of the experimental paradigm (116 days) mice were imaged using t 2 -weighted mri for brainstem pathology, and brain and spinal cord tissue was collected for histological analysis. all sod1 g93a animals showed a significant decrease in motor performance compared to baseline from $100 days. sod1 g93a animals showed a significant increase in signal intensity on t 2 weighted mr images, which may reflect the combination of neuronal vacuolation and glial activation in these motor nuclei. treatment with 2b3-201, but not free mp, significantly reduced t 2 hyperintensity, which correlated with significantly reduced histopathological manifestations in the brainstem motor nuclei, but not the spinal cord. interestingly, there was a significant reduction in astrogliosis but not microglial activation following treatment with 2b3-201. the results of this work indicated that the cns-targeted anti-inflammatory agent 2b3-201 has therapeutic potential in als. the toxic effects of new antiretroviral drugs on the central nervous system (cns) are unclear. because these drugs penetrate the brain even at low concentrations, it becomes crucial to determine the doses which can be toxic for the cns resident cells. moreover, after the recent introduction into clinical practice, it is unclear whether the efficacy of the antiretroviral drugs of new generation may also derive from their ability to exert extravirological effects on factors responsible for the development of hiv brain injury, e.g. matrix metalloproteinases (mmps). objective: to investigate on the toxicity of four different antiretroviral drugs and their ability to modulate the expression of gelatinase b (mmp-9) in astrocyte cultures. methods: primary cultures of rat astrocytes were activated by exposure to 10 mg/ml lipopolysaccaride (lps) (positive control) and simultaneously treated for 20 h with increasing doses (1-5-10-25. 50 mm) of: efavirenz (efv); darunavir (drv); maraviroc (mvc) or raltegravir (ral). mmp-9 mrna expression was assessed by rt-pcr. quantitative determination of mmp-9 expression was done by computerized scanning densitometry. single drug toxicity was assessed by the mtt test. each drug was considered toxic at the concentration able to induce a percentage of cell survival above 60%. results: the treatment with antiretroviral drugs inhibited mmp-9 mrna expression in lps-activated astrocytes in a dose-dependent manner. in particular, a statistically significant inhibition of mmp-9 expression was observed when astrocytes were treated with 25mm efv (54% of inhibition) or with 50 mm ral (43% of inhibition). as assessed by the mtt test, the toxicity of the antiretrovirals ranges from 10 and 50 mm. in particular, efv was toxic for astrocytes at the concentration of 25 mm, mvc at 10 mm, while drv and ral were toxic at the concentration of 50mm. conclusions: the present results indicate that efv and ral directly inhibit mmp-9 expression in lps-activated astrocytes with mechanisms that are independent from their antiviral activity. the toxic doses of antiretrovirals are much higher than those found in the csf of hiv-positive patients. our results highlight some beneficial/deleterious extra-viral effects of the antiretroviral drugs that may be useful to improve the development of new therapeutic strategies for the management of hiv infection. we used a parahippocampal kainate injection mouse model to induce mtle-hs and to study early expression patterns of kir4.1 and apq4 after se. mice were sacrificed 24 h, 72 h and 7d after kainate injection. immunhistochemistry of the hippocampus was performed to assess kir4.1 and aqp4 expression, and gfap staining was used for identification of astrocytes. gfap expression was increased compared to saline injected controls, suggesting that kainate injection induces astrogliosis. comparison of kir4.1 and aqp4 staining showed a similar change in the expression pattern. our data indicate a drop in kir4.1 and aqp4 expression within 24 hrs after se followed by an upregulation within the first week. altered expression of kir4.1 and aqp4 could contribute to dysregulation of potassium and water homeostasis and play a role in the early phase of epileptogenesis. c. l€ o€ ov, a. erlandsson uppsala university, neuroscience/neurosurgery, uppsala, sweden we have previously shown that astrocytes effectively engulf dead cells after trauma both in vitro and in vivo, but that they store the ingested material rather than degrade it. compared to macrophages, which degrade engulfed, dead cells within hours, our data show that astrocytes store the ingested material for weeks before the degradation is completed. to further study the routes of degradation and the possibilities to speed up this process we have used a cell culture model where uvtreated, dead cells are added to stem cell-derived astrocytes. by labeling the dead cells with the ph-sensitive dye phrodo prior to the engulfment, we demonstrate that the ph in the astrocytic phago-lysosomes is higher than in professional phagocytes. interestingly, lamp1 and lamp2, which are involved in the phago-lysosome fusion, are both highly expressed in the astrocytes, particularly around the ingested material. dendritic cells are known to express the inhibitory protein rab27a that slow down the degradation in order to preserve antigen for presentation. rab27a prolongs the actin coating around the phagosomes, which physically inhibit the phago-lysosome fusion, and interacts with nox-2, which leads to an increased consumption of protons. western blot analysis shows a high expression of rab27a in our cell cultures which may explain the slow degradation. moreover, dead cells in astrocytes are surrounded by actin rings for a long period of time after the ingestion. to counteract this, the astrocytes were treated with 0.1 or 1 mm of the actin inhibitor latrunculin b. the number of actin rings were significantly lower in the cultures treated with 1 mm of latrunculin b compared to controls, but the intensity of phrodo was unaltered indicating that the prolonged degradation may be due to a higher ph in the lysosomes rather than a delayed actin coating. next we will investigate the effect of rab27a expression on the degradation process by performing sirna experiments. one of the promising strategies for the treatment of spinal cord injury is the transplantation of glial cells obtained from the olfactory system; olfactory ensheathing cells (oecs). effective proliferation and migration of oecs are essential for optimizing clinical applications and there is a need for identification of small molecules that regulate glial cell biology. curcumin is a natural polyphenol compound found in the spice turmeric, which is known for its neuro-protective properties and has been reported to have an effect on neurogenesis and nerve regeneration. however, the effect of curcumin on oecs has not been determined. we have examined the effect of curcumin on oecs at the cellular level using fluorescence and timelapse microscopy. oecs were purified from s100b-dsred transgenic mice in which oecs express the fluorescent protein dsred. different treatments: (i) control medium, (ii) curcumin (0.5 to 20 mm), (iii) g5 commercial growth factor, or (iv) a combination of g5 and curcumin were used to determine the effect of curcumin on oecs proliferation and migration. cell proliferation was quantified at two different times by cell counting and mts proliferation assay. timelapse microscopy was used to visualize changes in cell morphology and cell migration. cell branching, number and area of lamellipodia and speed of migration per cell were measured for each treatment. we found that lower concentrations of curcumin (0.5 and 1 mm) increase oec proliferation. as well, combination of g5 and curcumin showed the highest proliferative effect on oecs suggesting a possible synergistic relation between g5 and curcumin. live cell imaging analysis showed that curcumin increased the number and area of lamellipodia resulting in faster migration of oecs. these results suggest that curcumin can regulate the proliferation, morphology and migration of oecs which could improve the therapeutic use of oecs for spinal cord injury repair. methods: the dams were divided in four groups and had received 50 ml of nonalcoholic or alcoholic beer solution -control, vehicle (nonalcoholic solution), etoh 5% (nonalcoholic solution 1 5%v.v ethanol) or etoh 10% (nonalcoholic solution 1 10%v.v ethanol) during gestational day 1 up to weaning (postnatal day 22). male offspring (30 -35 days old) were sacrificed and hippocampal acute slices were prepared. we analyzed gfap content, s100b and glutamate uptake as astrocyte parameters. we tested the animals in the plus-maze discriminative avoidance task to check anxiety and memory. results: gfap content decreased after pnee for etoh 5% and etoh 10% treatment (f 3,59 5 17.75, p < 0.0001). interestingly, we found that only the etoh 10% treatment showed an increase of hippocampal s100b content (t 5 2.38, p 5 0.02) and in the csf (f 3,18 5 3.249, p 5 0.0462), but no difference in secretion. glutamate uptake was significantly decreased for etoh 10% group (f 3,20 5 4.069, p 5 0.0208). to perform the plus-maze discriminative avoidance task we selected pups from control, vehicle and etoh 10% treatment on pnd 30. two-way anova revealed significant effects of treatment (f 2,46 5 4.32, p 5 0.019), arm type (f 1,46 5 103.9, p < 0.0001) and treatment x arm type interaction (f 2,46 5 6.66, p < 0.01). in the test session, only a significant prenatal treatment group effect (f 2,46 5 6.44 p < 0.01). bonferroni's post-hoc test revealed that only control group presented significantly less time in the enclosed aversive arm than nonaversive one. conclusions: in this study we showed that pnee with moderate doses could alter the structure and functional astrocytes balance. thus, high levels of s100b are indicated in some neurodegenerative disorders. taken this data together we could demonstrate that moderate ethanol exposure can be harmful to fetal brain. aging has been associated to neuroinflammation in the central nervous system, however it is not known whether microglial changes induced by aging are affected by early effects, such as litter size and sedentary life style. in addition, the lateral septum has been recognized by its complementary role in the memory processing for tasks of object recognition for identity and spatial location. in the present report, we investigated whether aging cognitive decline and microglial morphological changes in the lateral septal region are influenced by changing litter size early in life and by a sedentary life style. to assess these questions, wistar rats suckled in litters of either six or 12 pups per dam were raised sedentarily in groups of 2 up to 3, from the 21 st post-natal day onwards. at 4 (young adult) or 23 (aged) months-old, half of the sedentary rats underwent progressive daily treadmill exercise for five weeks, while the others remained sedentary. after performing the tests of recognition for spatial localization and object identity all of the animals were sacrificed and their brains were processed for selective microglia/macrophages immunolabeling with anti-iba-1 antibodies. a representative sample of the immunolabeled cells in the lateral septum was analyzed after three-dimensional reconstruction with neurolucida software (microbright field inc.) and morphological features of each cell were quantified by neuroexplorer (microbright field inc.). it was found that sedentary life style of wistar rats maintained in standard laboratory cages is associated with spatial memory deficits in both mature and aged subjects no matter the litter size, and that exercise decreased these effects in aged subjects raised in small but not in large litters. on the other hand, all sedentary aged rats, despite of the litter size, presented impairment of object recognition memory for identity, and exercise decreased this effect in animals from both large and small litter size groups. microglial morphological analysis revealed that cell soma area, perimeter and branches volume seem to be more intensely affected by aging and that these changes are mainly associated with animals raised in large litters. furthermore, it was observed important shrinkage and thickening of the microglial branches in aged individuals on a higher proportion in the sedentary group suckled in large litters. taken together the results suggest that litter size and sedentary life style may affect object recognition and spatial memories in both adult and aged rats and that exercise minimizes these effects on animals suckled in small but not larger litters. in addition, we suggest that these changes are related to distinct effects on the soma and branching patterns of septal microglia from young and aged subjects. huntington disease (hd) is a dominant inherited neurodegenerative disorder caused by an unstable expansion of a cag repeat within the gene encoding for huntingtin protein (htt). this mutation induces formation of a poly-glutamine tract in the mutant htt (mhtt) protein that prones its aggregation into the cells. hd is characterized by an initial and massive degeneration of the medium spiny neurons in the striatum with later neuronal loss in cortex, globus pallidus, and other structures leading to motor and cognitive symptoms. alterations of energy metabolism contribute to hd pathogenesis but the mechanisms responsible for these alterations are not well understood. a recent pet study provided evidence for a selective impairment of striatal glycolytic and not oxidative metabolism of pre-symptomatic hd patients. in the brain, glycolysis is predominantly an astrocytic metabolic process, whereas oxidative metabolism is primarily neuronal raising the question that metabolic defects in hd could originates from astrocytes. the purpose of our study was to characterize (1) the metabolic alterations in vivo in a transgenic mouse model of hd (bachd mice) and (2) the respective roles of astrocytes and neurons in metabolic defects occurring in hd using an in vitro strategy. bachd mice express the human full length htt protein with 97 glutamine repetitions under the human htt promoter into a bacterial artificial chromosome. bachd mice present a slow disease progression with motor deficits occurring at 6 months and progressive neuronal aggregates from 12 months, reflecting human pathology. first, we performed [14c]-2-deoxyglucose autoradiography experiments in vivo to assess energy metabolism in 14 months bachd (n 5 6) and wt (n 5 6) mice. we performed a 3d analysis of the whole brain glucose uptake without any regional a priori. using statistical parametric mapping (spm), a voxel-wise statistical analysis approach, we showed that compared to age-matched wt mice, 14 months bachd mice present hypometabolism in the striatum, like pre symptomatic hd patients, and also in the hippocampus. hypermetabolism was also detected in the hypothalamus. second, we performed an in vitro study to dissect out the cellular mechanisms responsible for these metabolic changes. by using cell insert, we realized neurons/astrocytes co-culture without any physical contact. this culture system allowed us to grow neurons in presence of wt or bachd astrocytes. we showed that both bachd and wt neurons have a decreased [14c]-2-deoxyglucose uptake when cultured in presence of bachd astrocytes but not wt astrocytes. these results collectively suggest that energy defects in bachd mice are due to noncell autonomous mechanisms by which astrocytes expressing mhtt induce neuronal metabolic dysfunction. naag and naa are abundant metabolites in the brain which can be utilised by oligodendrocytes to make myelin. but elevated levels of naag and naa are associated with myelin loss in the leukodystophies canavan and pelizaeus-merzbacher-like disease, whereas lower levels of naa are observed in brain tissue of ms patients. currently, the physiological function of these two metabolites as well as their role in white matter pathology is unknown. naag and naa can also act as signalling molecules as they can affect glutamate receptors. oligodendrocyte precursor cells (opcs) express nmda receptors and activation of these receptors may be important for their differentiation and efficient myelination. in disease opcs are recruited to demyelinating lesions where they proliferate and differentiate into new myelinating oligodendrocytes. we therefore investigated the effects of both physiological and pathological concentrations of naa and naag on opc biology in vitro. we used calcium imaging to see whether naag or naa can induce intracellular calcium changes in opcs as they are known to act on neuronal nmda receptors. 1mm naa or 1mm naag evoked intracellular calcium changes in opcs (df[340/380] 44.6 6 0.7 3 10 23 and 9.0 6 3 3 10 23 respectively), but only one third of the cells responded which corresponds to the proportion of nmda responsive cells in the cultures. however, not all opcs that responded to nmda did also respond to naag or naa. therefore, it seems that the naa and naag evoked calcium raise is via an nmda receptor independent pathway. naag and naa evoked a small but significant increase in intracellular calcium in opcs, which even after prolonged application of pathological levels of naa and naag did not affect the cells' viability as propidium iodide staining of opcs showed no significant difference in viability. over 90% of the opcs survived 4hrs application of 1mm naa and 1mm naag. naag and naa are produced and released by neurons but it is not known how opcs take them up from the environment and how it affects them. we therefore addressed which possible transporters opcs express and the effect naa and naag have on opcs' proliferation and differentiation. it is unlikely that naa and naag even at high concentrations have a detrimental effect on opcs. perhaps these high concentrations of naa and naag found in canavan and pelizaeus-merzbacher-like disease indicate rather opcs' deficiency to transport and utilise naa and naag for energy and myelin formation. vanishing white matter (vwm) disease is a severe leukoencephalopathy, classically starting in childhood. the disease is characterized by neurological symptoms, including uncoordinated movements and balance disturbances which are slowly progressive. episodes of rapid deterioration are triggered by febrile infections and minor head trauma, which can lead to coma. a treatment is unavailable and eventually all patients die within a few years upon diagnosis. the disease is caused by mutations in any of the five genes encoding the subunits of the eukaryotic translation initiation factor 2b (eif2b). although this protein has an important function in all cells, mainly the cells in the brain white matter (astrocytes and oligodendrocytes) are affected by these mutations. pathologically the brain shows cystic degeneration with meager gliosis, dysmorphic astrocytes, lack of myelin and an increased number of oligodendrocyte and astrocyte progenitor cells. we have developed two mouse models for vwm to further unravel the disease mechanisms at molecular level and to develop and test possible treatments. the 2b4 mouse line is homozygous for a mutation in the eif2b4 gene, encoding the eif2bd subunit, and the 2b5 mouse line is homozygous for a mutation in the eif2b5 gene, encoding the eif2be subunit. both mutations have been described in human patients and are associated with a severe form of vwm. preliminary data show that these mice models recapitulate the clinical and neuropathological phenotype observed in vwm patients. pathologically, the central nervous system white matter is vacuolated and shows lack of myelin while astrocytes are dysmorphic and immature. these transgenic mice appear to be a representative model for vwm. microglial cell lines were differentiated from ips cells and represent in vitro models for human microglia. rt-pcr and flow cytometry analysis of ipsdm showed gene transcription and protein expression of cd33 respectively. the protein expression was increased by breaking the cis-interacting sialic acids on the microglial glycocalyx using sialidases. cd33-fc fusion protein was designed and purified via the fc tag to study binding patterns of cd33 to various cell types expressing different glycocalyx structures. additionally, ipsdm exhibiting either an over expression or knockdown of cd33 will be used to determine protein functions. furthermore, preliminary immunohistochemical analysis of control brain tissue compared to ad patients showed increased cd33 protein expression in cd68 positive cells, in the latter. in summary, data show that cd33 is expressed by human microglial cell line enabling further characteristic and functional analysis of the protein. furthermore, preliminary analysis on primary brain tissue corroborates the association of cd33 -expressed on microglia -to lateonset ad. the neurotrophins are essential for peripheral nervous system development and myelination. we have previously demonstrated that the neurotrophin bdnf exerts contrasting influences upon myelinationacting through neuronal p75 ntr to enhance myelination, but inhibiting it via neuronal trkb. recently we have generated a small peptide called cyclo-dpakkr that structurally mimics the region of bdnf that binds p75 ntr . here we aim to investigate whether utilising cyclo-dpakkr to selectively target p75 ntr is an approach that could exert a unified promyelinating response. like bdnf, cyclo-dpakkr promoted myelination of ngf-dependent neurons in vitro, an effect dependent on the neuronal expression of p75 ntr . importantly, cyclo-dpakkr significantly enhanced the myelination of bdnf-dependent neurons in vitro, whereas bdnf inhibited it. local injection of cyclo-dpakkr adjacent to the neonatal sciatic nerve in vivo significantly enhanced myelin protein expression and increased the number of myelinated axons. we found that injection of cyclo-dpakkr also significantly upregulated the expression of neuregulin 1 type-iii, a key factor required to induce peripheral myelination. thus, our data demonstrate that cyclo-dpakkr promotes peripheral myelination in vitro and in vivo. to investigate whether cyclo-dpakkr promoted the remyelination of peripheral neurons, we utilized the ean, a rodent model of peripheral demyelinating neuropathy. our data show that administration of cyclo-dpakkr significantly delayed the disease onset and reduced clinical severity in ean. this improved disease phenotype is supported by reduced demyelination, as cyclo-dpakkr substantially reduced the extent of myelin damage in myelinated peripheral nerves subjected to ean. collectively, our data demonstrate that using cyclo-dpakkr to selectively target p75 ntr promotes peripheral myelin development, and is effective at ameliorating ean disease and protecting against myelin damage. our findings suggest that selective targeting of p75 ntr is a strategy worthy of further investigation for the treatment of peripheral demyelinating diseases introduction huntington's disease (hd) is a neurodegenerative disease characterised by huntingtin protein misfolding and the intra-cellular accumulation of mutant huntingtin (mhtt). accumulation of mhtt is most pronounced in neurons and is thought to underpin neuronal dysfunction. oligodendrocytes have been shown to accumulate mhtt aggregates, which may contribute to cellular dysfunction in these cells. evidence from diffusion tensor mri (dt-mri) studies shows that white-matter changes are amongst the earliest pre-symptomatic changes observed in hd. these findings have been taken to implicate axonal and or glial aberrations prior to disease onset. methods we have used a variety of techniques, which include, emulsion in situ hybridisation, whole brain sub-fractionation, immunohistochemistry, meso scale cytokine assay, and western blots. results our investigations using the r6/2 mouse model show that mhtt aggregates result in a specific down-regulation of the small heat shock protein (shsp) hspb5 (alpha-b-crystallin). localisation of hspb5 by whole brain sub-fractionation shows that hspb5 is enriched in the myelin fraction. emulsion in situ hybridisation further shows localises hspb5 in oligodendrocytes. detergent extraction of myelin fractions show differential hspb5 partitioning between soluble and insoluble fractions. cumulatively, these findings suggest that hspb5 is constitutively expressed in oligodendrocytes and is present in two distinct pools. recent studies implicate hspb5 as being a negative downregulator of inflammation. as such down-regulation of the shsp in r6/ 2 mice may promote increased oligodendrocyte vulnerability to mhtt cytotoxicity. to investigate the role of hspb5 as a negative regulator of inflammation, we obtained transgenic hspb5 knockout mice. the knockout mice are viable and show very little phenotypic difference against littermate controls. we have analysed the inflammatory profiles of these mice using the meso scale. to validate our findings in the r6/2 mice, we are currently investigating human tissue from distinct vonsattel stages of disease, to see if hspb5 downregulation is also observed in the human disease. conclusion as hspb5 has several critical cellular roles, its downregulation may compromise oligodendrocyte structure and function, which ultimately has negative consequences for neurons. it is therefore worthwhile to investigate it's specific role in the cns. the myelin sheath is attached to the axon through multiprotein complexes that form the axon-glial interactions. they consist of cell adhesion molecules and voltage gated ion channels and divide the axon into distinct domains: the node of ranvier, the paranode, the juxtaparanode and the internode. this molecular organization is crucial since recent studies identified distinct perinodal proteins as autoantigens in ms patients: nodal and paranodal neurofascin isoforms and juxtaparanodal tag-1/contactin-2. tag-1 is a cell adhesion molecule expressed both by axons and glial cells. in the adult nervous system, tag-1 organizes the juxtaparanodal domain of the fiber, where it interacts with caspr2 and the potassium channels (vgkcs). question: in this study we have analyzed the alterations of the juxtaparanodal proteins caspr2, tag-1 and vgkcs in relation to paranodal caspr and nodal sodium channels upon the onset and progression of experimental autoimmune encephalomyelitis (eae) and in the cuprizone model of toxic demyelination. methods: we immunohistochemically and biochemically analyzed both models of cns demyelination. results-conclusions: our results show a higher paranodal susceptibility to disruption compared to juxtaparanodes in eae. as eae progresses there is subsequent compensatory efforts for myelinated fiber restoration that include paranodal protein re-clustering and increased heminodal formation, without subsequent juxtaparanodal protein aggregation. in contrast, juxtaparanodal organization was observed only when remyelination occured in the cuprizone model of toxic demyelination. supported by: imbb-forth scholarship, manassaki scholarship (university of crete), the project "myelintag" (593) is implemented under the "aristeia" action of the "operational programme edu-cation and lifelong learning" and is co-funded by the european social fund (esf) and national resources. multiple sclerosis (ms) is a chronic disease of the human central nervous system that is characterized by focal lesions with inflammation, infiltration of immune cells, demyelination and axonal damage. activation of cannabinoid cb 1 /cb 2 receptors is considered a potential therapeutic strategy for the treatment of ms, based on the evidence that exogenous cannabinoid agonists exert neuroprotective and immunosuppressive effects in experimental models of the disease. nevertheless, the therapeutic use of synthetic and/or plant-derived agonists acting on brain cannabinoid receptors is limited by the possible adverse responses related to memory and learning impairment. an alternative approach that could avoid this limitation consists of enhancing the concentration of the main endocannabinoids (aea, 2-ag) by increasing their synthesis or decreasing their degradation. the main objective of this study was to analyze the effects of jzl184, a selective inhibitor of 2-ag hydrolyzing enzyme monoacylglycerol lipase (magl), in the chronic eae model of ms. mice were treated daily with jzl184 (8 mg/ kg and 32 mg/kg) or vehicle from onset of the motor symptoms to the end of the experiment. comparison of the motor score curves indicated that both doses of jzl184 ameliorated the deficits observed in vehicletreated mice during the disease course. nevertheless, the beneficial effect of the 32 mg/kg dose was no longer evident in mice scored at 40 dpi. chronic treatment with 8 mg/kg jzl184 reduced the conduction latency of the corticospinal tract measured at the end of the experiment, as well as the number and size of inflammatory lesions in spinal cord, whereas chronic administration of the high dose of the magl inhibitor had no effect on these parameters. importantly, treatment with the 32 mg/kg dose of jzl184 reduced the coupling ability of cannabinoid receptors to g i/o proteins, measured by [ 35 s]gtpcs autoradiography, in all the brain areas analyzed. finally, incubation with jzl184 prevented cytotoxicity by activation of ampa-type glutamate receptors in oligodendrocyte precursors in vitro. this protective effect of the jzl184 was blocked by the cb 1 receptor antagonist am281. our findings point to the involvement of cb 1 receptors in the in vivo therapeutic effects of jzl184, and suggest that chronic administration of magl inhibitors may be a promising strategy for the treatment demyelinating disorders in which oligodendrocyte excitotoxicity plays an important role as mechanism of white matter damage. under disease conditions, connexins can also release these signaling molecules into the extracellular milieu through hemichannels. since astrocytes are involved in the disease progression of als, we are exploring if abnormal gj activity can serve as a potential mechanism for the reported astrocyte-mediated toxicity. we used the sod1 g93a mutation as a als mouse model to address the role of connexins in als. we observed that at the end stage of the disease, spinal cords of sod1 g93a mice exhibited a significant increase in expression of connexin 43 protein compared to their spinal cords of control mice. this increase in cx43 co-localizes primarily to astrocytes in the gray matter of the spinal cord. importantly, human post-mortem tissue from als patients compared to control patients exhibited a significant increase in cx43 rna and protein levels in the motor cortex and a trend for increase in cx43 in the cervical spinal cord. this is an important finding in pursuing cx43 as a potential candidate contributing to astrocyte-mediated toxicity in als. in addition, when astrocytes were isolated from wt and sod1 g93a mice, endogenous levels of cx43 rna and protein were elevated in sod1 g93a mice compared to the wt derived astrocytes. we are currently investigating if functional properties of astrocytes from sod1 g93a mice are altered in addition to biochemical changes. in vitro studies using cx43 specific blockers to assess neuroprotection are also being conducted in co-cultures of neurons and astrocytes. these findings will be novel and an important step towards harnessing therapeutic strategies for als. the pdz protein omi/htra2 is localized in the intermembrane space of mitochondria. it is involved in mitochondrial homeostasis and may act as a chaperone. mutations in the serine protease domain of omi/ htra2 (mnd2 mice), leads to massive loss of striatal neurons and neuromuscular disorders in mice confirming the protective function within mitochondria. upon cellular stress, omi/htra2 is translocated from mitochondria into the cytosol when the mitochondrial outer membrane is permeabilised. in the cytosol, omi/htra2 blocks the action of the inhibitors of apoptosis proteins (iaps) by competitive binding or by degradation via the protease activity. this leads to caspase activation and apoptosis induction. omi/htra2 also regulates apoptosis in a caspaseindependent manner via its protease activity; recent studies have defined multiple substrates of the serine protease. in a yeast twohybrid screen we identified the ng2 proteoglycan as a binding partner of omi/htra2. ng2 is expressed by oligodendrocyte progenitor cells (opc). in the presence of hydrogen peroxide, which is a model of cellular oxidative stress, omi/htra2 is released from mitochondria and can bind to ng2 in opcs. binding of ng2 to omi/htra2 via the pdz domain may thus be a way to sequester or regulate the serine protease. oligodendrocyte-lineage cells are known to be particularly sensitive to cellular stress and white matter diseases of new borns is a major pathology in situations where premature infants are exposed to unphysiological oxygen concentrations. it has been recently shown that glioma cells often express the ng2 protein which may play a protective role. our results indicate that the interaction between omi and ng2 may help protect opcs from cellular stress. microglia are the immunocompetent cells of the cns that continuously survey the extracellular milieu for foreign antigens and play an instrumental role in brain inflammation. in contrast, astrocytes extend highly ramified processes between neurons and synapses and play a vital role in regulating brain homeostasis, synaptic function and plasticity. interestingly, both microglia and astrocytes adopt a specialized 'activated' or 'reactive' state when exposed to adverse brain conditions. the changes in their molecular profiles include the release a diversity of proteins such as pro-and anti-inflammatory cytokines and extracellular matrix molecules that could impact each other's behavior and regulate the properties of nearby neurons. in alzheimer's disease (ad), activated microglia and reactive astrocytes are detected around plaques in mouse models of ad and human ad tissues. however, exactly how these glial types contribute to the onset or progression of ad still remains an open question. to gain a better understanding of the bidirectional communication and response of microglia and astrocytes in the development of ad-like symptomatology, we investigated the properties of these cells types in the crnd8 transgenic ad mouse model by applying confocal imaging and western blot analysis. crnd8 is an early-onset model of ad-like symptomatology showing ab deposits present at 2 months of age and neuritic pathology by 5 months. our results show complex spatio-temporal changes in the interaction of microglia and astrocytes around ab plaques during the progression of the disease in both cortex and hippocampus. we found that subtle rearrangements of microglial morphology and astrocyte reactivity were detected as early as 1 month when ab plaque deposition is absent. by 2-3 months, low numbers of activated microglia and reactive astrocytes begin to surround small ab deposits. the establishment of complex 'reactive glial domains (rgds)' can be observed at 4 months, when amoeboid microglia fully encompass large ab plaques and become surrounded by reactive astrocytes forming an elaborate outer shell-like structure. intriguingly, after 6 months, the domains become progressively disrupted due to the reorganization and recruitment of additional microglia and astrocytes. we also observed gradated inflammatory phenotypes of glial cells. these are first restricted to rgds in the early and middle stages of ad but spread locally in late stages when disorganized rgds are observed. interestingly, neuronal dystrophy was correlated with the severity of the inflammation. we conclude that communication between microglia and astrocytes may be a key process in the ability of the brain to surmount a reparative response to early neurodegenerative conditions but may be disrupted or become overwhelmed in later stages of ad. supported by alzheimer society of canada (db) and by cihr (kkm and rq). poster topic 04 extracellular matrix and cell adhesion molecules opcs exist in mature rodent and human central nervous system (cns; around 5-7% of the total cells) and constitute an interesting source regenerative therapies in demyelinating diseases, like multiple sclerosis (ms). different molecules are involved in opc morphofunctional development during embryogenesis and postnatal stages, and some of them are upregulated in ms lesions, suggesting their involvement in the pathogenesis of the disease. this is de case of anosmin-1, an extracellular matrix glycoprotein coded by the kal1 gene and responsible for the x-linked form of kallmann syndrome. the best known mechanism of action of anosmin-1 seems to be mediated through the interaction of this protein with fibroblast growth factor receptor 1 (fgfr1) and the modulation of the activation of this receptor by fgf2. this protein participates in the adhesion, migration and differentiation of various cell types in the cns; among others, anosmin-1 promotes the adhesion of neurons, neurite outgrowth, axonal guidance and branching of different cns projection neurons, as well as having a role in the migration of different types of neuronal precursors, immortalized gnrh-producing neurons and embryonic opcs. in addition, previous results of our group also suggest a role of anosmin-1 in demyelinated lesions from ms patients and point to the feasible pharmacological and genetic manipulation of the fgf2/fgfr-1/anosmin-1 system on endogenous and/or exogenous opcs in demyelinating lesions. in the present work we studied the functional implications of anosmin-1 and fgf-2 on neurobiology (cell death, proliferation, motility, migration) of postnatal/adult murine opcs isolated from cerebral cortex (p0, p15, p60) and of opcs isolated from adult human biopsies, using both in vivo (immunohistochemistry) and in vitro (chemotaxis chambers, migration, brdu uptake, tunel, immunocytochemistry) techniques. these results would be useful for the design of effective neuroreparative therapies in ms and other demyelinating diseases. funded we generated ipsc lines from fibroblasts isolated from pitx3-gfp knock-in mice which allowed facs-purification of mature midbrain da neurons upon in vitro differentiation based on the expression of the mature da marker pitx3. subsequently, mouse pitx3-gfp knock-in ips cells were transduced with lentiviral vectors containing genes encoding for hl1 and for stx, the enzyme that add psa onto ncam. next they were differentiated in vitro into da neurons. the effects of hl1 and psa-ncam on differentiation, survival and outgrowth of ipsc-derived da neuron were analyzed in vitro and in vivo. indeed, we were able to obtain a pure suspension of mature ipscderived da neurons upon in vitro differentiation and fac sorting. transfection of pitx3-gfp knock-in ips cells with hl1 or stx did not affect the pluripotent potential of these cells and did not interfere with the process of dopaminergic differentiation itself. we continued to establish the beneficial effect of l1 and psa-ncam expression on survival and outgrowth of ipsc-derived da neurons in vitro and after grafting in the striatum of parkinsonian rats. vascular endothelial growth factor (vegf) is a major regulator of neurovascular remodeling following brain injury. while much have been learnt about vegf functions on target endothelial cells, little is known about its intracellular trafficking, mode of secretion and matrix interactions in "source" cells, i.e. mainly reactive astrocytes. here, we generated a vegf::gfp fusion protein to follow the distribution of vegf165 during its trafficking in primary astrocytes and cos7 cells. we found that vegf::gfp forms dimers and gets glycosylated similarly to wild type, and as expected, the molecule follows the endoplasmic reticulum-golgi pathway. however, its post-golgi trafficking suggests a unique route with features that do not conform the classical constitutive secretory pathway: the secretion of vegf165 occurs even at 19 c and shows a ca21-and pkc-induced increase. we investigated the distribution of vegf in polarized primary astrocytes in an in vitro scratch wound assay. in these cells, vegf::gfp appeared to follow a vectorial distribution and accumulated behind the leading edge. the accumulation appeared to be on the extracellular surface, moreover, ultrastructural immunogold labeling revealed that extracellular vegf::gfp remains associated with discrete areas of the cell membrane and is accumulated in caveolae as well as in microvesicular shedding elements. this particular localization corresponds to fibrillar adhesions (fb), where vegf::gfp is co-localized with fibronectin. we also observed that vegf::gfp is targeted to adherens junctions between astrocytes, however, at these sites vegf::gfp was not co-localized with fibronectin. finally, we show in frap experiments that integrin turnover is decreased in vegf associated fbs, which raises the possibility of an autocrine/paracrine regulation of astrocytic functions. together, these findings have strong implications for understanding focal coordination of angiogenesis and vascular remodeling by astroglia derived vegf. after invading the embryonic cns, microglia migrate extensively to occupy their final positions. however, the cellular and molecular mechanisms of microglial migration are unknown. therefore, this study aims to elucidate the microglial migration behavior, cellular interaction partners and ecm-integrin interactions essential for migration in the developing neocortex. methods: immunohistochemical stainings and immunogold labeling for transmission electron microscopy were carried out on fixed brain tissue of c57bl/6 cx3cr1 1/egfp mice embryos (embryonic day (e) 10.5-17.5). multicolor facs analyses were performed on homogenized age-pooled cx3cr1 1/egfp embryonic cortici. microglial migration was recorded in cx3cr1 1/egfp -hgfap-cfp acute brain slices using multiphoton time-lapse excitation and analyzed using mtrackj in imagej. results: laminin (lm) is expressed as punctuate dots near the pia and in the ventricular zone at e10.5 and disappears at e15.5 to remain only in blood vessels and the pia. fibronectin (fn) is present as aggregates from e10.5 until e17.5. both ecm proteins follow the course of radial cells. preliminary data indicate a transient upregulation of the lm receptor (lmr) on e15.5 and e16.5. in addition, the percentage of microglia expressing the fn receptor (fnr) tends to decrease during development. ex vivo time-lapse recordings show that embryonic microglia are dynamic cells that migrate in random patterns. these cells seem to make brief contacts with the processes of radial glia. conclusions: embryonic microglia express laminin and fibronectin receptors and possibly change the expression level during development. they are dynamic cells that migrate in random patterns, and possibly make occasional contact with radial glia. knowledge about ecm-integrin interactions during microglial migration could reveal targets for intervention in pathological settings, such as multiple sclerosis and alzheimer's disease. multiple sclerosis (ms) is a chronic demyelinating disease of the central nervous system in which astroglial cells become hypertrophic, produce various extracellular matrix (ecm) proteins which become aggregated when secreted. these aggregated ecm proteins contribute to a non-permissive environment for repair. tissue transglutaminase (tg2) expression is enhanced in astrocytes in ms lesions. this enzyme is well-known for protein cross-linking capacities. moreover, tg2 can act as a co-receptor for b-integrins to bind to ecm proteins, thereby mediating cell adhesion processes. in this study we questioned whether astrocyte-derived tg2 affects ecm protein production and/or aggregation, and if astrocyte-derived tg2 mediates cell adhesion onto various ecm proteins. primary rat astrocytes were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg2. alternatively, primary rat astrocytes were transduced with a lentiviral vector expressing scrambled shrna or tg2 shrna to knockdown endogenous rat tg2. the rat primary astrocytes overexpressing human tg2 showed an increased production of fibronectin and collagen v. conversely, knocking-down tg2 showed a decrease in fibronectin and collagen production. interestingly, overexpression of tg2 resulted in enhanced aggregation of extracellular laminin. also, human astrocytoma cells (u373) were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg2 and allowed to adhere onto ecmcoated wells. astrocytoma cells overexpressing human tg2 showed increased adhesion onto laminin-, and fibronectin-coated wells. we conclude that astrocyte-derived tg2 affects production/aggregation of fibronectin and laminin. moreover, the astrocyte-derived tg2mediated elevated production/aggregation of fibronectin and laminin coincides with more adherence of astrocytes onto these ecm proteins. we hypothesize that tg2-mediated enhanced production/aggregation of fibronectin and laminin is related to increased astrocyte adhesion which together could contribute to the non-permissive environment for repair in the central nervous system of ms patients. a. tripathi, z. parikh, p. pillai the m.s. university of baroda, vadodara, india background: oligodendrocytes originate as progenitor cells (opcs) in discrete areas of the developing brain which undergoes a complex and precisely timed program of proliferation, migration, differentiation, and myelination in cns. during migration it interacts with its surrounding environment through integrins. purpose: to evaluate the interactions between integrins and growth factors (gfrs) that promote the molecular events such as proliferation, cytoskeletal reorganization and migration in oligodendrocytes leading to oligodendrocyte mediated myelination. methods: cortical cells were prepared from p0 rat cerebral cortex following standard protocols. immunocytochemistry (icc), western blot (wb), immunoprecipitation (ip), migration assay and real-time rt-pcr analysis were performed. for dissecting out the roles of different intracellular signalling proteins in the regulation of events downstream of receptor activation, use of pharmacological inhibitors was carried out. results: tnfa, pdgfa and estradiol significantly increased cell proliferation and cell processes. data also suggest differential effects of extracellular matrix (ecm) on signalling component activation. significant increase in erk expression was found following gfrs-integrin interactions. ecm proteins bind to integrin family members and mediate bidirectional signalling between the cellular interior and the external environment through the activation of focal adhesion complexes. conclusion: integrin switching, oligodendrocyte proliferation, cytoskeletal reorganization and differentiation profile is highly influenced by ecm and gfrs. integrin governs the signalling cascades underlying oligodendrocyte differentiation and myelination. this study was performed in strict accordance with the recommendations for the care and use of laboratory animals. all the protocols were duly approved by the institutional ethical committee, the m.s. university of baroda. topography, roughness and mechanical properties of micro environment are crucial parameters influencing cell adhesion/motility, morphology and mechanics as well as the development of cells e.g. stem/progenitor cells, and neuronal cells [1] [2] [3] [4] [5] . atomic force microscopy is a powerful tool not only to study the morphology in terms of high resolution imaging and roughness measurements, but also to map mechanical and adhesive properties of the sample/cells and tissues. combining these remarkable abilities with advanced optical microscopy allows for extensive characterization of biomaterials and tissue slices [6] [7] [8] . however, commercially existing technical solutions are either time consuming, or only limited practicable for soft, sticky, or fragile samples. therefore, we developed a new force curve based afm mode -quantitative imaging (qi tm ). the novel qi tip movement algorithm prevents lateral forces and controls the vertical forces for nondestructive imaging. to demonstrate the performance and flexibility qi tm mode we investigated topography, adhesion properties, and young's modulus local distribution of living dorsal root ganglion cells. a specialized platform -cellhesion v r -has been developed to run single cell force spectroscopy (scfs) with a need for long-range cellsurface and cell-cell binding experiments with up to 100 microns pulling length. using single cell force spectroscopy (scfc), cell-cell adhesion can be quantified [e.g. 3], and the contribution of different components e.g. from the extra cellular matrix, can be assessed [9] . a new technical solution will be demonstrated if a cantilever attached cell can be transferred through the liquid-air interface. this approach allows to measure the adhesion of the same individual single cell on different materials within different dishes as it will presented for cho cell adhesion on plastic as well as albumin coated surfaces. we want to present the progress in automatic data processing and display to investigate topography, young's modulus and local adhesion phenomena on transparent and non-transparent biomaterials like titanium, peptide functionalized surface or cell layers, and tissue slices. l. vargova 1 , m. cicanic 1,2 , e. sykova 1,2 1 charles university, 2nd faculty of medicine, prague, czech republic 2 institute of experimental medicine, v.v.i., department of neuroscience, prague, czech republic bral-2 is a link protein that is localized in distinct brain regions, where it stabilizes the perineuronal nets of the extracellular matrix (ecm). our previous studies showed that qualitative and quantitative ecm changes, might evoke changes in the diffusion properties of the extracellular space (ecs), thus affecting the movement of neuroactive substances in the cns. in the current study, we determined by the real-time iontophoretic method the extracellular space volume fraction a (a 5 ecs volume / total tissue volume) and the geometrical factor tortuosity k (k 2 5 free / apparent diffusion coefficient) in coronal brain slices obtained from the sensorimotor cortex and the ventral posteromedial thalamic nucleus of sixteen-month-old bral-2 deficient mice (bral-2 -/-), age-matched bral-2 1/1 controls and young adult (5-month-old) bral-2 1/1 controls. the diffusion parameters in the cortex of young adult bral-2 1/1 mice were: a 5 0.21 6 0.01 (mean 6 s. e. m.) and k 5 1.48 6 0.02, n 5 6, n 5 5 (n -number of slices, n -number of animals). in the thalamus, there was a smaller a and a larger k (0.16 6 0.01 and 1.53 6 0.01, respectively, n 5 26, n 5 10). in young adult mice, we found no significant differences in the ecs diffusion parameters between bral2 positive and negative mice in either the cortex or the thalamus. in the cortex of aged bral-21/1 mice we found a smaller a (0.18 6 0.01, n 5 10, n 5 7) than in the cortex of young adult mice. during aging, there was no significant difference in the thalamic diffusion properties in wild-type animals, but in the thalamus of aged bral-2 -/-mice, a significantly decreased (0.13 6 0.01, n 5 28, n 5 11) in comparison to the younger bral2-/-animals as well as to age-matched controls. immunohistochemical analysis of the ecm in the ventral posteromedial thalamic nucleus revealed no positivity for bral2 protein in either group of bral2-/-mice. in the youger mice, bral deficiency was associated with a small attenuation in the positivity of brevican and another link protein, ctrl1; however, in aged bral2-/-animals, brevican and ctrl1 positivity was increased in comparison with the age-matched controls. in contrast to the cortex, we found no decrease in a in the thalamus of aged wt animals, suggesting that the process of aging may differ between the cortex and the thalamus. a decrease in the ecs volume fraction in the thalamus of bral-2 -/-aged mice may result from a disruption of the perineuronal nets and rearrangements of the molecular assembly, which seem to differ in young adult and aged animals. here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of camp pathways. a bulk of astroglial transcripts were differentially regulated by camp signaling. camp analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. moreover, genes typically activated in reactive cells, such as immunological mediators and scar components, were repressed by camp. gene set enrichment analysis and evaluation in situ of gene expression in astrocytes in different states showed that camp signaling conferred a mature and in vivo-like transcriptional profile to cultured astrocytes. these results indicate that camp signaling is a key pathway restricting developmental and activation features of astrocytes and promoting their maturation. a positive modulation of camp signaling is suggested to suppress the mechanisms of activation driven by pathological situations and to promote the physiologically normal state of differentiated astrocytes. question: microglia, the innate immune system of the central nervous system, are crucial to maintain homeostasis by cleaning debris and attacking stressors. in the ageing brain, increased numbers of microglia with ameboid morphology and increased inflammatory cytokine levels are reported. the altered microglial phenotype in the ageing brain is characterized by hyperresponsive to immune stimuli, which is called immune priming. immune priming could be a potential cause of age-associated neurodegeneration. the aim of this study is to characterize the immune pathways and biological processes that are altered in ageing-induced immune primed microglia and to find genes potential drivers of this process. design/methods: the effect of ageing on microglia has been addressed in naturally aged mice (24 months old) and in ercc1 transgenic mouse model in which accelerated ageing is induced by dna damage accumulation. from each of these models a pure population of microglia was isolated. microarray technology was used to obtain information about the genome wide gene expression profile. weighted gene co-expression network analysis (wgcna), uses correlational structures to find clusters of genes to make optimal use of a dataset in biological interpretation. results: using weighted gene co-expression network analysis, a very robust microglial immune priming gene module was discovered. in this module, there is a significant enrichment of the complement system, antigen presentation, interferon type-1 signaling and phagocytosis-machinery. the average expression of this module is stronger up-regulated in ercc1 transgenic than in normally physiologically aged mice, supporting the notion that ercc1 is characterized by excessive immune priming. conclusion: microglial priming which occurs in normal ageing or in accelerated ageing models is marked by increased expression of several immune signaling pathways. methods: we investigated three single nucleotide polymorphisms (snps) (rs1042713 (arg16gly), rs1042714 (gln27gly) and rs1042711) and five haplotypes in adrb2 to explore whether these polymorphisms were associated with ms progression in 436 well phenotyped ms patients. genotyping was done using illumina bead microarray and missing genotypes were imputed using beagle. results: a trend towards a decreased frequency of rs1042714c allele (27gln) was in people with progressive ms than in people with relapsing remitting ms. this was seen in both sp-ms (p (uncorrected) 5 0.0072) and in pp-ms (v 2 5 6.85, p (uncorrected) 5 0.0325). however, those p values did not reach statistical significance after correction for multiple testing. neither haplotypes nor snps were significantly associated with altered msss, age of onset, time between fist and second relapse, processing speed (sdt) or brain atrophy. conclusion: we found no evidence that polymorphisms in adrb2 influence severity in multiple sclerosis. here we examine the expression of these brain-specific pgc-1a isoforms in neurons and glia cells. we characterize the abundance of the different isoforms of pgc-1a in primary murine cell cultures of neurons, glia cells, including oligodendrocytes, astrocytes and microglia under control conditions and metabolic stress. we show that pgc-1a is differentially expressed in the different cell types, e.g. neurons and oligodendrocytes express much higher levels of the novel brain-specific isoforms of pgc-1a. the different expression levels might reflect the different metabolic needs of the different cell types. s. marques, g. castelo-branco karolinska institutet, stockholm, sweden oligodendrocytes (ol) are neuroepithelium-derived cells that insulate neuronal axons through myelin-containing membranes, essential for axonal integrity and impulse transmission. defects in this process are present in several diseases, including the highly debilitating multiple sclerosis (ms). the process of remyelination can be promoted by ol precursor cells (opc) endogenously and occurs during a short window of time at earlier stages of ms, ultimately failing as the disease evolve. the understanding of development and differentiation regulation of ol is essential to clarify the reasons behind the defective myelination during pathologies and to open the pave to new remyelinating cell therapies. opcs start to be specified early during embryogenesis but regardless of the origin, their terminal differentiation and functional maturation occurs only at post-natal stages. in this project, we are characterizing the transcriptome of opcs during development, which will allow us to better characterize the heterogeneity of these cells and point out what might be the key factors involved in transition between states. recent studies suggest that non-coding rnas can interact with proteins complexes containing chromatin modifying enzymes and transcription factors, regulating their activity, acting as scaffolds and/or recruiting them to specific loci in the genome. therefore, we are investigating their role in opcs by rna interference and how they might modulate the function of key transcription factors. neural stem cells (nscs) represent a potentially valuable resource when considering repair strategies for cns damage. unlike their embryonic counterparts, adult neural stem cells (anscs) in the two neurogenic niches have an astrocyte-like identity, raising the question of what regulates this ansc state. some early postnatal astrocytes in the parenchyma also retain some nsc-like characteristics and in vitro display self-renewal and multipotency, though these properties are lost following in vivo maturation. interestingly, following injury, adult astrocytes can become reactive and reacquire nsc-like properties, whilst others can generate new neurons through forced expression of specific transcription factors. we are aiming to elucidate the gene regulatory networks underlying specific nsc and astrocyte states and transitions between them using transcriptomic and epigenomic tools alongside computational methods in different nsc and astrocyte populations. we will describe our latest findings from an in vitro system that aims to model immature versus mature astrocytes derived from a common progenitor. from microarray data, we have identified candidate genes and pathways involved in regulating astrocyte potential versus differentiation, including a role for specific pro-inflammatory signalling. we will also describe the associated epigenetic signatures that accompany cell state and discuss the implications of our latest findings. microglia are the cns immune-related cells and associated with the pathogenesis of several types of neurological diseases. following peripheral nerve injury (pni), spinal microglia become activated and have crucial roles in generating neuropathic pain. we have previously shown that interferon regulatory factor-8 (irf8), upregulated in spinal microglia after pni, regulates gene expressions that switch to a reactive phenotype. here we identified irf5 as a crucial factor of reactive microglia that works cooperatively with irf8. we found that pni increased expression of irf5 in the spinal cord, the expression of which was restricted to microglia. forced expression of irf8 in cultured microglia markedly increased expression of irf5 in a manner that depended on its ability to bind dna. furthermore, irf8-deficient mice failed to increase the expression of irf5 in spinal microglia after pni, indicating irf8-dependent expression of irf5 in microglia in vivo. interestingly, upregulation of p2x4 receptor (p2x4r; an atp-gated channel crucial for producing neuropathic pain) by forced expression of irf8 in cultured microglia was markedly suppressed by knockdown of irf5 expression. in a model of neuropathic pain, suppressing upregulated irf5 protein by spinal administration of sirna targeting irf5 alleviated pain hypersensitivity. altogether, the irf8-irf5 axis drives a program of gene expression that promotes p2x4r hi microglia gating neuropathic pain. astrocytes are the most abundant glial cell type. they express a range of neurotransmitter receptors localized along their processes that cover synapses and constitute "microdomains" for signalling pathways. to investigate astroglial purinergic p2y1 and glutamatergic nmda receptors we generated knockout mice in which gene recombination of "floxed" receptors was controlled by an astrocyte-specific and tamoxifen-inducible cre recombinase (glast-creert2). here, we present data on the dna recombination efficiencies of glast-creert2 x floxed p2y1 mice and glast-creert2 x floxed glun1 mice in different brain regions (cerebellum, hippocampus, brainstem, optic nerve and cortex). recombination is determined by quantitative real-time pcr of genomic dna using primers across the loxp sequences flanking exon 1 of the p2ry1 gene and exon 11-21 of the grin1 gene, respectively. dna recombination was induced in young adult mice by intraperitoneal tamoxifen injection for three consecutive days and analyzed three weeks later. primers were designed such that reduction of the pcr signal reveals increasing recombination (i.e. gene excision and knockout) compared to control. the degree of reduction in the investigated brain regions is expected to be similar in both mouse lines; allowing conclusions (1) about the reliability of the glast-creert2/loxp system in general (i.e. recombination efficiency at different chromosomal locations), and (2) gives a percentage of glastpositive cells (predominantly astrocytes) in the given brain area. first data demonstrate 30% recombination in the cortex, which is well in line with the percentage of astrocytes in this brain region. these results could be confirmed by using a reporter mouse line expressing the red fluorescent protein tdtomato (r26-tdtomato) and using immunohistochemistry with astrocyte markers. in conclusion, the combination of quantitative real-time pcr analysis of gene recombination and cell-specific cre mouse lines represents a reliable approach to reveal the percentage of a given cell type in a given tissue region. the transcription factor creb is protective in injury and neurodegeneration, but the contribution of neuronal vs astrocytes is unknown. we have generated a mouse with targeted over-expression of a constitutively active creb (vp16-creb) in astrocytes by crossing teto-vp16 and tta-gfap mice. vp16-creb/gfap mice show expression of vp16 in astrocytes -as detected by immunohistochemistry -restricted to cerebellum, hippocampus and outer layers of cortex. we determined the role of astrocytic creb in the outcome of brain injury by subjecting wild type (wt) and vp16-creb/gfap (tg) mice to a focal cryolesion (c) in the frontal cortex. at 3 days post-lesion, wt mice presented a necrotic region filled with macrophages (labeled with lectin) surrounded by a rim of tissue with robust gliosis (labeled with gfap). tg mice showed reduced macrophage infiltration as compared with wt mice. a dna array analysis of the damaged zone (agilent) has revealed differentially expressed genes in paired comparisons. wtc/wt: 13,984; wtc/tgc: 5,821; tgc-t:913; tg/wt:0 (statistical linear model in limma., p values computed by empirical bayes moderated t-statistics at 0.05). there were 159 significantly enriched kegg pathways regulated by the cryolesion, according to gsea and hypergeometric tests set at p < 0.05. vp16-creb had an effect on 127 of the pathways. upon increasing the analysis stringency by setting the cut-off at p < 0.0001, the number of significantly enriched kegg pathways regulated by vp16-creb was reduced to 16. we selected 10 of these pathways related to 5 functions: energy metabolism, inflammation, structural reorganization, genomic stability and apoptosis, which we define as the core signature of the vp16-creb-elicited change. the differential expression of representative genes related to this signature was validated by real-time pcr. thus, vp16-creb rescued the decreased expression of enzymes regulating energy metabolism, while decreasing the expression of pathways related to apoptosis, extracellular matrix and inflammation. the latter is consistent with the decreased macrophage infiltration detected by immunohistochemistry in tg mice. we posit that vp16-creb protects the brain against bionergetic failure and secondary damage due to macrophage infiltration, while rendering the tissue more conducive to neurite regeneration by reducing extracellular matrix components. the study supports the notion of astrocytes as therapeutic targets in brain injury. furthermore, ca 21 /cn/nfat dependent mmp3 expression was confirmed in pure astrocyte cultures derived from neural stem cells (ast-nsc), demonstrating that the induced mmp3 expression occurs in astrocytes, and not microglial cells. in an in vivo stab-wound model of brain injury, mmp3 expression was detected in nfatc3-positive scarforming astrocytes. since [ca 21 ] i increase is an early event in most brain injuries, these data support an important role for ca 21 /cn/ nfat-induced astrocyte mmp3 expression in the early neuroinflammatory response. understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain. the posterior hypothalamus (ph) is crucial for maintaining wakefulness whereas the anterior hypothalamus (ah) constitutes a sleep-promoting region. consequently, the energy needs of ah and ph should probably change throughout the sleep-wake cycle. the glycogen mobilization altogether with the astrocyte-neuron lactate shuttle (anls) likely constitutes two major mechanisms by which astrocytes ensure the neurometabolic coupling. therefore, we hypothesized that astrocytes of ah and ph may display differences in genes expression involved in these mechanisms during the sleep-wake cycle. to investigate this, we measured the gene expression levels in astrocytes obtained from transgenic mice expressing the fluorescent protein egfp under the control of the astrocyte-specific gene gfap promoter. mice were sacrificed at four time, zt0, zt6, zt12 and zt18 (zt0 corresponding to light-on). after brain dissection, "punches" of ah and ph were micro-dissected from slices. cell suspensions were obtained from a pool of 3 samples by enzymatic digestion and cell trituration. then, gfp-positive cells, which were highly enriched in astrocytes, were sorted by facs. total rna was extracted from these cells and gene expression levels were assayed by qrt-pcr and normalized by cyclophylin mrna levels. seven genes related to anls were tested: the alpha 2 sub-unit of the na-k atpase (atp1a2), the two sub-unit of the lactate dehydrogenase (ldha and ldhb), the glutamate transporters (glast and glt1) and two monocarboxylate transporters (mct1 and mct4). levels of mrna encoding genes related to glycogen metabolism such as ptg, the glycogen synthase (gs) and the glycogen phosphorylase (gphos) were also measured results showed that the levels of mrna encoding ldha, mct1 and mct4 expressed significant circadian variations which can reach 200% for mct4 at zt0, compared to zt6 levels in ah. the glt1 mrna levels as well as mct1 and mct4 mrna levels also displayed significant circadian variations in ph. levels of expression of genes encoding the gphos, gs and ptg did not display major regional circadian variation. this study shows that astrocytes displayed some transcriptional regulation in hypothalamic areas involved in the sleep-wake cycle. the difference in the pattern of expression of anls related genes in ah and ph, such as glt1, suggests that neuro-metabolic coupling capacity of each structure might be different and might play a functional role in the sleep-wake regulation. , are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter 1 (glt-1) with significantly increased levels of glt-1 in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. to study glt-1 regulation in more detail, we generated a family of transgenic mice using increasing lengths of the 5 0 non-coding region of the glt-1 gene controlling expression of tdtomato. these mice were crossed with full length bac-glt-1-egfp mice to compare astrocytic expression of the reporter genes. interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an 8.3 kb eaat2 promoter fragment mouse with the bac-glt-1 mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat2 expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato1/gfp1 and tdtomato-/gfp1 astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax6 and kir4.1 were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir4.1 mrna was noted to be selectively enriched in cortical tdtomato1/gfp1 cells, compared to gfp1 only cells. it suggests kir4.1 may serve as an astroglial subtype marker. . we have hypothesized that microglial c/ebpb is a potential target to attenuate the neurotoxic effects of neuroinflammation. in order to test this hypothesis in vivo, c/ebpb knockout mice are not suitable because they show multiple phenotypic alterations as a result of the widespread expression of c/ ebpb. mice with specific c/ebpb deletion in microglia would be a better model. unlike gfap promoter for astrocytes, there is not a gold standard promoter for cre expression in microglia. cd11b, cx3cr1 or lysozime m (lysm), among other promoters, have been used to this end. we have generated lysm-cre/c/ebpb fl/fl mice with two main aims: 1) to validate the use of lysm as a suitable promoter for microglial gene deletion by cre-lox recombination 2) to analyze the effects of the absence of microglial c/ebpb in vitro and in vivo lysm-cre/c/ebpb fl/fl mice which were fertile and viable and did not show the female infertility and high perinatal mortality described in c/ ebpb knockout mice. western blot experiments revealed the presence of c/ebpb in protein extracts from wild-type but not from lysm-cre/c/ ebpb fl/fl microglial cultures. immunocytochemistry experiments confirmed this observation. thus, c/ebpb immunoreactivity was present in all cells in wild-type microglial cultures and it was absent in all microglial cells in lysm-cre/c/ebpb fl/fl microglial cultures. the microglial specificity of lysm-cre recombination was demonstrated in primary mixed glial cultures. whereas in wild-type primary mixed glial cultures c/ebpb immunoreactivity was observed both in astrocytes and microglia, in lysm-cre/c/ebpb fl/fl mixed glial cultures it was detected in astrocytes, but never in microglia,. to assess the functional outcome of microglial c/ebpb deficiency, we analyzed lps1ifncinduced no production and we observed a marked decrease in lysm-cre/c/ebpb fl/fl microglial cultures. this decrease was also observed in mixed glial cultures in fitting with the microglial origin of no production in this model. in summary, these findings show that lysm-cre promoter causes recombination in 100% of microglial cells in primary culture and it is therefore suitable for microglial-specific gene deletion in vitro. experiments are in progress to determine the degree and specifity of microglial c/ebpb deletion in vivo in these mice. supported by grants pi10/378 and pi12/00709, instituto de salud carlos iii, spain. to study the functional role of cpeb3, transgenic mice overexpressing cpeb3 in astrocytes were generated. cpeb3 overexpression without the endogenous 3 0 untranslated region (utr) led to the downregulation of astrocytic connexins (cx43 & cx30). consistently, in the hippocampus of cpeb3 overexpressing mice, intercellular gap junction coupling was strongly impaired. cpeb3 overexpression also led to downregulation of glutamate transporter 1 (glt-1) and glutamine synthetase (gs), key players involved in extracellular glutamate clearance. we have now raised mice overexpressing cpeb3 with the endogenous 3 0 utr. in these mice, we observed the downregulation of cx43, cx30, glt-1 and gs. since interastrocytic coupling and astrocytic glt-1 and gs activities are downregulated in epilepsy patients with hippocampal sclerosis, we hypothesize that upregulation of cpebs in astrocytes may contribute to the pathogenesis of epilepsy by causing astrocyte dysfunction. to study the influence of the p53 gene in the structural and quantitative characteristics of astrocytes in the mice retina methods: adult mice of the c57bl/6 strain (12 months old) were distributed into two groups: 1) mice with two extra copies of p53 ("super p53"; n 5 6) and 2) wild-type p53 age-matched control, as the control group (wt; n 5 6). retinas were immunohistochemically processed with gfap. gfap1 astrocytes were quantified. results: in comparison con wt: i) retinal-astrocyte distribution followed established patterns; however, morphological changes were seen through the retinas in relation to p53 availability: astrocytes were more robust and secondary processes were more evident; ii) astroglial density was significantly higher in the sp53 retinas, both in the whole-retina (p < 0,01 student's t-test) and in the intermediate and peripheral concentric areas of the retina (p < 0.05 student's t-test). conclusion: the astrocyte changes in the sp53 retinas might improve the resistance of the retinal cells against oxidative stress and its downstream signalling pathways. however, neuronal damage caused due to excessive microglial activation is a well known phenomenon in neurodegenerative diseases. mirnas are novel regulators of gene function, controlling several biological processes. recent reports show altered mirna expression in immune-mediated pathologies, suggesting that mirnas have roles in modulating immune responses. thus, the present study was initiated to identify micrornas and their target mrnas which can modulate microglial inflammatory responses. a pilot study was designed to understand the role of mirna-200b in modulating microglia-mediated immune response as this was found to be localized in the microglia. recently, mirna 200b has been shown to target several proteins including c-jun, the substrate of jnk mapk (mitogen-activated protein kinase) which mediates the release of proinflammatory cytokines in activated microglia. qrt-pcr revealed the decrease of mir-200b expression level in activated bv2 microglia. loss-of-function and gain-of-function studies confirmed c-jun to be the target of mir-200b in microglia. overexpression of mir-200b in activated microglia resulted in a decrease in c-jun and jnk expression and activity, thereby dysregulating the mapk-jnk pathway and proinflammatory cytokines. further, treatment of neuronal cells, mn9d with conditioned medium obtained from activated microglial cells, resulted in increased inflammatorymediated cell death upon knockdown of mir-200b. overexpression of mirna-200b also reduced the phagocytic ability in activated microglia. taken together, these results demonstrate the important role of mir-200b in modulating the mapk pathway via c-jun which in turn affects different aspects of the inflammatory process accompanying microglia activation including cytokine response, no production, phagocytosis and neuronal cell death. thus, mir-200b may prove to be a useful target for developing therapeutic strategies to control microglial mediated inflammation in neurodegenerative diseases. further in order to understand the global mirna changes contributing to microglial activation, a global mirna microarray was carried out using control, lps-activated and amyloid b-activated primary microglia. this screen identified several families of mirnas differentially expressed between the different treatment groups enabling us to analyze the mirna signature in activated microglia. national university of singapore, singapore, singapore an inflammatory process in the brain as a result of an injury or infection is mediated by the microglia, the prime immune effector cells of the central nervous system (cns). microglia have also been shown to display chronic inflammatory responses leading to neurodegenerative disorders. small ubiquitin-like modifier-1 (sumo-1) is a post-translational protein modifier, which has been shown to be associated with nuclear factor kappa b (nfjb)-mediated inflammatory pathway. in this study, we showed the expression of sumo-1 in microglia and characterized the roles of sumo-1 in activated microglia. we found that sumo-1 is specifically expressed in the amoeboid microglial cells of the early postnatal rat brain. secondly, lipopolysaccharide-mediated activation of microglia led to a significant increase in the expression as well the nuclear translocation of sumo-1 in microglia in vitro. in order to establish a function for sumo-1 in activated microglia, we sought to silence its expression using sirna-mediated gene knockdown. sumo-1 knockdown in lps-treated bv-2 microglia in vitro subsequently showed the decreased nuclear translocation of nfjb and expression of tumour necrosis factor-alpha (tnf-a) level. further, we performed an in silico screening to identify the targets of sumo-1 in the nfjb pathway. our study thus, suggests that sumo-1 regulates nfjb translocation into the nucleus for the transcription of pro-inflammatory genes including tnf-a in activated microglia. microglia are the main resident immunological cells the cns. in the healthy brain, microglial cells are in a surveillance state whereas upon rupture of cns homeostasis they enter activated states. in ischemic stroke, activated microglia is one of the most important cellular components of post-stroke neuroinflammation, which mainly occurs in and around the area of the infarct. microglia activation is characterized by morphological alterations, functional and transcriptional remodeling, which account for the acquisition of immune phenotypes. purinergic receptors are known to play important roles in microglia activation, and p2x4r have been suggested as potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain diseases. in the present study, we have developed an experimental approach based on laser micro-capture to isolate microglia from the penumbra area at different post-lesion times (from 4h to 7 days post-lesion). the repertoire of genes expressed by microglial cells was determined through cdna microarray analysis. with this approach we have been able to determine clusters of genes that are co-regulated thus revealing the time course of microglia activation in this model. in addition, we have compared the transcriptional remodeling of microglia in wild-type and p2x4-deficient mice. our results suggest that wild-type and p2x4r ko mice present specific transcriptional profiles, which only partially overlapped. thus our data suggest that p2x4r play significant roles in the regulation of microglial cells functions. z. chen 1 , m. ghosh 2 , r. sattler 1 , m. robinson 2 , j. rothstein 1 1 johns hopkins university, baltimore, united states 2 university of pennsylvania, philadelphia, united states question: astrocytes, the most abundant cell type in the central nervous system (cns), are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter 1 (glt-1) with significantly increased levels of glt-1 in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. methods: to study glt-1 regulation in more detail, we generated a family of transgenic mice using increasing lengths of the 5 0 non-coding region of the glt-1 gene controlling expression of tdtomato. these mice were crossed with full length bac-glt-1-egfp mice to compare astrocytic expression of the reporter genes. results: interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an 8.3 kb eaat2 promoter fragment mouse with the bac-glt-1 mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat2 expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato1/gfp1 and tdtomato-/gfp1 astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax6 and kir4.1 were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir4.1 mrna was noted to be selectively enriched in cortical tdtomato1/ gfp1 cells, compared to gfp1 only cells. it suggests kir4.1 may serve as an astroglial subtype marker. conclusions: our data clearly suggest the existence of molecular subtypes of astrocytes. factor expressed by activated astrocytes and microglia. studies with non-glial cells have shown that c/ebpd is able to regulate the expression of proinflammatory genes. however, the role of c/ebpd in neuroinflammation has not been established. we have here analyzed the effects of c/ebpd absence on glial activation in vitro and in vivo. in primary mixed glial and microglial-enriched cultures the expression of the pro-inflammatory genes nos-2, cox-2 and il-6 induced by lps1ifnc, but not by lps alone, was attenuated in the absence of c/ebpd. chip experiments revealed binding of c/ebpd to the promoters of these genes in activated, but not in control, mixed glial cultures. in neuronal-microglial co-cultures the neurotoxicity elicited by lps1ifnc-activated microglia was completely abolished when microglial cells were devoid of c/ebpd suggesting that this transcription factor plays a key regulatory role of the expression of potentially detrimental proinflammatory genes by activated microglia. to analyze the role of c/ebpd in neuroinflammation in vivo mice were treated systemically with lps (100 ug/mouse; i.p.). this treatment induced a marked increase in c/ebpd mrna and protein brain levels. double immunofluorescence experiments showed the presence of c/ebpd in astrocytes and microglia in lps-treated mouse brain. in c/ ebpd -/-mice, systemic lps-induced brain expression of nos-2, tnfa, il-1b and il-6 was attenuated. finally, in mouse experimental autoimmune encephalitis (eae) c/ebpd was upregulated in the spinal cord and the severity of eae symptoms was reduced in c/ebpd -/-mice. altogether these findings demonstrate that c/ebpd plays a major role in the regulation of proinflammatory gene expression in neuroinflammation and point to microglial c/ebpd inhibition as a novel strategy to reduce the detrimental effects of neuroinflammation. supported by pi10/378 and pi12/709 from instituto de salud carlos iii, spain gene targeting strategies have become a powerful technology for elucidating mammalian gene function. the recently generated knockout (ko)-first strategy produces a knockout at the rna processing level and also allows for the generation of conditional ko alleles by combining flp/frt and cre/loxp systems, thereby providing high flexibility in gene manipulation. however, this multipurpose ko-first cassette might produce hypomorphic rather than complete knockouts if the rna processing module is bypassed. moreover, the generation of a conditional phenotype is also dependent on specific activity of cre recombinase. here, we report the use of an efficient molecular biological approach to test pannexin1 (panx1) mrna expression in global and conditional panx1 ko mice derived from the ko-first mouse line, panx1 tm1a(komp)wtsi . using qrt-pcr, we demonstrate that tissues from wild-type mice show a range of panx1 mrna expression levels, with highest expression in trigeminal ganglia, bladder and spleen. unexpectedly, we found that in mice homozygous for the ko-first allele, panx1 mrna expression is not abolished but reduced by 70% compared to that of wild-type tissues. thus, panx1 ko-first mice present a hypomorphic phenotype. crosses of panx1 ko-first with flp deleter mice generated panx1 f/f mice. further crosses of the later mice with mgfap-cre or nfh-cre mice were used to generate astrocyte-and neuronal-specific panx1 deletions, respectively. a high incidence of ectopic cre expression was found in offspring of both types of conditional panx1 ko mice. our study demonstrates that panx1 expression levels in the global and conditional panx1 ko mice derived from ko-first mouse lines must be carefully characterized to ensure modulation of panx1 gene expression. the precise quantitation of panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of panx1 under physiological and pathological conditions. agarwal, e. hughes, d. bergles johns hopkins university, baltimore, united states astrocytes elevate intracellular ca21 in response to stimulation of metabotropic receptors. these ca21 transients are linked to processes as diverse as synaptic plasticity and functional hyperemia, but the relevance of this signaling remains uncertain. although most studies of ca21 signaling in astrocytes have focused on somatic events, anatomical and functional studies indicate that ca21 signaling in astrocytes may be compartmentalized into functionally isolated "microdomains." to facilitate analysis of ca21 signaling, we generated transgenic mice in which the cytosolic and membrane anchored variant of genetically encoded calcium indicator gcamp3, referred as cgcamp3 and mgcamp3 respectively, can be expressed conditionally in a cell specific manner. a ubiquitous cag promoter is used to control cgcamp3 and mgcamp3 expression, and a "stopper" sequence flanked by loxp sites was placed upstream of the coding sequence, preventing expression until cre excises this dna. these constructs were targeted to rosa26 locus to ensure widespread expression. to evaluate whether cgcamp3 and mgcamp3 were expressed at levels sufficient to resolve ca21 transients, we bred r26-lsl-cgcamp3 and r26-lsl-mgcamp3 mice to glast-creer mice. double transgenic offspring were injected with tamoxifen at 3 weeks of age and analyzed 2-3 weeks later. histology revealed that cgcamp3 and mgcamp3 were expressed in astrocytes throughout the brain. astrocytes in acute cortical slices exhibited large amplitude, spontaneous increases in both cgcamp3 and mgcamp3 fluorescence. the signal to noise ratio of the fluorescence intensity was greatly improved in astyrocytes expressing mgcamp3, partially due to the ability of mgcamp3 to better resolve near membrane ca21 flux. ca21 transients were typically restricted to small regions of an individual astrocyte, consistent with ca21 elevations within microdomains. spontaneous ca21 transients were occasionally observed in the soma of astrocytes expressing cgcamp3 but not mgcamp3, and were not coincident with activity in the processes, suggesting that these regions are functionally uncoupled. to determine if gcamp3 expression is sufficient to visualize ca21 signaling in vivo, we made cranial windows over the somatosensory cortex and performed 2-photon imaging. microdomain ca21 transients were prominent in cortical astrocytes, indicating that these mice provide new opportunities for understanding the significance of this form of signaling. in the trigeminal ganglion (tg), satellite glial cells (sgcs) surround neurons and are able to release substances that may affect sensory transmission through the tg and contribute to mechanisms underlying craniofacial pain. the aims of this study were 1) to investigate whether sgcs could be provoked to release glu in vitro and 2) to study the in vivo response properties of trigeminal afferent fibres upon artificial elevation of glu concentration in the tg. methods: confocal microscopy was used to assess glu content in and the expression of excitatory amino acid transporter 1 (eaat1) and eaat2 by sgcs of the tg. glu release was investigated in vitro, where sgcs were isolated from the tg of 9 adult male sprague-dawley (sd) rats and treated with control medium or medium containing 10 mm kcl together with 0, 0.1, 1, 10 mm of the eaat1/2 inhibitor tfb-tboa. in vivo electrophysiology experiments were conducted in 6 additional anaesthetized adult male sd rats to determine the effect of repeated intraganglionic injections of glu (500 mm, 3 ml 3 2, 30 min apart) on the response properties of 6 tg neurons that innervated either the temporalis or masseter muscle. cumulative afferent discharge was calculated as the sum of action potentials (ap) 10 min post injection subtracted from the sum of ap 10 min prior to injection. an electronic von-frey hair was used to measure afferent mechanical threshold (mt) at 1 min intervals for 10 min prior to the first and again 20 min after the second microinjection of glu. results: most sgcs were found to be glu positive and express eaat1 and eaat2. treatment with 10 mm kcl resulted in a $2-fold increase in glu concentration, compared to control (10.6 6 1.1 vs. 4.8 6 0.1 mm, respectively; p 5 0.038). treatment with 1 mm tfb-tboa significantly reduced the glu concentration to 5.8 6 1.4 mm (p 5 0.047), which was further lowered to 3.0 6 0.8 mm when 10 mm was applied (p 5 0.001). cumulative afferent discharge evoked in tg neurons by the second injection of glu (20 6 9 ap) was not significantly different from that evoked by the initial injection (21 6 10 ap; p 5 0.92). glu injections significantly reduced muscle afferent mt (baseline: 35 6 19 g) by 30 6 3% (p 5 0.03). these results indicate that glu can be released from sgcs through eaats, and that increased glu concentrations in the tg excite neurons and induce a state of peripheral sensitization. this may hypothetically be a mechanism, whereby activated tg sgcs could contribute to e.g. migraine headache. in an accompanying poster, we present data demonstrating that in cultured neurons l-lactate induces plasticity-related genes expression through a nmda-dependent mechanism (i.e. arc, zif268 and c-fos). here, we describe the effect of l-lactate on neuronal excitability by performing electrophysiological recordings of cultured cortical neurons. we observed that application of l-lactate (10 mm) triggers an inward current with an amplitude of 20.55 1/-0.1 na and slow kinetics, with a peak reached at 189 1/-55s (called ilac). the membrane current decreases gradually to reach a plateau, with a mean inward current of 20.13 1/-0.04 na persisting up to 780s (13 minutes) after addition of l-lactate (called ipyr). in order to assess l-lactate specificity on the generation of these currents l-pyruvate, another monocarboxylate, as well as d-lactate, the non-metabolized enantiomer of l-lactate) were tested. in contrast to l-lactate, l-pyruvate (10 mm) induced a low-amplitude sustained current (20.07 1/-0.03 na) similar to the late-phase slow current evoked by l-lactate (i.e ipyr), but no current similar to ilac. dlactate on its side did not elicit any specific current. pharmacological characterization of ilac and ipyr currents demonstrate that ilac is generated by nmda receptors (as it is blocked by mk801) and relies on active nmda receptors (as it is blocked by either glutamate or glycine binding sites inhibitors). in contrast, generation of ipyr by both l-lactate and lpyruvate is prevented by diazoxide, a katp opener, whereas ilac was unaffected by the treatment. in order to determine the importance of katp for plasticity-related gene expression, the katp closer glibenclamide (200 um) was applied to neurons and mrna gene expression of arc and zif268 quantified. results obtained demonstrate that arc and zif268 mrna expression are unaffected by glibenclamide. as a whole this set of data demonstrates that l-lactate generates two types of inward currents in neurons i.e. nmda-dependent (ilac) or katp-dependent (ipyr). moreover, they highlight the nmda-dependent ilac current as the key mediator, in contrast to katp, of l-lactate-induced plasticity gene expression. this is further sustained by the observation that l-pyruvate does not reproduce the effect of l-lactate on gene expression (see accompanying poster). this set of results provides novel insights into the mechanisms of action of l-lactate as an inducer of plasticity gene expression, and an important signaling molecule for neuronal plasticity. the reliance on drg neuron co-culture for sclcs fate commitment stands as a major hurdle for the therapeutic application of this finding. thus, we aim to unravel the mechanisms underlying sclcs fate commitment in order to develop a drg neuron-free condition for generating fate committed schwann cells. we hypothesised that the switch is brought about by the membrane bound neuregulin (nrg), nrg 1 type iii, but not the other soluble isoforms of neuregulin. as our results show that sclcs treated with soluble neuregulin did not show significant changes in morphology nor marker expression when compared with untreated sclcs. nrg 1 type iii expression was observed on purified drg neurons by immunocytochemistry, western blot analysis as well as reverse transcription pcr for the mrna. mammalian expression constructs for nrg 1 type iii were made by transfection into human embryonic kidney cells, hek 293t, and mouse embryonic fibroblasts (mef) with the aim of generating surrogate cell types for co-culture with sclcs to pursue cell-specific effects of nrg 1 type iii on differentiation of sclcs. the findings promise a way for generating fate committed schwann cells for autologous transplantation for recovery after nerve injury. (2) the co 2 /hco 3 buffer system. the latter being an open buffer system, because biomembranes are usually permeable to co 2 , and, in addition, most cells express hco 3 transporting membrane proteins. the enzyme family of carbonic anhydrases (cas) catalyses the reversible reaction from co 2 and h 2 o to hco 3 and h 1 , and thereby modulates the intracellular h 1 buffer dynamics.we have performed calibrated in situ live-cell imaging with the proton-sensitive fluophore bcecf in glial cells and neurons of acute cerebellar slices of wild-type and knockout mice. the studies were used to quantify the intracellular buffer capacity and to dissect the contribution of the intracellular caii and the extracelluar caiv by comparing intracellular h 1 shifts in glial cells and neurons from wild-type mice and mice deficient in their caii or caiv gene. we found that only one proton in 400000 is unbound and thereby chemically active, and that about 50% of this buffer capacity is mediated by the co 2 /hco 3 buffer system and the other 50% by intrinsic buffers. the rate of co 2 -induced change in intracellular h 1 concentration was increased by intracellular caii in both glial cells and neurons. the extracellular caiv on the other hand affected primarily neurons, but also showed unexpected intracellular activity (schneider et al. 2013, pnas 110). the rates of acidification and alkalinisation in wild-type and knockout mice were significantly reduced in the presence of the ca blocker 6-ethoxy-2-benzothiazolsulfonamid (10 mm). we confirmed ca protein expression by western blot and could also show that the expression of the remaining ca is not upregulated. these results provide new insights into cellular proton-coupled processes in acute neural tissue, which shows some new complex roles of carbonic anhydrases in shaping proton dynamics in cells and tissues. objective: it is becoming increasingly evident that inflammatory mechanisms promote neuronal hyper-synchronization and epileptogenesis following brain insults. our recent studies identified serum albumin as a key player in the inflammatory cascade leading to epilepsy, at least partially through the activation of tgf-b signaling pathway in astrocytes. in this study we set out to explore the mechanisms by which tgf-b1 induces glial activation and epileptogenesis. methods: primary cultures of microglia and astrocytes were treated with tgf-b1 and gene expression analysis along with protein quantification were performed by quantitative pcr and elisa. electrophysiological response to il-6 was obtained in acute brain slices (field potential recordings) and in-vivo (sub-dural corticoencephalography) following to a continuous administration of il-6 into the lateral ventricle for 7 days. results: in glia cultures tgf-b1 induced early and rapid up-regulation of il-6 at both mrna and protein levels whereas the upregulation of other pro-inflammatory cytokines such as il-1b and tnf-a was milder and at a later time point. notably, smad2/3-dependent tgf-b1 signaling induced the expression of il-6 primarily in astrocytes and to a significantly lesser extent in microglia. il-6 was sufficient to induce epileptiform activity in acute brain slices and recurrent seizures within 3-4 days a following continuous intracerebroventricular injection of il-6. conclusions: we thus suggest astrocytic release of il-6 as a potential key mechanism in epileptogenesis. okayama univ., dept. of brain science, okayama, japan dopamine transporter (dat) is expressed not only in catecholaminergic neurons but also in astrocytes. we previously showed that repeated l-dopa treatment markedly induced expression of dat and apparent dopamine (da)-immunoreactivity in the reactive astrocytes in the striatum of animal models of parkinson's disease. it is thought that astrocytes can uptake both l-dopa and da via neutral amino acid transporter lat and dat, respectively. therefore, uptake and metabolism of l-dopa and da in the striatal astrocytes may influence their availability in dopaminergic system of parkinsonian patients. to clarify uptake and metabolism of l-dopa and da in striatal astrocytes, the contents of l-dopa, da and their metabolites were measured after the l-dopa/da treatment using primary cultured astrocytes. first, we revealed the expression of neutral amino acid transporter lat and aromatic amino acid decarboxylase (aadc) in the striatal astrocytes. the level of l-dopa in astrocytes was markedly increased after 4-hr l-dopa exposure, but da was not detected in the astrocytes 4 or 8 hr after the treatment. on the other hand, the da treatment for 4 hr increased levels of da and its metabolites, especially dopac. these results indicate that uptaken da into astrocytes is rapidly metabolized and that uptaken l-dopa never be converted to da in astrocytes. furthermore, level of uptaken l-dopa in cultured striatal astrocytes was rapidly decreased after removing extracellular l-dopa. taken together, the present results suggest that striatal astrocytes act as a reservoir of l-dopa to uptake or release l-dopa depending on the extracellular l-dopa concentration, but have less ability to convert l-dopa to dopamine. s. limmer, c. kl€ ambt universit€ at m€ unster, m€ unster, germany neuronal function consumes a large amount of energy and thus the brain requires a constant but regulated supply of metabolites. in invertebrates, such as drosophila, the nervous system is floating in the hemolymph and all metabolites that enter the brain have to be transported across the blood brain barrier (bbb). as in primitive vertebrates, the drosophila bbb is generated by glia. a layer of so-called subperineurial glial cells completely encapsulates the nervous system and thus, all metabolites reach the neurons en route through glia. the main energy supply of the inner organs of drosophila is provided by trehalose that is found in high concentrations in the hemolymph. trehalose is a nonreducing disaccharide, in which two dglucose units are linked via a a,a-1,1-glycosidic bond. the uptake of trehalose is mediated by two trehalose transporters, which are members of the slc2a gene family. the subperineurial glial cells then either secrete c 6 sugars to the neurons -or alternatively supply energy to neurons by secreting c 3 metabolites as described for the bee retina. to discriminate between these possibilities we have followed a number of different approaches. the relevance of trehalose transporters in the different cell types has been determined by mutant analysis and rnai studies. furthermore, we have studied the role of all other putative sugar and monocarboxylate transporters in glial cells by rnai knockdown. to determine whether c 6 or c 3 carbohydrates are secreted by glial cells we suppressed the expression of core enzymes regulating glycolysis or components of the respiratory chain in either glial cells or neurons. moreover, we have ablated mitochondria specifically in glia or in neurons through expression of a restriction enzyme targeted to mitochondria. we will summarize our results and discuss approaches towards identifying neuronal signals that control glial carbohydrate secretion during energy homeostasis of the brain. microglia have long been known to respond to virtually any insult to the central nervous system. they quickly migrate to the site of the insult, and play many important roles, such as barricading the injury site, phagocytosing debris, and releasing cytokines. the role of microglia in the normal brain, however, has only recently begun to be appreciated. with the advent of in vivo imaging, microglia have been shown to be constantly surveying their environment with rapid extensions and retractions of their processes. subsequent studies have shown that these 'resting' microglia (now known as 'surveying microglia') are indeed active throughout development and adulthood. during development, microglia have been shown to be involved in synaptic monitoring and pruning as well as synaptic stripping after injury. thus far, many studies have focused on the role of microglia at the synapse. however, we report here, for the first time, that in the cortex of normal adult rodents, a small percentage of microglia are specifically associated with the axon initial segment (ais). the ais is characterized by a high density of voltage-gated ion channels and plays an important role in initiation of the action potential and maintenance of neuronal polarity. this interaction seems to be limited to the 'surveying' phenotype of microglia and much less frequent in 'activated' microglia. this overlap of processes appears early in development and continues throughout adulthood although the function of microglia at the ais is still currently unknown. x. liu 1 , j.-m. petit 2,3 , p.j. magistretti 2,3 , c. giaume 1 1 cirb, collège de france, paris, france 2 lndc, brain mind institute, epfl, lausanne, switzerland 3 centre de neurosciences psychiatriques, chuv, prilly, switzerland astrocytes act as modulators of neuronal activity through mechanisms such as neurotransmitter uptake, 'gliotransmitters' release, and metabolic supply. recently, astrocytes were shown to modulate sleep by vesicular release of atp. besides the cellular mechanism, we are interested in the network behavior of astrocytes in sleep-wake cycle. indeed, astrocytes form networks of communicating cells via gap junction channels constituted by connexins (cxs). recently, we have demonstrated that astroglial networking is regulated by neuronal activity and that neuronal activity is affected by deletion of the two main astroglial cxs (cx43, cx30). to investigate how astroglial networks behave in sleep-wake cycle, we used pharmacological treatments and sleep deprivation to manipulate sleep-wake status of mice and determined whether astroglial networks would change in response. astroglial networks in acute cortical slices were revealed by "dye coupling", i.e. by loading of a patch-clamped astrocyte with sulforhodamine b that diffuses into neighboring astrocytes through gap junction channels. also, cxs expression at mrna and protein levels was measured as an index of connections among astrocytes. the results are as follows. first, i.p. injection of modafinil, a potent wakefulness-promoting drug, increased both mrna and protein expression of cx 30 in the mouse cortex. superfusion of modafinil also enhanced dye coupling among astrocytes in acute coronal slices of the mouse somatosensory cortex. this effect was abolished by ttx treatment, which demonstrates neuronal activity is necessary for the effect of modafinil. in contrast, c-hydroxybutyric acid (ghb), a sleep-promoting agent, and propofol, a general anesthetic, decreased astroglial coupling in cortical slices. interestingly, the effect of ghb was not affected by ttx, suggesting a different pathway of action from modafinil. next, we used a "gentle" sleep deprivation model to sleep deprive mice for 6 hours from the onset of the light period. as a result, mrna of cx30, but not cx43, was increased in both the cortex and hippocampus. finally, dye coupling in cortical astrocytes was enhanced in mice after sleep deprivation compared to mice after normal sleep. this increase in dye coupling, however, was not observed in slices from cx30 knock out mice after the same sleep deprivation treatment. altogether these results indicate that astroglial networks are bidirectionally regulated by perturbations in sleep-wake cycle and that cx 30 is the major cx sensitive to such perturbations. in neurons, synaptic and extrasynaptic gaba a receptors (gaba a rs) differ in their subunit composition, conferring them distinct functional and pharmacological properties. oligodendrocyte precursor cells, also called ng2 cells, are contacted by bona fide neuronal gabaergic synapses. however, we recently showed a synaptic to extrasynaptic switch in the mode of transmission between gabaergic interneurons and ng2 cells during postnatal development of the somatosensory cortex. we therefore hypothesized that the postnatal switch of gabaergic transmission in ng2 cells is accompanied by changes in the expression of gaba a r subunits. to test for this hypothesis, we stimulated neuronal fibers to evoke gaba a r-mediated responses in ng2 cells recorded in acute slices of the somatosensory cortex of ng2-dsred transgenic mice. the effect of zolpidem and a5ia on evoked gabaergic responses reveals the predominance of functional a1-and a5-containing gaba a receptors, respectively, at interneuron-ng2 cell synapses early in development. however, the expression level of a5 decreases when responses rely exclusively in extrasynaptic transmission at more mature developmental stages. more importantly, specific pharmacology for the c2 subunit, a crucial molecular component for the clustering of gaba a receptors at postsynaptic sites, demonstrated a down-regulation of this subunit in ng2 cells prior to the complete loss of gabaergic synaptic activity. in keeping with the synaptic nature of the c2 subunit in neurons, this molecular change in ng2 cells correlates with the switch from synaptic to extrasynaptic transmission. to corroborate these functional data, we performed single cell multiplex rt-pcr of a1-5, b1-3, c1-3 and d subunit mrnas in ng2 cells of the second and fourth postnatal weeks. despite the large heterogeneity of subunit mrna expression at both developmental stages, we found that the number of cells expressing c2 mrnas dramatically decreases in the fourth postnatal week. in conclusion, the expression loss of the c2 subunit is an important molecular determinant impacting the change of transmission modes between interneurons and ng2 cells during cortical development. financial support: frc, anr blanche, arsep, dfg (sfb/tr3) and eu (fp7-202167 neuroglia). university of rochester, medical center, rochester, united states synaptic plasticity is a critical process throughout the lifespan for achieving and maintaining normal and efficient operation of the nervous system. while much is known about the functional and structural changes that occur at the synapse during synaptic plasticity, the mechanisms implementing these changes are poorly understood. despite being classically characterized as immune cells, microglia have recently been shown to play a role in normal brain function by restructuring and removing synapses. given the novelty of this role, few details are known about how microglia behave to implement such a role during periods of plasticity. to characterize the changes in microglial behavior during ocular dominance plasticity in the developing visual cortex, we examined microglial morphology in fixed brain sections as well as in vivo using two-photon microscopy. for analysis of microglia in fixed sections, c57/bl6 mice were monocularly deprived for either 12 hours, 1, 2, 4, or 7 days during the visual critical period (p23-p30). fixed coronal sections were stained with iba1, a specific marker for microglia, imaged on a confocal microscope and analyzed using imagej. microglial density decreased rapidly (within 12 hours) and remained low for the 7 day deprivation period. characterization of microglial morphology revealed an increase in the complexity of the microglial process arbor after 12 hours, which steadily decreased back to baseline by 7 days. this suggests that microglia do undergo a rapid change in morphology during plastic events that is reminiscent but distinct from the traditional activation pattern observed during pathological events. for analysis of microglia in vivo, heterozygous cx3cr1/gfp (jung 2000) mice were monocularly deprived directly preceding the first imaging session, the binocular segment of primary visual cortex was imaged, and the same cortical area was re-imaged 2 and 4 days later. while no change in microglial density was observed, analysis of microglial process motility showed a decrease in motility after periods of monocular deprivation. these results, combined with the observed morphological changes, show that microglia undergo behavioral changes after a period of monocular deprivation that are inconsistent with pathological activation, suggesting that microglia take on different roles and activities during normal brain function. g. baltazar, j. oliveira, t. roxo, c. fonseca faculty of health sciences, cics-ubi, covilhã, portugal neuroinflammation is a pathological hallmark in patients and experimental models of parkinson's disease. both present the classical features of inflammation, with evidence of an uncontrolled process. moreover, microglia may become activated early in the disease process and remain primed, responding strongly to subsequent stimuli, and thereby enhancing inflammation-induced oxidative stress and cytokinedependent toxicity in vulnerable neuronal populations. we have previously demonstrated that glial cell line-derived neurotrophic factor (gdnf), released by ventral midbrain astrocyte cultures, potently prevents microglial activation induced by a pro-inflammatory agent. therefore, gdnf is an important mediator in the astrocytemicroglia crosstalk keeping microglia in a resting state. however, in the brain, microglia and astrocytes are not alone and other cell types, e.g. neurons, may interfere and influence this astrocytic control of inflammation. therefore, it is important to investigate if the presence of neurons alters gdnf-mediated astrocytic control of microglial activity. in this work we aimed at investigating whether the presence of neurons, injured or not, changes the ability of astrocyte-derived gdnf to prevent microglial activation. for that purpose, we used primary cultures of ventral midbrain astrocytes and microglia, as well as neuronastrocyte mixed cultures from the same brain region. neuron-astrocyte cocultures were exposed to vehicle (control) or to the da neurotoxin mpp 1 . the media conditioned by control and mpp 1 -challenged neuron-astrocyte mixed cultures, or by astrocyte cultures, was collected and transferred to ventral midbrain microglia cultures, which were then exposed to the pro-inflammatory agent lipopolysaccharide. the effect of conditioned media obtained from the different cell culture systems on microglial activity was determined by analyzing the production of nitric oxide by the griess reaction and of the phagocytic activity by determining the engulfment of fluorescent microspheres by fluorescence microscopy. university of lausanne, lausanne, switzerland lactate is produced by astrocytes and used by neurons as metabolic substrate during activity. recent evidence indicates that lactate may not only serve as energy substrate but also as modulator of glutamatergic or gabaergic neurotransmission. we tested this hypothesis using primary cultures of cortical neurons obtained from wild type and gad67-gfp knock-in mice (c57bl/6). neuronal excitability was monitored by electrophysiological recordings and calcium imaging using the fluorescent probe fluo-4 am. spontaneous action potentials and rhythmic calcium transients were present in more than 50% of cultured neurons, which were either gabaergic or glutamatergic cells. in the presence of 5mm glucose, l-lactate application reversibly diminished calcium transient frequency in a concentration-dependent manner in both cell types (glutamatergic neurons ic50 4.23 6 1.9mm and gabaergic neurons ic50 4.18 6 2.8mm). to test whether lactate effects were dependent on metabolism, we applied the closely related substrate pyruvate (5mm) or switched to different glucose concentrations (0.5 or 10mm). none of these conditions altered the calcium transient frequency. in contrast, the application of d-lactate, thought not to be metabolized by neurons, decreased the spiking activity in the same concentration range as l-lactate (ic50 4.58 6 1.2mm). we determined that d-lactate was taken up by neurons, however more than two-fold less efficiently than l-lactate. these results suggest that the mechanisms of lactate sensitivity are not purely metabolic. since d-lactate has the same potency as l-lactate but it is less internalized, it is likely that lactate acts as an extracellular ligand that triggers the reduction of neuronal activity. this hypothesis is currently under investigation. this modulating effect of lactate represents a novel role for the compound that has to be taken into account in the context of the interactions between astrocytes and neurons. ben-gurion university, ber-sheva, israel microglia integrate within the neural tissue with a distinct ramified morphology through which they scan the surrounding neuronal network, a process which appears to contribute to the integrity, maintenance and functioning of the brain. since microglia are long-lived, they are subjected to senescence processes which may severely compromise their function with age. here we used a digital tool for the quantitative morphometric characterization of fine cortical microglia structures in mice. we thus followed the morphological changes microglia underwent with aging and with the progression of alzheimer's-like disease. compared with microglia in young mice, microglia in old mice are less ramified and possess less branches and fine processes, a phenomenon which was associated with increased expression of pro-inflammatory genes. notably, a similar microglial pathology appeared 6-12 months earlier in mouse models of alzheimer's disease (ad). we thus demonstrate that in addition to promoting neurotoxic inflammation, amyloid plaques attract the microglia and modify their structure. this, in turn, causes a severe microglial process deficiency, which possibly results in compromised neuronal function and repair. blood-borne glucose is the main energy substrate for the brain under physiological conditions. however, the question remains unresolved if the two main cell types in the brain -neurons and astrocytes -consume glucose upon their individual needs or if glucose is mainly taken up by one cell group and glycolytically degraded to energy-rich substrates (e.g. lactate), which are then shuttled to the other cell group. a novel genetically encoded fret glucose sensor (bittner et al., 2010, 2011) together with two-photon microscopy now provides for the first time the opportunity to determine intracellular glucose concentrations in single cells in the intact organism. cortical microinjections of an adenoviral vector (aav9 and aav6) coding for the sensor flii 12 pglu600md6 with the promoter sgfap for astrocytes and synapsin for neurons were performed in mice and chronic windows were implanted above the somatosensory cortex. after two weeks, specific expression in astrocytes and neurons was observed. the ratiometric fret signal in both cell types increased after intraperitoneal glucose injection and decreased after insulin application demonstrating the functionality of the construct in vivo. the astrocytic sensor displayed a large linear range at blood glucose levels ranging from 3 to 20 mmol/l. in a second set of experiments, we examined the dynamic behavior of cellular glucose concentrations upon increased brain activity. during electrical hindpaw stimulation 21% of examined astrocytes showed a 0.5% decrease of the fret signal, whereas neuronal glucose levels did not exhibit activity-dependent fluctuations. to be able to draw conclusions about metabolic rates of substrate oxidation we recently started with experiments where cellular glucose uptake was transiently inhibited using different blockers of glucose transport. drugs were applied systemically and intracortically. we could demonstrate this principle on first measurements on astrocytes. glucose fret sensors allow measurements of the dynamics of glucose concentrations of single neurons and astrocytes in the intact organism. measurements of cellular glucose transients during transport blockade hold the potential to compare glycolytic rates of neurons and astrocytes. this tool may substantially contribute to uncover the complex organization of brain energy metabolism. neuron-glial communication in the cns is fundamentally important for many brain processes including synaptic transmission and plasticity. perisynaptic astrocytic processes (pap) contact excitatory synapses, forming tripartite structures with neurons. in the hippocampus, the morphology of pap has been shown to remodel rapidly and continuously but the mechanisms and roles of this form of structural plasticity remain unknown. this study investigated the physiological mechanisms driving pap movements and their role during long-term potentiation (ltp). pap and spines contacts were labeled by viral gene delivery of farnesylated fluorescent proteins in hippocampal slice cultures and imaged by confocal microscopy. electron-microscopy of infected slices confirmed that pap-synapse morphology is preserved in organotypic cultures. pap movements adjacent to dendritic spines were evaluated with an index of motility (mi). increasing neuronal activity by schaffer collaterals (sc) stimulation elevated pap mi and this was prevented by both ttx and mglur inhibition, suggesting that neuronal activity modulates pap movements. we next investigated whether intracellular calcium (ca 21 i ) in astrocytes influenced pap motility. bapta-am bulk-loading specifically chelated ca 21 i in astrocytes and reduced pap movements. when exogenous gq-coupled receptors mrga1 or mrgc11 were specifically targeted to astrocytes, their agonist fmrfa induced ca 21 i increases and accelerated pap motility. moreover, fmrfa delivery at the synaptic level by two-photons flash photolysis was sufficient to elevate pap mi. we then investigated pap dynamics in relation to ltp. application of theta-burst sc stimulation initially increased pap motility, but then resulted 30min later in a decrease of pap motility. interestingly, the reduction of pap movements correlated with both spine enlargement and increased pap's spine coverage. to assess the consequences of these changes, we then specifically triggered elevation of pap movements at the synaptic level by flash photolysis. spine stability analysis 24 hour after uncaging revealed that spines in contact with activated paps were more stable than others. this study suggests that excitatory synapses control the motility of surrounding paps through the triggering of neurotransmitter-evoked astrocytic ca 21 i elevations. increased pap motility seems to be necessary to elevate their coverage of the synapse during ltp, leading to higher synapse stability. astrocyte reactivity occurs in response to many pathological brain situations. reactive astrocytes display morphological and functional changes that could result in concentration variations of some brain metabolites localized both in astrocytes and in neurons. such changes could be detected by magnetic resonance spectroscopy (mrs), allowing non-invasive monitoring of astrocyte reactivity and thereby neuronal dysfunction in vivo. one of the major peak detected by mrs in the brain corresponds to neuronal metabolite n-acetyl-aspartate (naa). although its function is unclear, it is considered as a biomarker for neurodegeneration and a decrease in its concentration is interpreted as neuronal death or dysfunction. however, there is not clear evidence that other cell types, specifically astroglia, could not contribute to change in naa levels in the brain. in this study, we used a rat model of astrocyte activation by stereotaxic injections of lentiviral vectors encoding for either the cytokine ciliary neurotrophic factor (lenti-cntf) into the right striatum or betagalactosidase (lenti-lacz) into the left striatum, as a control. mrs data were acquired on a 7t magnet from two voxels placed over each injected striatum. a diffusion-weighted laser sequence with echo time te 5 40 ms was used. concentrations were measured using lcmodel software for total n-acetyl-aspartate (naa 1 naag), myo-inositol (ins), total choline (cho), glutamate and taurine relative to total creatine. lenti-cntf injection promotes a sustained, extensive and selective activation of astrocytes, as evidenced by overexpression of gfap and vimentin and cellular hypertrophy (fig.1) . importantly, neurons displayed unaltered morphological, molecular and electrophysiological features, as described previously (escartin et al., 2006; beurrier et al., 2010). mrs evidences changes in metabolite concentration: in the lenti-cntf injected striatum, levels of the astrocyte metabolites ins and cho were increased, whereas levels of the neuronal metabolite naa and glutamate were decreased. increased levels of ins and cho are generally found associated with astrocyte reactivity, however, the decrease in naa is unexpected given that neurons are not dysfunctional with cntf. to understand how astrocyte reactivity can influence naa levels, we are currently characterizing the molecular changes occurring in the naa metabolism using quantitative pcr, biochemistry, histology and hplc. our data suggest that reactive astrocytes alone result in alterations of metabolite concentrations detectable by nmr. although the mechanisms by which activated astrocytes regulate neuronal metabolism remain to be elucidated, our work challenges the mrs dogma that decreased neuronal metabolites can be used as a biomarker for neurodegeneration. microglial phagocytosis is a vital phenomenon for the clearance of damaged and death cells or infectious agents in a context of brain injury or infection, respectively. in addition, microglia can boost synaptic plasticity by the phagocytosis of axon terminals and dendritic spines. therefore, it is crucial to better understand the mechanisms involved in microglia clearance in order to devise new strategies to promote an efficient brain repair. recently, we showed that histamine modulates microglia motility and cytokines release. in this work, we aimed to investigate the role of this molecule and its receptors in microgliainduced inflammation by evaluating microglial phagocytic activity and reactive oxygen species (ros) production. for that purpose, an iggopsonized latex bead assay was performed in n9 murine microglial cell lines exposed to lipopolysaccharide (lps) or histamine 1, 10 and 100 mm. we showed that histamine significantly stimulated phagocytosis of opsonized latex beads via h1 receptor activation. this effect was accompanied by the rearrangement of cytoskeleton analyzed by phaloidin and acetylated tubulin expression and cellular distribution. the phagocytic activity was significantly reduced after pretreatment with apocynin, a putative napdh oxidase (nox) inhibitor. all nox isoforms were expressed by microglial cells, but only the expression of nox1 was increased by treatment of histamine. rac1, an important subunit for the activation of nox1, was also activated by histamine. in addition, histamine induced ros production via h1 and h4 receptor activation. in conclusion, histamine plays an important role in the regulation of microglial phagocytosis, which is mediated by nox1 and rac1 activation. multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal damage, involving both white (wm) and gray matter (gm) areas. furthermore, cortical pathology correlates with ms progression and cognitive dysfunction. we previously found altered expression of gap junction (gj) proteins connexin32 (cx32) and cx47 in oligodendrocytes and cx43 in astrocytes in wm lesions and normal appearing wm (nawm) in brain samples from ms patients, and in a mouse model of experimental autoimmune encephalomyelitis (eae), implicating uncoupling of myelinating cells as an important aspect of ms pathology. here we studied cx32 and cx47 and their astrocytic partners cx30 and cx43 in and around cortical lesions and the normal appearing cortex (nacx) in ms samples and non-ms controls. we examined their expression by real-time pcr, quantitative immunoblot and immunohistochemichal analysis. compared to non-ms controls, expression levels of cx32 and cx47 mrna were reduced within lesions whereas cx30 and cx43 were increased. in nacx, all four connexins were upregulated with cx47 being significantly increased. immunoblot analysis of ms cortex revealed reduced cx32 and increased cx43 levels, while cx30 and cx47 showed no significant change. immunohistochemical analysis showed severely reduced cx47 gj plaques within lesions with gradual increase toward nacx. in contrast to nawm, cx47 gj plaques were not increased in nacx. both astrocytic gj proteins were increased in ms, but in distinct patterns: cx30 gj plaque counts were higher within lesions and perilesional areas. cx43 was increased in ms cortex compared to non-ms controls, but in contrast to nawm, cx43 gj numbers were higher in nacx than in lesions. similar alterations were found in the eae mice, in which cx32 and cx47 gj counts were reduced whereas cx43 and cx30 gjs were increased. our results indicate that oligodentrocytic as well as astrocytic gjs are affected not only in ms wm but also in the gm. loss of oligodendrocyte gjs and in parallel increase of astrocytic gjs reflecting astrogliosis occurs throughout the ms brain and may negatively affect the prospects of repair and remyelination, worsening disease progression. acknowledgements: this project was funded by the cyprus research promotion foundation (grants access/0308/11 and health/bios/ 0308/01 to kak) and by cyprus telethon (2010-14 grant to kak). post-mortem human brain tissues were kindly provided by the uk multiple sclerosis society tissue bank, imperial college london, funded by the uk multiple sclerosis society. the mainly astroglialocated enzyme glutamine synthetase is required to synthesize glutamine from the re-uptake of either glutamate or gaba. thereby, this enzyme significantly contributes to the control of brain glutamate and gaba levels. there is good evidence that both neurotransmitter systems are dysregulated in depression. hence, we decided to study the cellular expression of this enzyme in subjects with major depression and bipolar disorder. material and methods: we investigated the numerical density of glutamine synthetase immunoreactive astroglial cells in different brain areas (prefrontal cortex, anterior insula) of 14 subjects with major depression, 15 subjects with bipolar disorder and 19 matched control cases. results and discussion: we found that compared with controls in cases with major depression there was a significant reduction of the density of immunostained glial cells in all brain regions studied, whereas bipolar cases showed a normal expression of the enzyme. our findings point to a profound disturbance of the glutamateglutamine-gaba cycle in major depression, which is in good agreement with neuroimaging observations and molecular biologic data of others. m.-c. marx, d. billups, b. billups university of cambridge, cambridge, united kingdom perisynaptic astrocytes are thought to play a key role in the recycling pathway of glutamate by supplying the precursor glutamine to the presynaptic terminal. using fluorescent ph i measurements of astrocytes and direct patch-clamp recordings from the calyx of held synapse in rat brain slices, we have investigated the transport mechanisms that may mediate this release of glutamine from astrocytes in situ and assessed the role of glutamine in maintaining glutamatergic neurotransmission. glutamine transporter currents were elicited by puff application of 10 mm glutamine from a nearby pipette. for imaging astrocytic ph i , hpts was included in the patch pipette. system a (sa) transporter function was probed using the substrate inhibitors meaib, alanine and serine, while system n (sn) function was isolated using its unique ability to transport li 1 in place of na 1 . histidine was used as a substrate inhibitor of both sa and sn. glutamine-induced membrane currents i gln (220.0 6 2.4 pa, n 5 21) were blocked by bath applied alanine (10 mm) and meaib (20 mm), as well as substitution of external na 1 with li 1 . fluorescent ph i measurement revealed an alkalinisation during puff application of glutamine. while bath application of histidine (20 mm) abolished the ph change, meaib and serine (20 mm) only attenuated it. most of the ph change (63 6 11%; n 5 7) was insensitive to the substitution of na 1 with li 1 . in presynaptic terminals, puff application of glutamate did not induce a membrane current indicating a lack of direct glutamate uptake; however, puff application of glutamine resulted in a i gln which was eliminated by the removal of external na 1 and by meaib. miniature epscs recorded from postsynaptic neurons (238.8 6 3.5 pa) were inhibited by meaib (228.6 6 3.4 pa; n 5 5) following turnover of the presynaptic glutamate pool. single epscs were unaffected by meaib indicating no direct effect on postsynaptic ampa receptors. together, these data identify two glutamine transport systems within astrocytes. properties of the glial i gln indicate expression of sa while ph imaging reveals a non-electrogenic glutamine transporter with properties identifying it as sn. sn is known to be readily reversible under physiological conditions and we propose that this mediates the astrocytic glutamine release mechanism within the glutamate-glutamine cycle. furthermore, system a mediates glutamine transport in glutamatergic nerve terminals and is important in maintaining the supply of glutamate for excitatory neurotransmission during periods of high frequency stimulation. a. briens, m. schwalm, f. docagne, d. vivien inserm u919, caen, france astrocytes are key players in the brain, cooperating with neurons to modulate crucial processes. in particular, they are able to uptake and release neurotransmitters and neuromodulators to control their synaptic level. in our study, we hypothesize that astrocytes can regulate some effects of the serine protease plasmin in the cns through a mechanism of uptake. conversion of the zymogen plasminogen to the serine protease plasmin by the activators (tpa or upa) is the basis of fibrinolysis in the vasculature. but cerebral functions for the plasminogen activator/plasmin system have recently arisen. it has been shown that plasmin was essential to activate the brain derived neurotrophic factor (bdnf) in its mature form, promoting long-term hippocampal plasticity. in alzheimer's disease, plasmin plays a protective role by participating in beta-amyloid clearance. plasmin can also lead to neuronal injury by degrading extra-cellular matrix proteins. in animal models of multiple sclerosis, plasmin can also help removing intraparenchymal deposits of fibrin, thus limiting axonal damage. these data suggest that a system of regulation of plasmin effects in the brain is necessary. in this study, we showed that astrocytes can actively and specifically uptake plasminogen and plasmin in a time-dependent and a dose-dependent manner. this mechanism involves clathrin pits formation in astrocytes and the lysine-binding site of plasminogen/plasmin. plasminogen and plasmin, following endocytosis are found in early endosomes, late endosomes, lysosomes and recycling endosomes. finally, we noticed that astrocytes uptake plasmin more efficiently than plasminogen. this study brings out a mechanism of glial uptake of plasminogen and plasmin. this could control their extra-cellular levels and regulate their effects within the brain. further investigations should determine whether this mechanism can modulate plasminogen activation and synaptic plasmin activity. besides, plasminogen activators (tpa) and cerebral substrates of plasmin (pro-bdnf) are also taken up by astrocytes. a link could thus exist between these processes to regulate the amount of synaptic mature bdnf. this would reveal a cross-talk between neurons, releasing precursors in the synapse (pro-bdnf and plasminogen) and astrocytes, regulating their activation and their clearance. understanding and modulating plasmin uptake could be very important in pathological conditions where the plasminogen/plasmin system is involved, such as alzheimer's disease, multiple sclerosis or stroke. healthy brain aging is characterized by neuronal loss and cognitive decline being inflammation a major causative factor. microglial activation is considered to be a major driver for the neuropathological findings in alzheimer's disease (ad). we evaluated whether young vs. aged microglia responded differently to ab soluble oligomers (abo) and to conditioned media from abo-treated hippocampal neurons. microglia from 1 day cd1 pups were incubated for 24 h at 2 (young) and 16 (old) days in vitro (div) with abo (ab1-42 at 50 and 1000 nm). in parallel, e16 mice hippocampal neurons were treated with abo at 4 (young) and 18 (old) div for 24 h, and the incubation media used as conditioned media to treat microglia, at respective div, for further 24h. cell migration (boyden chamber), extracellular content in glutamate (commercial kit), and activity of matrix metalloproteinases (mmp)22 and 29 (gelatin zymography) were assessed. an increased migration towards abo and atp was observed in young microglia but almost undetectable in aged cells (p < 0.01). interestingly, microglia exposure to conditioned media from 1000 nm abotreated neurons markedly decreased both young and old microglia migration, while treatment with media from 50 nm abo-treated neurons enhanced migration, mainly in the young microglia (p < 0.05). concerning glutamate, aged cells released less than young ones upon abo treatment (p < 0.01). in addition, when exposed to neuronal conditioned media, only the young microglia were able to exert a neuroprotective role by reducing the media glutamate content. curiously, while abo promoted a microglia release of mmp-9, independently of cell age, it only induced mmp-2 secretion in old cells in an abo concentrationdependent manner (p < 0.05 for 1000 nm abo). in contrast, conditioned media from abo-treated neurons elicited mmp-2 release only from young microglia. together, our data point to a loss of microglia function towards abo with ageing and are unique in suggesting that senescent microglia may release high levels of mmp-2 in ad. in addition, our results indicate that abo-treated neurons enhance the release of mmp-2 even by young microglia. whether this finding may contribute to enhance inflammatory stress and the onset of ad will be the aim of further studies. human immunodefeciency virus-1 (hiv-1) productively infects microglia within the central nervous system (cns). viral replication within microglia and the resulting immune response and neurotoxicity leads to significant cognitive impairments not limited to dementia, neurocognitive abnormalities, and memory deficits. combination antiretroviral therapy reduces the cognitive impairments associated with hiv-1, but the regulatory hiv-1 tat protein is still produced in the cns during treatment, even in the absence of productive infection. our laboratory analyzes the effect of tat on the murine cns by stereotactically injected into cortex, which mimics in part the neuroinflammation and neurodegeneration characteristic of hiv-1 associated neurocognitive disorders (hands). additionally, in vitro tat-treated bv-2 microglial cells exhibit activation associated with increased cytokine expression and phagocytosis. mixed lineage kinase 3 (mlk3) has recently been implicated in the tat-mediated activation of microglia. mlk3 activity is increased by the presence of tat protein, which results in microglial activation and increased production of inflammatory cytokines. conversely, the brain penetrable, small molecule, new chemical entity urmc-099 inhibits the mlk3 pathway. we hypothesize that urmc-099 attenuates tat-induced microglial activation as measured by microglia process length, phagocytosis assay and cytokine expression. in vitro application of urmc-099 decreased tatinduced production of ccl2 and il-6, microglia process length, and phagocytosis of charged and uncharged beads in bv-2 microglia cells. these data, in aggregate, strongly support a role for the mlk3 pathway in neuroinflammation and suggest that inhibition of this pathway with urmc-099 may have significant anti-inflammatory effects in an inflammatory cns milieu. in conclusion, this work will advance our understanding of mlk3 activity and may provide a viable drug therapy for hands. the contributions of glial cells to the modulation of adult synaptic plasticity are becoming increasingly more appreciated. astrocytes in the supraoptic nucleus (son) of the hypothalamus demonstrate prominent temporary structural changes during physiological events such as dehydration and lactation. such structural remodeling is characterized by a reduction in the astrocytic coverage of oxytocin neurons and their synapses. these astrocyte morphological changes also contribute to the observed functional changes in synaptic activity during dehydration and lactation. in order to elucidate the underlying mechanisms regulating the astrocytic contributions to synaptic plasticity we have established an astrocyte protein expression profile for structural changes in the son during lactation and hyperosmotic challenge. for this, son from virgin, lactating, and hyperosmotic rats were collected from acute hypothalamic slices and analyzed by mass spectrometry to identify proteins differentially regulated by the changes in synaptic structure induced by lactation and hyper-osmolarity. our mass spectrometry data analysis has placed particular focus on regulation of astrocyte-specific proteins and the roles of these proteins in molecular pathways known to be involved in son plasticity, such as cytoskeleton remodeling, cell adhesion, and cell signaling. gene ontology analysis revealed over representation of pathways known to be highly active in astrocytes including metabolism and antioxidant activity. promising target proteins for future functional studies will be discussed. here we elucidate a novel merlin isoform 2 (merlin-iso2)-specific function in maintaining axonal integrity and propose that reduced axonal nf2 gene dosage leads to nf2-associated polyneuropathy. we identify a merlin-iso2-dependent complex corresponding to activation of the gtpase rhoa, enabling downstream rho-associated kinase to promote neurofilament heavy chain phosphorylation. in vivo, specifically merlin-iso2 knockout mice exhibit impaired locomotor capacities, delayed sensory reactions, and electrophysiological signs of axonal neuropathy. sciatic nerves from these mice and sural nerve biopsies from nf2 patients show reduced nf-h phosphorylation, decreased interfilament spacings and irregular-shaped axons. a. catenaccio, p. silva, f. court pontificia universidad cat olica de chile, santiago, chile axonal degeneration is an active process involved in a variety of neurodegenerative conditions triggered by diverse stimuli. this degenerative process can cause permanent loss of function, so it represents a focus for neuroprotective strategies. we have recently shown that axonal degeneration triggered by distinct mechanical and toxic damage is dependent on the activation of the mitochondrial permeability transition pore (mptp), which probably represents a central execution program of axonal degeneration, upon which several pathways converge (barrientos et. al., journal of neuroscience 2011). nevertheless, the participation of other cellular types in this degenerative process has been largely overlooked. after axonal damage in the peripheral nervous system (pns), schwann cells enveloping axons have been regarded as responsible for clearing degenerating axonal debris and to mount an inflammatory response for tissue clearance by invading macrophages. nevertheless, a possible function of glial cells in modulating axonal degeneration has not been addressed so far. here we explored the possibility that glial cells actively participates not only in the clearance of degenerating axons, but directly in the axonal degeneration program during wallerian degeneration. we found that axonal injury activates an early response of schwann cells that results in the fragmentation of their associated axons by actin-rich cytoplasmic domains of schwann cells known as schmidt-lanterman incisures (sli). this first step of schwann cell fragmentation of axons is dependent on the erk signaling pathway, wich control the dedifferentiation program initiated in schwann cells after nerve injury (napoli et. al., neuron 2012). in the axonal compartment, injury triggers a parallel cell autonomous degenerating program dependent on mitochondrial mptp activation, which is cell non-autonomously assisted by schwann cells through the activation of a cell extrinsic pathway of axonal destruction by tnf-alpha secretion. we showed for first that time that axonal degeneration in the pns proceeds by axonal autonomous mechanisms, as well as cell non-autonomous ones dependent on scs. therefore, effective targets for neuroprotection should consider not only the axonal compartment, but also the associated glial cell. glutamate is the major excitatory neurotransmitter in the brain. its concentration is the synaptic area is highly regulated in order to maintain point-to-point transmission and to prevent excitotoxicity associated with chronic activation of glutamate receptors. as there is no endogenous extracellular enzyme to degrade glutamate, the clearance of glutamate is ensured by transporters located on the surface of astrocytes. among these surface transporters glt-1 ensures almost 90% of glutamate uptake at synapses. it is widely accepted that many receptors and transporters on the surface of neurons and glia display surface trafficking properties and are not simply static proteins. the basic surface trafficking properties of glutamate receptors at the synapse have been studied in depth and have allowed us to understand many important trafficking events that occur during synaptic plasticity. the action of glt-1 is fundamentally important in synaptic communication. surface trafficking of this transporter may play a pivotal role in the spatial and temporal properties of glutamate diffusion at the synapse. however, it is still unknown whether glt-1 is dynamically regulated on the surface of astrocytes. thus we set out to uncover the surface trafficking of glt-1 in astrocytes by using a combination of high-resolution imaging, such as single particle (quantum dot [qd]) tracking, molecular biology and electrophysiological approaches. here we have demonstrated that glt-1 is indeed subject to surface trafficking on astrocytes. using pharmacological approaches we have shown that this diffusion is subject to regulation by the activity of the transporter itself as well as neuronal activity. furthermore, we have demonstrated that the speed of glt-1 diffusion is greatly reduced at excitatory synapses. this leads us to the conclusion that glt-1 surface diffusion is highly regulated at the synapse where it can effectively carry out its role as the principal glutamate transporter at the synapse. finally, to elucidate the physiological role of glt-1 surface diffusion, we have carried out electrophysiological experiments in which glt-1 trafficking is effectively reduced using a cross-link (x-link) protocol. we observed that a reduction in the surface trafficking of glt-1 resulted in an increase of neuronal excitability. thus glt-1 surface trafficking on astrocytes has an implicit role in regulating basal neuronal activity through glutamate uptake at the synapse. o. chever, e. dossi, n. rouach collège de france, paris, france astrocytes play crucial roles in brain physiology by dynamic interactions with neurons. they form plastic and extensive networks mediated by gap junction channels. it has recently been shown that astroglial networks limit neuronal network activity. however, it is currently unclear how astroglial networks influence neuronal excitability and population activity. to investigate how astroglial assembly regulates neuronal network activity, we performed electrophysiological recordings in hippocampal slices (ca3 and ca1 areas) from mice with disconnected astrocytes, in which both astroglial gap junction forming proteins, connexin 30 (cx30) and connexin 43 (cx43), are knocked out (gfap-cre cx30 -/-cx43 fl/fl , dko). synchronized excitatory discharges were recorded in an acute pharmacological model of epileptic-like activity. in this model, spontaneous bursting discharges are characterized by a sharp and large amplitude shift in field potential, generated by a major depolarization and firing of all putative neurons. pyramidal cells depolarized up to 40 mv during seconds and such depolarization is followed by a long-lasting undershoot of the membrane potential. we found that the frequency of bursting activity in dko hippocampal slices is drastically increased. however, the duration of neuronal depolarizing bursts is severely reduced. furthermore, pyramidal neurons are more depolarized due to an increase of synaptic bombardment. this suggests that increased synaptic background promoting resting membrane potential depolarization facilitates triggering of neuronal bursts, but compromises the strength of synchronized events. to investigate local dynamics of bursts generation, we performed multielectrodes arrays recordings in the ca3 area of the hippocampus. in slices from dko mice, bursting activity in the ca3 region is generated within a more restricted area, indicating that the number of neurons to be recruited is highly decreased. altogether, these results indicate that gap junction-mediated astroglial networks strengthen the coordination of neurons during synchronized events. acutely loaded hypoxia increases ventilation, and increased ventilation does not immediately return to the pre-hypoxic level, but persists for a while after resuming room air inhalation. this phenomenon is referred to as short term potentiation or plasticity of breathing, and is thought to contribute to the stabilization of ventilation. although extensive studies have been conducted to clarify the cellular mechanism of respiratory plasticity focusing on neurons and synapses, its precise mechanism remains unknown. on the basis of the recent discovery that astrocytes are actively involved in neural plasticity in various brain regions, we hypothesized that the post-hypoxic potentiation of breathing is mediated by astrocytes. we used unanaesthetized unrestrained adult mice. ventilatory parameters (respiratory frequency, tidal volume and minute ventilation) were measured by whole body plethysmography. to test the hypoxic response, mice breathed room air, hypoxic gas (7% o 2 , 93% n 2 ), and again room air. we also analyzed the hypercapnic response that was conducted under a hyperoxic condition to eliminate the influence of the carotid body. to test the hypercapnic response, mice breathed pure o 2 , hyperoxic hypercapnic gas (95% o 2 , 5% co 2 ), and again pure o 2 . after recording of the hypoxic and hypercapnic responses with their recovery time courses, arundic acid (ono-2506) was injected intraperitoneally. arundic acid is a compound that suppresses the function of astrocytes through the inhibition of s100b production in astrocytes. arundic acid did not affect ventilatory augmentation induced by either hypoxia or hypercapnia. however, arundic acid suppressed the short term potentiation induced by hypoxia (but not by hypercapnia) in a dose-dependent fashion. high dose arundic acid completely eliminated hypoxia-induced short term potentiation. these results suggest that the post-hypoxic potentiation of breathing is mediated by astrocytes with the involvement of s100b. the mechanism of hypercapnia-induced potentiation is different from that of hypoxia. we propose a model to explain the cellular mechanism of post-hypoxic potentiation: hypoxia stimulates the respiratory neuronal network in the brainstem through the excitation of the carotid body. neurotransmitters spilled from excited neuronal synapses activate nearby astrocytes. the activation of astrocytes is sustained, and these astrocytes persistently excite respiratory neuronal network by releasing gliotransmitters. monosynaptic c-fibre evoked excitatory postsynaptic currents (epscs) were recorded in lamina i neurons using whole-cell patchclamp in rat transverse spinal cord dorsal-root slice preparations. spontaneous epscs were recorded simultaneously. in all experiments bicuculline and strychnine were added to the bath solution to block gaba a -and glycine-mediated synaptic currents. the superfusion of spinal cord slices in vitro with fkn results in a rapid facilitation of evoked epscs in spinal lamina i neurons receiving monosynaptic input from primary afferent c-fibres. both the microglial cell inhibitor minocycline and a fkn neutralising antibody, completely prevented fkn-induced changes in synaptic transmission. in addition, the inclusion of the ca 21 chelator bapta or the nmda receptor antagonist mk801 in the pipette solution completely prevented fkn-induced enhancement of synaptic strength, suggesting a post-synaptic ca 21 -dependent mechanism of ltp induction. analysis of paired-pulse ratio (ppr) indicated that fkn-induced facilitation of synaptic strength is accompanied by a decrease in ppr suggesting a pre-synaptic mechanism of ltp expression. in support of this finding, fkn increases the number of spontaneous epscs recorded from lamina i neurons. these data indicate that stimulation of microglial cells in order to induce a pro-nociceptive activity state is sufficient for facilitation of synaptic strength within the dorsal horn. both pre-and post-synaptic mechanisms contribute to fkn-induced facilitation of synaptic strength. this work was funded by a wellcome trust flexible travel fellowship to akc. the organization and connectivity of the developing neocortex remains largely unresolved. the establishment of neuronal microcircuits is a complex process that partly depends on the diversity of neuronal cell types and their ultimate role in the mature brain. this complexity is increased by the existence of bona fide synaptic connections between neurons and oligodendrocyte precursor cells, also called ng2 cells, which properties and function remains largely unknown. to unravel the physiological characteristics of gabaergic synapses between interneurons and ng2 cells, we performed paired recordings in the developing somatosensory cortex. experiments were carried out during the second postnatal week in vgat-venus;ng2-dsred double transgenic mice that allowed us to identify interneurons (venus 1 ) and opcs (dsred 1 ) in acute slices. our results demonstrated that ng2 cells are synaptically contacted by fast-spiking (fsi) and non-fast-spiking (nfsi) interneurons. however, a predominant input was received by fsi, a class of interneuron that constitutes only a minor population in the second postnatal week. on the contrary, only a small proportion of the abundant nfsi were connected to ng2 cells. all the connections showed paired-pulse depression, low probability of response, and preliminary analyses suggest that each interneuron form a single synaptic contact onto ng2 cells. paired recordings, extensively used to examine the synaptic properties of neuronal microcircuits, may open a new perspective to understand in detail the mechanisms of gaba release and signaling between interneurons and ng2 cells in the healthy and pathological developing brain. first, we characterized a microglial cell line obtained from cd1 mouse cortex (n9), in order to clarify the mechanisms involved in the activation pattern of these cells. for that, we incubated n9 microglia with 300 ng/ml of lipopolysaccharide (lps) for 24 h. morphology (anti-iba1 staining), phagocytic ability (fluorescent latex beads) and chemotaxis to atp (boyden chamber) were evaluated. we observed that n9 microglia is ramified in control conditions and after lps treatment they change to an amoeboid morphology, which is accompanied by increased phagocytosis and decreased migratory ability, indicating that lps induces activation of n9 cell line. next, we investigated the role of the released factors from a mn-like cell line expressing human sod1 with g93a mutation (nsc-34/ hsod1g93a) on microglia functions. nsc-34 expressing human sod1 wt were used as control. thus, n9 microglia were incubated for 4 and 24 h with nsc-34 conditioned media that had been collected at 1 and 4 days of differentiation (div) and reactivity tests were performed as above. although after 4 h of incubation no significant changes were observed, data evidenced that incubation for 24 h with nsc-34/ hsod1g93a conditioned media collected at 4 div changed the bipolar morphology of the n9 microglia into an active amoeboid state, decreased phagocytic ability (22%, p < 0.05) and induced apoptosis (50%) (fig.1) . moreover, microglia have shown to not be attracted by the released factors from degenerating mn, as determined by the chemotaxis assay. together, our results evidence that released factors from nsc-34/ hsod1g93a degenerating cells although inducing microglia morphological activation trigger a reduction in cell phagocytic ability and loss of viability, suggesting a role for microglia along als progression. organotypic explants culture has a pivotal role in studying the complex structure of neuronal tissue. although embryonic tissue explants and slices are used to obtain long term culture, most applications such as drug testing require adult tissue for reliable results. in this study, we show that nanostructured metal-oxide arrays can be employed to yield long-term organotypic culture of adult neuronal tissue explants as demonstrated for the adult guinea pig retina and adult murine brain slices. even after 14 days of culture, the thickness and the layered structure of the retina were well maintained. quantitatively, the number of nuclei within the inner and outer nuclear layers was comparable to freshly isolated retinae and no significant change in the densities of the nuclei within the layers was observed. the outer plexiform layer, as the site of synaptic connection between photoreceptors and neurons, was still well preserved. moreover, the thickness of the photoreceptor layer was conserved, indicating that the inner and the outer segments of the photoreceptors survived well during two weeks culture. this is hardly observed in long-term cultures since the outer segments were cultured without retinal pigment epithelium. concerning the adult murine brain slices from neo-cortex, after 9 days of culture, the neurofilaments and cell nuclei were still well preserved and the neuronal network displayed the typical arrangement of long axons. however, preservation of the tissue depends strongly on the geometry of the nanostructured array surface such as roughness and spacing, whereby organotypic brain slice culture requires different geometries than retina culture. since organotypic culture of adult tissue could only be obtained for about 7 days with conventional techniques, our new substrate material could be the breakthrough for in vitro studies on tissue regeneration, neuroplasticity, as well as drug testing. the intrinsic optical signal (ios) is widely used for mapping neuronal activity in afferent activated brain areas. ios evoked by afferent stimulation in vitro is generally attributed to glial cell swelling via activation of na 1 /k 1 /clcotransporter that follows postsynaptic activation. despite the phenomenon is known for decades, the relative contribution of different cell types and the underlying molecular mechanisms have not been disclosed in detail. our goal was to explore the glial and neuronal correlates underlying in vitro ios genesis. we characterized ios initiated by schaffer collateral stimulation in the rat hippocampal slice using simultaneous high-frequency imaging and field potential recordings. we used a 464-element photodiode-array device (pda) that enables ios detection with 0.6 ms time resolution, making it achievable to align optical and electrophysiological signals. ios was primarily observed in the proximal region of the stratum radiatum and stratum pyramidale of the hippocampus. ios was decreased by blockade of neuronal activity by voltage-gated na 1 channel inhibitor tetrodotoxin and was significantly enhanced by suppressing inhibitory signaling with the gaba a antagonist picrotoxin. we found that ios was predominantly initiated by postsynaptic glutamate receptor activation and progressed by the activation of astroglial glutamate transporters and mg 21 -independent astroglial nmda receptors. in agreement with previous studies, furosemide, the blocker of both the neuronal k 1 / clcotransporter kcc2 and the glial na 1 /k 1 /clcotransporter nkcc1 decreased the ios amplitude. to decide which cotransporter is responsible for this effect of furosemide, we applied selective blockers of nkcc1 and kcc2, bumetanide and (1)[r]-dioa, respectively. we evidenced, that nkcc1 did not contribute to the ios generation, in contrast to previous suggestions. enhancement and slight inhibition of ios through anion and volume-regulated anion channels, respectively, were also depicted. major players of ios mechanisms disclosed by high-frequency ios imaging imply that spatiotemporal ios reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. our model may help to better interpret in vivo ios and support diagnosis in the future. question: a poor x3 polyunsaturated fatty acids (x3 pufa) status, favored by the low x3/x6 ratio in western diets, seems to contribute to cognitive decline in the elderly, but mechanistic evidence is lacking. we therefore explored the impact of x3 status on the evolution of glutamatergic transmission, astroglial regulation and neurogenesis in the hippocampus during ageing in rats. these processes are involved in memory formation and their dysregulation participates to the agerelated brain damage leading to cognitive decline. methods: we have compared 6 groups of rats aged 6 to 22 months fed x3-deficient, x3/x6-balanced, or x3 (fish oil) supplemented diets: young x3 balanced (yb), deficient (yd) or supplemented (ys), and old x3 balanced (ob), deficient (od) or supplemented (os) rats. we have evaluated synaptic efficacy and plasticity (electrophysiological recording), astroglial regulations (glutamate uptake and gfap expression), neuronal markers (glutamate transporters and receptors), neurogenesis (proliferation of neuronal precursors in the sub granular zone), and analyzed brain fatty acids composition. results: dietary modulation of x3 intakes efficiently modified the incorporation of docosahexaenoic acid (dha, the main x3 in cell membranes) in brain (250% deficient vs balanced, 110% supplemented vs balanced). ageing induced a 35% reduction of synaptic efficacy due to decreased pre-synaptic glutamate release, and a 30% decrease in the astroglial glutamate uptake associated to a marked astrogliosis (1100% gfap). x3 deficiency further decreased these hallmarks of ageing (od vs ob rats: 235% synaptic efficacy, 215% glutamate uptake, 130% gfap). on the opposite, x3 supplementation increased synaptic efficacy (125% os vs od) and seems to abolish astrogliosis (os vs ys : no change in gfap). neurogenesis was altered by x3 deficiency but not by supplementation. conclusion: our results characterize some specific age-related alterations of the glutamatergic synapse in the hippocampus that are aggravated by a dietary deficit in x3 and attenuated by x3 supplementation. methods: sgcs were isolated from the trigeminal ganglion (tg) of adult male wistar rats (n 5 9). animals were deeply anaesthetized and both tg were extracted and digested first in 5 mg/ml collagenase for 15 min and then in 0.125% trypsin containing dnase for 10 min. in initial experiments, isolated sgcs were treated with control medium or medium containing 16.5, 33, or 66 mm cis for 8h. in the subsequent experiments, sgcs were pretreated for 24h with control medium or medium containing 100 mm ibu or 1 mm skf. afterwards, sgcs were stimulated for 8 hours with medium containing 66 mm cis. pge 2 concentration was determined by elisa. results: cis caused a concentration-dependent increase in pge 2 release from sgcs. treatment with 33 mm cis significantly increased pge 2 concentration to 354.8 6 87.3 pg/ml, compared with control 211.3 6 39.0 pg/ml (p 5 0.018). pge 2 concentration was further increased to 464.1 6 77.0 pg/ml (p 5 0.002) following 66 mm cis. pretreatment with ibu significantly lowered the pge 2 concentration compared with cis-stimulated sgcs (197. 5 6 85.2 pg/ml vs. 415.8 6 143.8 pg/ml, respectively; p 5 0.05). a more pronounced inhibitory effect was observed following skf pretreatment, where it reduced the pge 2 concentration to 19.7 6 1.8 pg/ml (p 5 0.003, compared with cis-stimulated sgcs). conclusion: these findings suggest that cis contributes to an inflammatory response in the tg by provoking release of pge 2 from sgcs, which may lead to development or maintenance of peripheral sensitization involved in cipn. the data also suggest that treatment with agents that target pge 2 release cascade may prove beneficial for preventing development or maintenance of cipn. functional alterations in synaptic contacts have often been described as a hallmark of major depressive disorder (mdd). antidepressants (ads) have been shown to restore neuronal circuits through an enhancement of synaptogenesis in some regions of the brain. nevertheless, the underlying mechanisms are still unclear. glia cells have been since long acknowledged as active partners of neurons in orchestrating molecular signals crucial for the proper arrangement of neuronal circuits in the developing and adult brain. therefore, understanding how both neurons and astrocytes respond to antidepressants (ad) is of high interest. using rat c6 glioma cells and primary cultures of astrocytes and neurons, we showed a time-dependent cell-autonomous modulation of the erk/mapk signalling pathway after acute treatment with various classes of ads in both cell types. specifically, glia cells responded to ads with the simultaneous and fast activation (after 10 min treatment) of both erk1/2, in contrast to treatment with antipsychotics or mood stabilizers. this activation induced 48 hrs later an increased release of gdnf, a factor involved in synapse formation and axonal wiring, which was mapk-dependent. on the contrary, hippocampal neurons showed a reduction in erk activity that correlated with neuronal activity inhibition after short term (10 min) ads administration, as demonstrated by quantification of c-fos expression after kcl stimulation. interestingly, this inhibitory effect was reversible after long term (48 hrs) ads treatment. to further identify whether gdnf released from astrocytes treated with ads might be responsible for long term changes in neuronal synapses, we examined how ads influenced synaptic densities in neurons alone or co-cultured with astrocytes. strikingly, we found that the number of synapses was reduced at 48hrs after ad treatments, but only in the presence of intact astrocytes and not in cultures of neurons alone or neurons treated with astrocyte-conditioned media. this effect was reversed after 120 hrs treatment and rescued by the soluble form of the specific gdnf receptor gfralpha1, but not by gdnf alone. moreover, live imaging of astrocytes showed dramatic morphologic changes of their processes in response to ads that might underlie the observed synaptic remodelling. our analysis of changes occurring at the neuronglia interface upon ad treatments might elucidate novel mechanisms of ads action that may open the avenue for unravelling the role of astrocytes in psychiatric diseases and implement pharmacologic treatment regimens for mdd patients. here we extend these findings showing that the neurotrophin nt-3, which also belongs to the putative factors secreted from glial cells after t3-stimulation, also increases the navd in neuron enriched cultures. the effects of nt-3 and fgf-2 are, however, not additive. antibodies against nt-3 potentiated the effects of fgf-2, suggesting that by converging signal cascades nt-3 could reduce the effect of fgf-2. in neuron-glia mixed cultures antibodies against nt-3 enhanced the t3-induced up-regulation of navd, suggesting, that a co-secretion of nt-3 from glial cells could limit the effects of fgf-2. in a second series of experiments we investigated, whether the well-known effect of t3 on the expression of na 1 /k 1 -atpases, thought to contribute to the effect of thyroid hormone on metabolism, is also influenced by glial cells. we found, that the effect of t3 on 3 [h]ouabain-binding and on the expression of na 1 / k 1 -atpase alpha2 subunits detected by western-blots was highest in neuron-mixed cultures, suggesting that a neuron-glia interaction is also involved in the t3 effects on na 1 /k 1 -atpase expression. in the healthy brain microglia engage in bi-directional interactions with neurons and other brain cells, which is crucial for maintaining normal physiology and cognitive function of the brain. disturbances in homeostasis and cell-cell communication may predispose to age-related dysfunction and neurodegenerative disease. for decades it has been speculated that microglia exhibit phenotypic diversity allowing specialisation to a variety of brain microenvironments. previous observations of regional variations in microglial densities and morphologies support the existence of microglial heterogeneity. however it is clear that a more comprehensive understanding, in particular of the molecular basis of microglial phenotypic and functional diversity on a global scale, is required. the limitation of recent gene expression studies is the use of mixed whole brain or dissected mixed cell extracts which preclude the detection of cell-specific gene expression profiles. thus, our aim was to validate an efficient extraction process for microglia, ideally minimising the impact on the resting state. the method developed is based on existing procedures and was refined to ensure consistency and to maximise cell yield and purity. the extraction of highly pure microglia as a single cell type was achieved by a two-step isolation technique using density-gradient and magnetic bead separation. purity between 90-95% was verified by flow cytometry and immunohistochemistry. quantitative pcr also demonstrated expression of specific microglial genes indicating that isolated cells are microglia. cytokine assays suggest that no or only minimal activation of cells was induced by the isolation. hence, the microglia retained their ability to respond to immunogenic stimuli and produce il-1b as a key inflammatory cytokine. overall, the purification procedure was shown to achieve consistent high purities and retain crucial functional properties. thus, the extracted microglia are likely to adequately represent the real in vivo state and enable the study of microglial phenotypes and functionality in the healthy and ageing whole brain and accordingly brain regions. early diversity experiments showed region-specific microglia phenotypes in the brain of healthy and immune challenged animals as well as differences between unchallenged and challenged animals. question: activation of nmda receptors increase the respiratory frequency in mammals. astrocytes are in intimate contact with neurons, specially in glutamatergic synapses, and are able to sense neuronal activity, react to, and even influence nearby neurons. furthermore, they can sense changes in h 1 or pco 2 , and in response to these stimuli, they can release molecules like atp and d-serine, which will activate neuronal circuits. then d-serine, an agonist of the glycine site of the nmda receptor, can affect the respiratory rhythm during hypercapnia. our aim was to study the probable role of d-serine in the modulation of the respiratory rhythm in mice neonates. methods: fictive respiration was recorded with suction electrodes from c4-c5 ventral roots in "en bloc" (brainstem-spinal cord) preparations from p0-p3 cf1 mice. superfusion was done with artificial cerebrospinal fluid equilibrated with o2:co2 5 95%: 5%, (ph 7.4,28 c) containing different d-serine concentrations (0.1-100 mm) in presence or absence of d-amino acid oxidase (daao), which degrades d-serine. in addition, hypercarbic acidosis (switching equilibration from 5 to 10% co2, changing ph from 7.4 to 7.2) was done in absence or presence of daao. results: application of d-serine increased the frequency of the respiratory rhythm in a concentration-dependent way. application of dao attenuated the effects of d-serine application. likewise, daao reduced slightly the effect of hypercarbia on the respiratory rhythm. conclusions: our preliminary experiments indicate that d-serine is a potent agent increasing the respiratory rhythm frequency in in vitro neonatal preparations, and likely, in association with atp, is mediating the respiratory response to hypercarbia. there, we showed that mac-2 positive microglia accumulate within the motoneuron area and phagocyted apoptotic bodies at the onset of motoneuron developmental cell death (from embryonic day 12.5-e12.5). motoneuron developmental cell death and microglia accumulation in the motoneuron area increased at e13.5, decreased at e14.5 and disappeared at e15.5. since it was supposed that microglia promote developmental cell death in the developing central nervous system, at least in vitro, we determined to what extent it was also the case in the embryonic spinal cord in vivo. to address this issue, we analysed in vivo the progression of developmental neuronal death in the spinal cord of transgenic mouse embryos lacking microglia (pu.1-ko mice). as opposed to what is observed in the sc of wild type mouse embryo, we found that activated caspase-3 staining was detectable before e12.5 in pu.1-ko mouse embryos. in pu.1-ko mouse embryos, activated caspase-3 staining was not restricted to the motoneuron area, as several positive cells are also present in the dorsal region of the spinal cord and in the progenitor zone. at e13.5, activated caspase-3 staining was 10 times greater in the spinal cord ventral area of pu.1-ko mouse embryos when compared to wild type littermates. moreover, and opposed to what occurs in wild type animals, caspase-3 staining in the motoneuron area of pu.1-ko mouse embryos increased at e14.5 and persisted at e15.5, being likely partly due to the accumulation of apoptotic bodies in the absence of microglia. our results suggest that, in addition to their phagocytic role, embryonic microglia are likely to protect neuron from death at the onset of developmental cell death and to promote survival of progenitor-like cells in the embryonic sc in vivo. accordingly, microglia could have an opposite effect on cell survival in the embryonic sc when compared to postnatal snc developmental stages and pathological conditions in the adult. we recently demonstrated a key role of the a-secretase tace, also known as adam17, in peripheral nervous system (pns) myelination by tace-mediated cleavage and subsequent inhibition of neuregulin1 (nrg1) type iii activity. unlike the pns in which nrg1 type iii is an essential instructive signal for myelination, oligodendrocyte (ol) development and myelination in the central nervous system (cns) are likely controlled by several growth factors some of which undergo cleavage by secretases. to study the role of tace on opc development, immunopanned a2b51 opcs were cultured in proliferating or differentiating medium and treated with a soluble form of tace (rhtace). when ols were scored according to their morphology, we observed enhanced opc differentiation upon addition of rhtace to proliferating opcs. addition of rhtace to differentiating opcs also increased the number of ols with a complex morphology and already presenting membrane sheets, suggesting that tace might promote opc differentiation. to further investigate the role of neuronal tace in cns, we used an in vitro ol myelinating coculture system. when cocultured with tace null drg neurons, rat wild type opcs never myelinate, suggesting that neuronal tace might control opc differentiation. in agreement, preliminary ultrastructural analyses of transgenic mice lacking tace in cns neurons showed hypomyelination and signs of myelin degeneration. accordingly, conditional transgenic mice lacking tace in ol are normally myelinated. these studies suggest that in the cns the a-secretase tace promotes opc differentiation and might regulate cns myelination. a better understanding of the role of tace will provide novel insights into the mechanism regulating cns myelination. in the excitatory network glutamate is released in the synaptic cleft during synaptic activity. glutamate clearance by astrocytes is mainly performed by na 1 -dependent glutamate transporters. the na 1 load during glutamate uptake evokes an activation of the na 1 /k 1 atpase, which dramatically increases energy demands in astrocytes. astrocytes are also involved in the regulation of extracellular potassium (k e 1 ) which significantly increases during the repolarization of excitatory neurons. in this study we evaluated how these fundamental functions of astrocytes coexist and if they interact. we investigated the impact of altering k e 1 concentrations on glutamate transporter activity measuring intracellular sodium (na i 1 ) using the na 1 sensitive fluorescent dye asante sodium green loaded in cultured astrocytes. we observed that glutamate uptake caused a reversible rise in na i 1 , which was tightly modulated in amplitude, slope and recovery rate by k e 1 levels. in order to evaluate the impact of physiologically relevant k e 1 fluctuations on the kinetics of the glutamate transporter the na 1 /k 1 atpase was inhibited. therefore we found that low k e 1 evoked a significantly faster influx of na 1 , whereas high k e 1 markedly slowed down glutamate capture, indicating that k e 1 directly modulates the kinetics of glutamate transporters. we then investigated the energy demands of astrocytes caused by k e 1 alterations. atp hydrolysis was indirectly measured through the changes in intracellular free mg 21 using the mg 21 sensitive fluorescent probe magnesium green. we found that low k e 1 led to higher energy demands of astrocytes during glutamate uptake. overall these results indicate that k e 1 acts as a negative feedback on glutamate uptake in astrocytes. the physiological consequences of these findings have to be considered in the context of k 1 buffering and of maintenance of neurotransmitter and energy homeostasis. recovery was significantly improved by nf fractions, as well as tubulin (tub), added after lpc removal: compared to cultures treated with lpc alone the number of olp (a2b51 cells), and their proliferation increased significantly, as well as the number of differentiated (cnp1) and mature (mbp1) ol. moreover, nf and tub protected ol from lpc toxicity when added at the time of lpc treatment: they increased olp proliferation, as well as the number of cnp1 and mbp1 ol significantly, compared to cultures treated only with lpc. on the contrary irrelevant proteins (actin, and skin proteins) were ineffective, demonstrating the specificity of the cytoskeleton proteins effects. importantly, nf and tub increase olp survival and proliferation when challenged with lpc, without delaying differentiation and maturation. we hypothesize that release of nf and tub following axonal damage in ms could participate in the regulation of remyelination through this process. this putative phenomenon might be complexified by alterations of axon protein expression, or of proteolysis, known to occur in ms models. purpose: microglial proliferation is commonly accepted as a hallmark of glial activation. an automatic method that allows assessing the number of microglial cells to analyze the proliferative microglial behavior in a laser-induced ocular hypertension (oht) model has been developed. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n 5 6) and lasered (n 5 6). retinal wholemounts were immunostained with anti-iba1 and images of iba-11 microglias were recorded with a fluorescence microscope. new algorithms of segmentation and control of distances were developed in matlab to obtain the number of iba-1 microglial cells. automatic results were compared with those obtain by direct human observation of the samples. results: the automatic method detected the number of iba-11 cells in the inner and outer plexiform layers of the retina in both, na€ ıve and oht-retinas. the number of cells present in the samples was not an obstacle for the program to run properly. the time required for counting iba-11 cells decreased from the human guide to the program based counting method from days to one hour. results show a strong correlation between both automatic and manual method (pearson correlation test, r 5 0.979; p 5 0.000 and r 5 0.942; p 5 0.000 for outer and inner plexiform layer respectively) indicating the consistency of the automatic counting. conclusions: a new, reliable and quick algorithm was developed with matlab to obtain the number of iba-11 microglial cells and also cellular density maps through retinal wholemounts in both na€ ıve and other proliferative conditions (i.e. glaucoma). due to this new automatic method, a bigger set of images or samples could be included in future studies to analyze the behavior of microglial cells. we are interested to understand whether scs in the peripheral nerves of mammals express functional ionotropic glutamate receptors, and whether scs use these receptors for communication with axons. to start addressing this question, we established a preparation of mouse live sciatic nerve slices employing "know-how" of live brain slices technique. using this preparation, we performed whole-cell patch-clamp recordings of scs at different stages of development (embryonic and early postnatal). we included a fluorescent dye into the pipette solution in order to perform post-recording morphological analysis of single scs. first, we recorded current responses of scs to a series of depolarizing voltage steps, aiming to test whether all scs in the nerve are electrophysiologically akin. we found that mouse scs differ in their passive membrane properties and expression of voltage-gated k1 channels: depending on the age, 3 to 4 cell types could be distinguished. post-recording morphological characterization of the recorded cells showed that scs in the mouse sciatic nerve differ in their size, as well as in the number and length of their processes. next, we used fast pressureinduced application of glutamate to investigate whether scs possess ionotropic glutamate receptors, and whether electrophysiologically distinct sc types differ in their expression of glutamate receptors. application of 1 mm glutamate caused an inward current in at least two types of scs, and preliminary analysis revealed a peak current amplitude in the range of 14-250 pa (n 5 18). glutamate-induced currents in scs were blocked by gyki53655, indicating that mouse scs carry ionotropic glutamate receptors of ampa/kainate type. currently we are studying which subunits of ampa receptors are present in scs, and how these receptors get activated in situ. although cx32 is expressed by myelinating schwann cells in the peripheral nerves, patients with cmt1x develop relatively early axonal degeneration, which correlates best with chronic disability. we found in previous studies of gjb1-null mice, a model of cmt1x, that the diameter of myelinated axons was progressively reduced at the age of 2 months compared to wild type (wt) littermates, resulting from progressive dephosphorylation of axonal neurofilaments and increased packing density. fast axonal transport was slower in distal axons of mutant compared to wt animals, with impaired mobility of synaptic vesicleassociated proteins and accumulation of beta-amyloid precursor protein and tau. how cx32-formed gjs are involved in the regulation of axonal cytoskeleton dynamics remains unclear. methods-here we investigated the signaling mechanisms regulating axonal cytoskeleton focusing on major kinases and phosphatases involved in neurofilament phosphorylation by immunohistochemistry and immunoblot. in addition we examined the localization of axonal mitochondrial, which also depend on axonal transport. we focused on nerve pathology in 2 month-old gjb1-null mice, when demyelination and inflammation is minimal, in order to identify the direct effects of gj loss on the axon. results-our results show that erk1/2 kinase and pp2a phosphatase were localized in non-compact myelin areas, including the schmidt-lantermann incisures and paranodes, where gjs are normally formed by cx32. another kinase, cdk5, was more diffusely localized along the axon. biochemical analysis showed altered amounts of erk1/ 2, pp2a and cdk5 as well as their upstream activators in gjb1-null nerves. the pp2a inhibitor phapi was also localized in non-compact myelin areas and significantly increased in gjb1-null nerves. assessment of axonal mitochondria by porin immunostaining showed significantly increased density in gjb1-null nerves, consistent with slower axonal transport. conclusion-our findings clarify some mechanisms of early axonal pathology that are independent of demyelination in this mouse model of cmt1x. several components of the signaling pathway regulating axonal cytoskeleton and axonal transport appear to be functionally related to gjs at the non-compact myelin sheath. loss of myelin gjs results in impaired regulation of axonal cytoskeleton, energy supply, and axonal transport. we have recently discovered that oligodendrocytes secrete exosomes containing a distinct set of proteins as well as mrna and microrna. intriguingly, oligodendroglial exosome release is stimulated by the neurotransmitter glutamate indicating that neuronal electrical activity controls glial exosome release. in this study we examined the role of exosomes in neuron-glia communication and its implications in glial support. results: to analyze the transfer of oligodendroglial exosomes to neurons, we exposed cultured cortical neurons to fluorescently labeled oligodendroglial exosomes. indeed, cultured cortical neurons internalized and accumulated oligodendroglial exosomes in the neuronal cell soma in a time-dependent manner. addition of endocytosis inhibitors or expression of dominant negative dynamin interfered with neuronal exosome internalization indicating that exosome uptake is mediated by clathrin-dependent endocytosis. furthermore, neuronal internalization of exosomes resulted in functional retrieval of exosomal cargo in vitro and in vivo upon stereotactic injection of exosomes. to investigate the influence of oligodendroglial exosomes on neuronal gene expression we performed a microarray screen of neuronal mrnas after exosome exposure. interesting candidates were further validated by qrt-pcr. conclusion: taken together, our results represent a proof of principle of exosome transmission from oligodendrocytes to neurons suggesting a new route of horizontal transfer in the cns. astrocytes are essential for the regulation of neuronal excitability, synaptic plasticity, and the vascular coupling of brain metabolism. unlike neurons, they are electrically silent, but produce a complex repertoire of ca 21 events that coordinate their major functions. however, the canonical cellular mechanism for ca 21 integration in astrocytes is unknown. using cultured astocytes and astrocytes in brain slices expressing ca 21 sensor, gcamp2, we report a candidate principle for ca 21 integration in single astrocytes. analysis of the spatiotemporal dynamics of ca 21 signaling in cultured astrocytes demonstrated that ca 21 event distribution can be described by a power law. we show that metabotropic glutamate receptor activation or changes in extracellular ca 21 regulate the power law exponent. these findings demonstrate scale-free ca 21 dynamics in astrocytes that can provide a potential computational theory for brain operation and metabolism. the biogenesis of the axon-myelin unit is controlled by reciprocal interactions between oligodendrocytes and neurons, which continue throughout life. oligodendrocytes secrete endosome-derived microvesicles termed exosomes, implicated in intercellular communication. these exosomes carry a specific set of proteins as well as rna species. here, we show that the neurotransmitter glutamate stimulates exosome secretion from oligodendrocytes by provoking ca 21 entry through oligodendroglial nmda and ampa receptors. furthermore, active neurons evoke exosome release from oligodendrocytes, indicating that neuronal activity controls oligodendroglial exosome release. in turn, neurons internalize oligodendroglial exosomes and functionally retrieve the exosomal cargo. thus, oligodendrocytes may influence the neuronal metabolism by an exosome-dependent transfer of bioactive substances to neurons. axonal degeneration resulting from lack of glial support occurs in plp and cnp deficient mice. intriguingly, both proteins are components of oligodendroglial exosomes. a proteomic approach revealed that amount and composition of exosomes derived from plp -/and cnp -/oligodendrocytes are altered, supporting the hypothesis that disturbed intercellular transfer of substances by exosomes may contribute to axonal degeneration in these mouse models. a. shahraz, j. kopatz, h. neumann university of bonn, bonn, germany microglial cells are the resident macrophages of the brain, which are derived from the embryonic haematopoiesis of the yolk sac. recently, we established a protocol for in vitro differentiation of pluripotent stem cells into microglial precursors by mimicking the embryonic haematopoiesis in a neural microenvironment. we now succeeded to produce microglia from human induced pluripotent stem (ips) cells. these human microglial cells were responsive to amyloid-b by increasing reactive oxygen species (ros) production. in addition, we produced primitive neural stem cells (pnsc) from ips cells that differentiated with suitable growth factors towards neurons. in human microglia-neuron co-culture, amyloid-b treatment resulted in shorter neural branches length compare to the control group. furthermore, results showed that the inhibitory human lineage specific receptor sialic acid binding immunoglobulin-like lectin-11 (siglec-11) was expressed on human microglial lines. we are now studying the neuroprotective role of siglec-11 in the amyloid-b-mediated microglia-neuron co-culture model. by expressing the human-lineage specific receptors, these microglial cells will be a suitable model to investigate these receptors in a human microglianeurons co-culture system.x purpose: organotypic retinal culture is a useful tool to perform preclinical drug testing, in which cellular interactions as well as tissue threedimensionality are preserved. the present study aimed to provide a detailed characterization of the morphologic changes of macro and microglial cells in this culture system. methods: retinas were isolated from 7 days old crl: cd(sd) rats with the retinal pigment epithelium attached as described previously (arango-gonzalez et al. 2010). explants were cultured for 4, 10 and 14 days (div4, div10 and div14). glial cell populations were characterized by immunostaining cryosections and wholemounts of age-matched control and cultured retinas using specific antibodies against gfap, vimentin and cd11-b. results: in div4 and div10 cultures, gfap-positive astrocytes were more robust and not homogeneously distributed as observed in vivo: in some retinal areas the astrocytic network was thicker and in other regions astrocytes were sparsely distributed. at div14 only few thinned gfap-positive astrocytes remained. gfap immunoreactivity of m€ uller cells was up-regulated in culture. however, no major difference was observed in vimentin staining between both, in vivo and in vitro retinas. m€ uller cells extended processes from the inner to the outer limiting membrane were observed for all examined retinas. microglial cells stained with cd11-b at div4 and div10 had somas more robust than in vivo and cell processes were thicker and retracted. same cells at div14 exhibited mainly a rounded morphology. conclusions: progressive morphological changes were observed in glial cell populations in retina explants. these changes include reactive macrogliosis, rearranged astrocytic distribution and microglial activation. since glial response is a hallmark of several retinal diseases, our organotypic retinal culture is a valuable resource for future investigations in retinal degenerative processes and therapy. it is widely recognized that grouped housing in enriched environment (enr) impacts the animals' brain structure and function. for instance, rats reared in enr exhibit enhanced neurogenesis in the dentate gyrus, improved hippocampus-dependent spatial memory task performance, and spine density increases of ca3 and ca1 pyramidal neurons. we found that that enr housing for four weeks after weaning induces an enhancement in gamma band local field potential oscillation power and an increase in spine density in the right ca1 stratum radiatum of long-evans rats. the mean spine size, as assessed by serial measurements of postsynaptic density areas, did not change substantially by laterality or rearing condition. one standing question is how glial processes are organized in enr treated rats. unlike cerebellar parallel fiber synapses, the astrocytic coverage of a hippocampal synapse varies from synapse to synapse. our preliminary electron microscopic observation suggests that right ca1 str. radiatum excitatory synapses tend to have larger degrees of astrocytic coverage in enr treated rats than singly caged rats.we are currently making morphometric measurements to quantitatively assess the peri-synaptic glial changes. it is becoming more and more evident that proper functioning of the neuron-astrocyte-microglia triad is fundamental for the functional organization of the brain, and thus it is of the utmost importance to better characterize their interactions in physiological and pathological processes. we recently demonstrated, in rat models of normal brain aging and lps-induced acute inflammation, that astrocytes and microglia actively collaborate in the clearance of apoptotic neurons and neuronal debris associated with programmed cell death. here we studied the interactions between neurons, microglia and astrocytes within the ca1 region of the hippocampus after bilateral common carotid artery occlusion (bccao) in the rat, a valid model of chronic cerebral hypoperfusion which leads to persistent ischemic conditions and ultimately to neuronal death. male wistar rats were subjected to permanent bccao. a group of rats was infused into the jugular vein with dipyridamole (7 days, 4 mg/kg/day by an osmotic minipump). sham-operated animals were used as controls. three months after bccao, immunohistochemical studies were performed on brain coronal slices, focussing on the hippocampal ca1 region. using an antibody against glial fibrillary acidic protein (gfap) no astrogliosis was detected. we found a significant increase in the number of total microglia, visualized using the iba1 antibody, in bccao-treated rats in comparison to the sham group (1 18%, p < 0.01, one way anova and newman-keuls) and this effect was completely reverted by dipyridamole (p < 0.01, one way anova and newman-keuls). as exemplified in figure 1 , in the ca1 and str. radiatum of bccao-treated rats, many neurons (anti-neun antibody, red) showing signs of degeneration were closely apposed to and infiltrated by astrocyte branches (anti-gfap antibody, green) which appeared to be bisecting the cell body into cellular debris, and microglia cells (anti-iba1, antibody, blue) were actively phagocytosing the damaged neurons. this finding is consistent with the scavenging activity of microglia upon dying neurons or debris, a possible mechanism that prevents further injury to neighboring neurons. it will be interesting to investigate which intercellular communication mechanisms allow the recruitment and activation of different glial cells in a well-organized reciprocal interaction to scavenge the damaged neurons and to verify the mechanisms of the protective effects of dipyridamole found in this chronic cerebral ischemic model. astrocytes are glial cells which provide important metabolic support to neurons, actively tune synaptic activity and influence brain microcirculation [1] . one of the key processes which sustain astrocyte communication with neighbouring cells is regulated exocytosis mediating the release of gliotransmitters (peptides, amino acids, atp), delivery of membrane transporters, channels and other molecules to the plasma membrane. the exocytic gliotransmitter release is thought to be associated with snare complexes. these consist of four a-helices where one of these is contributed by syntaxin-1, one by synaptobrevin-2 (sb2) and two by snap-23 in astrocytes. out of these, sb2 is a trans-membrane protein present on the secretory vesicle [2] . however, the subvesicular nano-architecture and the number of sb2 molecules per vesicle are unclear. moreover, it is also unknown how many sb2 molecules are involved in the fusion between the vesicle and the plasma membrane in astrocytes. to study single vesicles and their association with sb2 in live astrocytes, we generated a ph-sensitive indicator yellow synapto-phluorin (ysph) as a marker for sb2 and as a functional readout for monitoring the properties of fusion pores, which are formed following the merger between the vesicle and plasma membranes. in an acidic environment ysph is non-fluorescent and becomes fluorescent upon alkalinisation, permitting the study of fusion of vesicles with the membrane during gliotransmitter release [3] . our preliminary results, obtained by confocal fluorescence microscopy (with the resolution limit of about 200 nm) and by a super-resolution microscopic technique structured illumination microscopy (sim; with the resolution limit of about 120 nm) show that the new probe (i.e. ysph) efficiently reports the fusion pore establishment in astrocytes and the configuration of sb2 molecules in a vesicle. the number of schwann cells is fitted to the axonal length in the peripheral nerves. this setting is lost when tumorigenic stimuli induce uncontrolled schwann cell proliferation, generating tumours such us neurofibromas and schwannomas. schwann cells proliferate as well during wallerian degeneration. in both cases proliferation is finally arrested. we show that in neurofibroma, the induction of jmjd3 removes trimethyl groups on lysine-27 of histone-h3 and epigenetically activates the ink4a/arf-locus, forcing schwann cells towards replicative senescence. remarkably, loss of function of this mechanism allows unrestricted proliferation, inducing malignant transformation of neurofibromas. interestingly, our data suggest that in injured nerves, schwann cells epigenetically activate the same locus and go as well into the senescence program. indeed, when this pathway is genetically blocked, cell proliferation results increased after nerve injury. we postulate that the ink4a/arf-locus is expressed as part of a physiological response that prevents uncontrolled proliferation of the de-differentiated schwann cells generated during nerve regeneration, a response that is also activated to avoid overproliferation after tumorigenic stimuli in the peripheral nervous system. university of rochester medical center, rochester, united states synaptic plasticity is critical for normal neurodevelopment and proper circuit function in the adult nervous system. recent evidence suggests that microglia, classically studied in neuroinflammation, play critical roles in neurodevelopment and synaptic plasticity. however, the mechanisms driving these roles are not well understood. to start elucidating the molecular players involved in microglial communication with neurons during plasticity, we decided to first explore the role of purinergic signaling in this process. while purinergic signaling has been implicated in microglial behaviors, studies have primarily focused on neuroinflammatory roles. however, noninflamed microglia highly express the purinergic receptor, p2y12, which has known functions in microglial chemotaxis in early inflammation and can stimulate the release of cytokines implicated in synaptic plasticity. thus, we posited that purinergic signaling could contribute to the microglial motility that underlies synapse surveillance in the non-inflamed brain. this in turn may be critical for microglial roles in synaptic refinement during development. to explore this possibility, we took advantage of the p2y12 knock-out (ko) mouse and examined the morphology and motility of microglia in the visual cortex as compared to microglia in wildtype mice. first, we used immunohistochemistry for the microglia specific ionized calcium-binding adapter (iba-1) protein to stain microglia in fixed sections. we then used confocal imaging and scholl analysis to investigate the complexity of the microglial processes. we also used 2-photon microscopy to monitor microglial motility in p2y12 ko mice by crossing them with cx3cr1-gfp knock-in mice that allow visualization of microglia including fine processes in vivo. our preliminary evidence suggests that p2y12 disruption alters basal microglial morphology and behavior making microglia less complex and altering their motility patterns. we are now investigating whether microglia-synapse interactions, as well as functional plasticity elicited by visual experience are altered in p2y12 ko mice. our studies will expand our understanding of emerging roles for microglia in neurodevelopment and will pioneer new non-pathological roles for microglial purinergic signaling. a. dvorzhak, a. wojtowicz, r. grantyn charit e -university medicine, berlin, germany in neurodegenerative diseases, the afflicted brain is both an important object of study and an opportunity to characterize a given cellular interaction from a pathophysiological perspective. this dual approach is particularly advantageous when human disease is based on a monogenetic defect and an appropriate animal model becomes available for detailed investigation, as in case of z_q175_ki (q175), a new knock-in mouse expressing a mutant form of murine huntingtin. electrophysiological recordings of gabaergic unitary ipscs from striatal output neurons (sons) in sagittal slices from wild-type and homozygous q175 challenged the current viewpoint that gabaergic transmission is enhanced in the hd striatum. quantal analysis in combination with high frequency stimulation and paired pulse tests revealed that synaptic gaba release is in fact tonically suppressed resulting in disinhibition of striatal output activity (dvorzhak et al j physiol 2013). the underlying mechanism involves a retrograde endocannabinoid signalling pathway linking postsynaptic mglur5 with presynaptic cb1 and gaba release. in addition to this deficit, z-q175_ki homozygotes exhibited a significant reduction of tonic inhibition via extrasynaptic gaba(a) receptors. using pharmacological tools to alter the ratio of ambient glutamate and gaba concentrations made clear that the hd-related depression of synaptic and extrasynaptic gabaergic actions depends on the state of both the astrocytic glutamate transporter glt-1 and the gaba transporter gat-3. in support of h eja and colleagues, our imaging experiments suggest that in normal striatal astrocytes gat-3 is in a releasing mode and driven by the activity of glt-1. however, the latter activity is much lower in the hd striatum. sbfi recordings of glt-1-related na transients and patch clamp recordings of glt-1 transporter currents revealed a marked reduction (less than 50% of wt level) in sr-labelled astrocytes from symptomatic hd mice. together, our results suggest that the deficiency of astrocytic glutamate uptake might represent a pathophysiological key mechanism underlying the observed disinhibition in the striatum of mice affected by huntington's disease. nodes of ranvier are highly enriched in voltage-gated sodium channels (nav), allowing rapid action potential propagation on myelinated axons. cell adhesion molecules (neurofascin-186 (nfasc186) and contactin) and scaffold proteins (ankyring and bivspectrin), which are also enriched at the nodes, have a critical role in assembly and/or stabilization of na v clusters. oligodendrocytes have been described to induce nav channel clustering at nodes in the central nervous system (cns); however, the nature of the signal(s) involved in nodal formation remains mostly unknown in the cns. to gain insight into cns node assembly, we developed hippocampal neuronal cultures from e18 rat embryos. during the first week, as expected, na v channel accumulation was detected at the axon initial segment (ais), co-localized with ankyring and nfasc186, whereas expression along the axon was diffuse and hardly visible. in contrast, at 14 days in vitro (div), regularly spaced clusters of na v channels, ankyring and nfasc186 were detected along the axon. the percentage of hippocampal axons with clusters increased from 4.1 6 3.1% at 14 div, to 15.8 6 3.5% and 17.167.1% at 17 and 21 div, respectively (mean 6 sd). importantly, these clusters were formed in the absence of myelin and no accumulation of the paranodal protein caspr was detected at this stage. to study the role of extrinsic versus intrinsic pro-aggregating factors, we analyzed nearly pure hippocampal neuronal cultures. these purified neurons still formed ais, while only few axons formed na v clusters. interestingly, when these purified neurons were co-cultured with oligodendrocytes or with conditioned medium from oligodendrocytes, the percentage of neurons with nascent nodes was greatly enhanced. taken together, these results confirm that some glial soluble factor(s) might be involved in na v channels clustering, as previously shown for retinal neurons (kaplan et al., 2001). furthermore, nascent nodes were detected on gabaergic neurons, but not on glutamatergic hippocampal neurons, thus arguing also for the role of intrinsic neuronal factors in their establishment. using hippocampal sections from gad67-gfp mice, we showed that some gabaergic interneurons were myelinated in vivo. moreover, we observed that nascent node formation prior to myelination also takes place in vivo, strengthening physiological relevance. the electrophysiological properties of neurons with nascent nodes will be studied. microglia in the degenerating or aging brain are primed by aspects of pathology to produce exaggerated pro-inflammatory responses to subsequent central and systemic inflammatory insults. however, the mechanisms by which microglia become primed, rather than adopting an m1 phenotype, are not clear. triggers for priming may include altered expression of complement, recognition of amyloid or neuronal/synaptic debris for phagocytosis, decreased engagement of microglial regulatory molecules such as cd200r, trem2 or fractalkine receptor. there is also evidence that loss of basal neurotransmitter tone may contribute to this state. microglia are known to express the nicotinic cholinergic a 7 receptor, which has been shown to influence macrophage and microglial reactivity. in the current study we performed limited lesions of the basal forebrain cholinergic system using the murine-p75-saporin immunotoxin (mu-p75-sap), to investigate the degree to which loss of cholinergic tone predisposes the microglia to subsequent inflammatory stimulation and to assess the consequences of this for cognitive function in the vulnerable brain. intracerebroventricular injection of mu-p75-sap (0.08 lg) depleted cholinergic neurons in the basal forebrain and decreased cholinergic innervation of the hippocampus, but left performance on hippocampal-dependent reference and working memory tasks relatively intact. however, decreased cholinergic innervation of the hippocampus conferred increased susceptibility to cognitive deficits induced by systemic lps (100 lg/kg) 40 days after lesioning, but microglia were not primed 40 days after 20% lesions. to investigate the regional, temporal and neurochemical basis of this we induced more severe lesions of the basal forebrain (using 1.2 lg mu-p-75 sap) and assessed microglial priming at 14 or 40 days post-lesion. these data demonstrate that microglia are primed by low dose challenge at 14 but not 40 days and, when more robust lesions are induced, priming remains even at 40 days. these responses are observed at the site of injury (medial septum) but more profoundly at the site of denervation, the hippocampus. indicating that neuronal injury and, particularly, loss of cholinergic tone have a profound effect on the reactivity of the microglia to subsequent inflammatory challenge. these data expand our knowledge of microglial priming and have clear implications for the interaction of cholinergic tone and inflammation in cognitive dysfunction such as dementia and delirium in which cholinergic dysfunction is implicated. ). however, glucose may also be metabolized to glycogen or oxidized to co 2 , so it is not obvious that the stimulation of glut1 and hexokinase leads to lactate release. the objective of this work was to investigate whether lactate may be released by cultured mouse astrocytes within the time frame of the interstitial lactate rise observed in vivo. a first approach with an enzymatic assay showed a significant increase in extracellular lactate after 1 minute of exposure to 12 mm k 1 or 50 lm glutamate. to obtain better time resolution we developed a "lactate sniffer", a hek293 cell that expresses the fret lactate nanosensor laconic (san mart ın et al., plos one 2013). seeded on top of an astrocytic monolayer, the sniffer cell responded within 10 seconds of exposure to elevated k 1 12 mm and a similar response was elicited by 3 mm ba 21 . the response to k 1 and ba 21 supports a role for the na 1 /bicarbonate co-transporter nbce1, previously shown to mediate the stimulation of astrocytic glycolysis (ruminot et al., j. neurosci., 2011). the sniffer cell detected an equally fast increase in extracellular lactate when the culture was exposed to 50 lm glutamate. previous work had shown that glutamate does not stimulate glucose consumption in the short-term (bittner et al., j. neurosci. 2011), and therefore the rapid release of lactate came as a surprise. this phenomenon may relate to glycogen mobilization or perhaps to inhibition of astrocytic oxygen consumption (azarias et al., j. neurosci. 2011). k 1 , ba 21 and glutamate did not produce detectable effects on the sniffer cell in the absence of astrocytes. the rapid release of lactate by astrocytes exposed to k 1 and glutamate supports a role for these cells in the fast increase in interstitial lactate concentration that accompanies synaptic activity. ] i ) are regularly occurring as a result of uptake of glutamate from the synaptic space. glutamate uptake occurs via the glutamate/na 1 co-transporters glast and glt-1, where one glutamate is accompanied by 3 na 1 and 1 h 1 in exchange for 1 k 1 . the salt pump, na,k-atpase (nka), is responsible for the maintenance of the trans-membrane na 1 gradient by exporting 3 na 1 and importing 2 k 1 for each atp. nka is dose-dependently inhibited by ouabain. astrocytes express two isoforms of the catalytic nka a subunit, the ubiquitous a1 and in the cns the glia-specific a2. the isoforms differ with regard to na 1 affinity, which is lower for a2 and with regard to ouabain affinity, which is higher for a2 than for a1. although a2 mutations give rise to neurological symptoms -familial hemiplegic migraine type 2, the relative roles of the a1 and a2 isoforms in astrocytes are still incompletely understood. we hypothesized that the low na affinity of a2 makes it more suitable to cope with transient large increases in na i . to test this, we compared the effect of 200 mm glutamate for 10 min on real time changes of [na 1 ] i in primary astrocytes, which, following transient transfection, predominantly expressed either the a1 or the a2 isoform. in a1 cells glutamate caused a significantly larger increase in [na 1 ] i and longer recovery time to base line [na 1 ] i than in cells expressing a2. glutamate uptake dependence on either a isoform was determined by aspartate uptake in astrocytes expressing endogenous a1 and a2. in the presence of ouabain concentrations that selectively inhibit a2, we found a modest (1.9mm) increase in [na 1 ] i , accompanied by a disproportionately large decrease (24%) in glutamate uptake. it has been suggested that astrocyte glutamate transporters are clustered in microdomains where they may interact with nka. in gst pull down assays on rat brain we found that both glt-1 and glast interacted with the 1st intracellular loop of both a1 and a2, but the interaction was significantly stronger for the a2 isoform. no interaction was found with other segments of the a molecule. the data indicate that the nka a2 isoform, which has a more restricted expression than a1, plays a specific role for the interaction with the glutamate transporters and for sodium homeostasis in astrocytes. this specificity may be attributed to low sodium affinity of a2 and to the relatively high capacity of a2 to interact with the astrocyte glutamate transporters. ozone, a major component of air pollution, has considerable impact on public health. besides its well described inflammatory and dysfunction effects on the respiratory tract, there is accumulating evidence indicating that ozone exposure also affects brain functions. however, the mechanisms through which ozone exerts toxic effects on the cns remain poorly understood. we previously showed that in addition to lung inflammation,ozone exposure caused a neuronal activation in the dorsolateral regions of the rat nucleus tractus solitarius (nts, a sensory nucleus involved in visceral information processing) overlapping terminal fields of lung primary afferent running in the vagus nerves to investigate this hypothesis, we used electron microscopy and immunoblot techniques. in ozone-exposed animals, the astrocytic coverage of nts glutamatergic synapses is increased while the astrocyte volume fraction and the astrocyte membrane densities are not significantly increased. moreover, the expression of specific astrocyte markers gfap, s100beta and ezrin did not change in ozone-exposed animals. altogether, our results indicate that ozone inhalation induces glial plasticity, which is restricted to the peri-synaptic coverage. . we recorded pp-gc synaptic activity after eae induction via adoptive transfert (at-eae) and found that mepsc frequency was increased compared to control animals. moreover synaptic alteration in at-eae mice depended on astrocytic tnfr1 because the effect was absent in tnfr1-/-mice but reappeared upon tnfr1 re-expression in astrocytes. nr2b receptors are also necessary because in vivo ifenprodil injection prevented synaptic alteration. overall, our study reveals a specific mechanism responsible for hippocampal synaptic dysfunction possibly relevant for cognitive impairment in ms. research supported by snsf grant 31003a-140999 and nccr "synapsy" to av. we patched ng2 cells in the hippocampal ca1 region of ng2-dsred mice (8-20 days old) and investigated in current clamp mode how sodium and potassium channels affected synaptic depolarizations. epsps and ipsps were evoked from a resting membrane potential of 285mv by injecting mock epsc or ipsc waveforms derived from miniature currents. epsps which depolarized ng2 cells to more than 245 mv were increasingly shortened by vgcs. at 216 6 1 mv, corresponding to a quantal content of 640, the half width of epsps was shortened to 47 6 3% while the amplitude was hardly altered. application of ttx, 4-ap and tea showed that most of the observed shaping of epsps was primarily due to activation of a-type k1 current rectifying the depolarizing input. na1 currents only slightly ($10%) amplified epsp input, but this effect was outcompeted by the dampening effect contributed by a-type current ($15%). tea was almost without effect on epsps. due to their slower time course ipsps were very differently shaped by vgcs: while recruitment of vgcs by mock ipsps started at a similar threshold voltage, the maximal suppression of ipsp amplitude was much stronger. when depolarizing ng2 cells to 235 6 1 mv, corresponding to a quantal content of 235, ipsp amplitudes were suppressed to 52 6 4% and the duration of ipsps was not shortened but increased to 193 6 5%. a-type k1 current was the primary contributor for rectifying as well. interestingly, if a-type k1 current was blocked, ipsps with a quantal content of 105 unmasked a small ttx-sensitive spikelike depolarization of 20 6 2 mv (duration: 7 6 1 ms). for comparison, while synaptic responses to simultaneous release of 100 glutamatergic vesicles were hardly altered by vgcs, the synaptic response of 100 gabaergic vesicles was substantially low-pass filtered by a-type potassium currents. altogether, our results suggest that vgcs in ng2 cells normalize the synaptic strength of excitatory and inhibitory inputs and support their differentiation by ng2 cells through further emphasizing their pre-existing kinetic differences. to explore the involvement of this pathway in pain and analyze its impact separately in sensory neurons and sgcs, we tested pharmacological inhibitors and transgenic mice in an orofacial pain model. transient inflammation in the submandibular region of mice was induced using complete freund's adjuvant (cfa) and tactile sensitivity was quantified using von frey filaments. although inflammation was resolved by 28 days after injection, tactile hypersensitivity persisted in wildtype mice for at least 53 days, consistent with chronic post-inflammatory pain. tactile hypersensitivity in mice was reversed by systemic injection of the panx1/gap junction blockers mefloquine or carbenoxolone at 7 days (peak inflammation) and at 28 days after cfa-injection, suggesting the contribution of these channels to tactile hypersensitivity. in dissociated trigeminal ganglia (tg), which innervate the submandibular skin, bzatp-induced yopro uptake into neurons and glia was prevented by mefloquine, demonstrating the functional presence of the p2x 7 r-panx1 complex in tg. moreover, tg of cfa-injected mice showed more atp release and immunostaining of tg revealed higher expression of panx1 at 1 week after cfa injection compared to controls. development of hypersensitivity was prevented in p2x 7 r-null and in panx1-null mice, and was attenuated in mice with glia-specific deletion of panx1 (gfapcre-panx1f/f) but not with neuron-specific deletion (mnfhcre-panx1f/f), emphasizing the importance of glial panx1 signaling in pain. because p2x 7 r and panx1 both have a key role in the immune response by activating the inflammasome, we are currently dissecting the relative importance of neuron-glial communication compared to inflammatory responses in the development and maintenance of orofacial pain. overall, our results show that the p2x 7 r-panx1 complex likely plays a major role in signaling events contributing to tactile hypersensitivity. synaptic plasticity is central to the process of learning and memory and is accompanied by strengthening of existing synapses and/or formation of new synapses. numerous studies have investigated the molecular mechanisms of learning and memory with neurons as the primary interest. given the tight coupling between synaptic activity and brain metabolism, it seems reasonable to assume that synaptic plasticity changes occurring at the synaptic level may also occur at the metabolic level in order to keep up with the augmented demand at the synapse. in this study we investigated the importance of genes involved in brain energy metabolism, and in particular those involved in neuronglia metabolic coupling during fear-motivated inhibitory avoidance learning. using ( 14 c) 2-deoxyglucose (2-dg) technique we first defined the brain areas that are involved in this learning process. context-dependent avoidance behavior was tested in c57bl/6 mice using the step-through inhibitory avoidance paradigm (ia). regional brain metabolic activity, as measured by the 2dg uptake, was quantified in several brain regions. brain metabolic mapping revealed increased glucose utilization in hippocampus, amygdala [(basolateral complex (bla) and the central nucleus (cea)], and anterior cingulate cortex and mammillary bodies. quantitative mrna levels were assessed in dorsal hippocampal tissue at different time points following ia learning. results obtained demonstrate that learning modulates the expression pattern of genes involved in brain energy metabolism, i.e. glycogen synthesis and degradation, pyruvate metabolism, the pentose phosphate shunt and the astrocyte-neuron lactate shuttle (anls) in a time dependent manner. we found a late-phase of enhanced dorsal hippocampal gene expression, particularly for anls related genes, including monocarboxylate transporter 1 (mct1). furthermore, we found that mice deficient in mct1 transporter display impaired long term memory in the inhibitory avoidance and spatial learning in morris water maze. these observations indicate that metabolic adaptation shown by neuron-glia metabolic coupling might be a relevant phenomenon following learning in conjunction with synaptic plasticity. in order to systematically as well as functionally analyse rmg reactivity to retinal degeneration and thus explore the rmg-derived signalling molecules in depth, we aim at comprehensively profiling proteomewide cellular responses to stimuli. we thus developed a proteomics approach screening specifically for cell surface and membrane proteins as well as secreted protein expression profiles and in addition monitor quantitative changes induced by prototype inflammatory inducer lipopolysaccharide lps. this workflow utilises sugar residues of cell surface proteins on intact rmg which are mildly oxidized and then covalently coupled to biotin. biotinylated proteins are affinity purified and glycosylated peptides are specifically released by pngasef followed by protein identification and quantification by label-free lc-msms. this workflow resulted in the identification of more than 500 proteins on cell surfaces complemented by more than 700 proteins identified in the rmg secretome. bioinformatic analysis allocates 75% of the cell surface proteins to be truly membrane or extracellular matrix proteins. this comprehensive cell surfaceome includes transmembrane receptors, transporters, adhesion molecules, signalling molecules and proteases, including 18 cd markers, 18 integrins, 41 solute carriers, two ephrins, five ephrin receptors and six plexins. treatment of cells with lps results in a highly reproducible significant shift of cell surface proteome with upregulation of 36 proteins and downregulation of 13 proteins. among those lps-induced cell surface expression changes are proteins that suggest an active role of these glial cells in inflammatory processes. the specific changes will be discussed in detail. cell surface biotinylation on glycosyl-residues in combination with label-free lc-msms is a sensitive and reproducible method to profile cell surface proteomics and sheds light on the biological properties of cells. background: neuronal activity alters calcium ion (ca 21 ) dynamics in astrocytes, but the physiologic relevance of these changes is controversial. to examine this issue further, we generated an inducible transgenic mouse model in which the expression of an inositol 1,4,5-trisphosphate absorbent, "ip 3 sponge", attenuates astrocytic ca 21 signaling. results: attenuated ca 21 activity correlated with reduced astrocytic coverage of asymmetric synapses in the hippocampal ca1 region in these animals. the decreased astrocytic 'protection' of the synapses facilitated glutamate 'spillover', which was reflected by prolonged glutamate transporter currents in stratum radiatum astrocytes and enhanced n-methyl-d-aspartate receptor currents in ca1 pyramidal neurons in response to burst stimulation. these mice also exhibited behavioral impairments in spatial reference memory and remote contextual fear memory, in which hippocampal circuits are involved. conclusions: our findings suggest that ip 3 -mediated astrocytic ca 21 signaling correlates with the formation of functional tripartite synapses in the hippocampus. they are the only non-neuronal cell type in the brain that receives synaptic input from neurons. ng2-cells exhibit a variety of ca 21 -signalling pathways, which might be triggered by pre-synaptic neuronal activity. however, no global increase in the intracellular ca 21 -concentration ([ca 21 ] i ) was detected in ng2-cells employing the minimal stimulation technique at neuron-glial synapses. therefore we assume that post-synaptic ca 21 -microdomains might be activated under physiological conditions. these ca 21 -signals are locally restricted to the plasma membrane. membrane bound ca 21 -sensors with a high signal-to-noise ratio, such as lck-gcamp3, are best suited for optimal visualisation of ca 21 -microdomains. here, we set out to express lck-gcamp3 in ng2cells together with the cytosolic reporter dye dsred. we generated transgenic mice which express a bidirectional promoter encoding both proteins under the control of a tet-off system. this strategy allows an exclusive expression of the transgenes only in the presence of a tet-responsive transcriptional activator (tta). the construct was successfully cloned and expressed in hek293 cells. as expected, lck-gcamp3 was located only at the inner plasma membrane while dsred was expressed all-over the cytosol. functionality of the ca 21 -sensor was tested in vitro. elevation of [ca 21 ] i reliably increased the fluorescence intensity of lck-gcamp3. after zygote injection seven positive founder animals could be identified and will be crossbred with a ng2-tta mouse line. taken together, the presented construct appears suitable for functional imaging of ca 21 -microdomains in post-synaptic ng2-cells. according to the "lactate shuttle" hypothesis, glycogen stored in astrocytes can be converted to l-lactate and exported to neurones as preferred energy substrate. we use optogenetics to investigate signalling between astrocytes and noradrenergic (naergic) neurones of the locus coeruleus (lc). to selectively activate g s -protein-mediated signalling in astrocytes, we generated a viral vector to express a chimera of rhodopsin and b 2 -adrenoceptor (optob 2 ar). we confirmed that this construct activates camp-mediated signalling in astrocytes. in cultured astrocytes, excitation of optob2ar resulted in acidification as detected using snarf-5 indicator, and this acidification could be blocked by pre-incubation with an inhibitor of glycogen breakdown 1,4-dideoxy-1,4-imino-d-arabinitol (dab), suggesting that the acidification was due to build-up of l-lactate. fast scan cyclic voltammetry (fcv) was used to measure noradrenaline (na) release in organotypic slices cut at the level of the lc in which astrocytes were expressing optob 2 ar. light stimulation of astrocytes powerfully induced release of na which could be prevented by pre-incubation with dab or application of d-lactate. bath application of l-lactate (!100 mm) in absence of optogenetic stimulation triggered the release of na. in patch clamp experiments, l-lactate depolarised lc neurones and evoked vigorous firing of action potentials. these experiments suggest that activation of g s -protein-coupled receptors in astrocytes could potentiate the release of na from lc neurones via l-lactate. the cellular and molecular mechanisms of this signalling mechanism are currently under investigation. supported by the british heart foundation. rgcs. in addition to this, the organization of astrocytes in the retina of a rat model of glaucoma was found to be significantly different to those of a healthy retina. these alterations in glial cell morphology may reflect changes in their relationship with rgcs and could signify profound changes in their secretome, which may influence rgc survival. in this study we investigated labeling of astrocytes by sr101 in acute slices from the ventro-lateral medulla and the hippocampus of transgenic mice expressing egfp under the control of an astrocyte-specific promoter. while sr101 efficiently labeled egfp-expressing astrocytes in hippocampus, we found that sr101-staining was very weak in the ventro-lateral medulla and rather unspecific. thus, sr101 is not a reliable marker for brainstem astrocytes. although carbenoxolone decreased the labeling of astrocytes in the hippocampus significantly, mefloquine which blocks pannexin and connexin hemichannels, was unable to prevent sr101 uptake in hippocampal astrocytes. time-lapse 2-photon imaging revealed that in hippocampus both astrocytes and neurons showed temporary sr101-loading. in astrocytes the rise of sr101 fluorescence was slower as compared to egfp-negative cells and sr101 was quickly removed from non-astrocytic cells during washout, while it was retained in astrocytes. in brainstem astrocytes, however, only a very weak and transient sr101-labeling was observed. to test if sr101 is actively removed from astrocytes in the brainstem, we applied mk-571 to block the multi-drug resistance transporters mrp-1. we did not observe any increase of sr101-labeling in brainstem astrocytes. in contrast, astrocytic sr101 labeling was significantly reduced by substrates of organic anion transport, probenecid, estron-3sulfate and dehydroepiandrosterone sulfate suggesting that sr101 is actively transported into hippocampal astrocytes by an organic anion transporting polypeptide (oatp). additionally, the data suggest that astrocytes modulate extracellular concentration of neurosteroids in the hippocampus. as an alternative approach we have used scanning electron microscopy (sem) to obtain high-resolution back scattered images (bsi) of serial ultrathin sections over a wide-area. in tem the size of the specimen is limited to less than 1 mm 2 , whereas in sem it can be expanded to 20-30 mm 2 , allowing us to mount and examine more than 100 serial ultrathin sections on the same stage, collecting 3d information about many nodal structures simultaneously. here we demonstrate a variety of nodal architectures assembled from bsi-sem (su8010,hitachi). in our preliminary study using this technique, the pattern of cellular processes approaching the perinodal axolemma is quite different among axons even in only four nodes examined in optic nerve. in the present study, we analyzed perinodal elements at 25 whole nodes in rat optic nerves and reconfirmed this variety of perinodal glial elements include the followings: 1) perinodal space with extracellular matrix (20-100%); 2) closely abutting astrocytic processes (0-80%) ; 3) unidentified glial cell, presumable ng21 glial processes (0-50%), but could not find 4) pre-synapse-like pseudo-neuronal processes, which was observed at the perinode in the cns grey matter (cerebral cortex) in the previous study. additionally, we found unexpected structures, small teardrop-like protrusion(s) of axolemma at 18 out of 25 nodes examined (72%). in 16 nodes out of these 18 nodes (88.9%), glial elements encircled or densely contacted with the teardrop protrusions. a typical case of 3d reconstructed image is shown in the figure. nodal axon (ax, red), unidentified glial process (yellow; ug, presumable ng2 cell) and teardrop-like protrusion (asterisks) are demonstrated. outer part of the protrusion from the position indicated two arrows is encircled by the process of ug cell. based on these observations, we propose that some glial cells, including astrocytes and/or ng2 cells, might contribute to remove wasted membranous elements from the axon at the node of ranvier, which is a new type of neuron-glial interaction. to understand what makes this ssc network more synchronous and excitable, we performed a morphological study of neurons and astrocytes by estimating densities of neun and s100-positive cells respectively. although we found similar densities of neurons and astrocytes between gaers and non-epileptic controls, the thickness of the ssc was 30% smaller in gaers. western blotting quantification of gfap, another astrocytic marker, was significantly higher at p15-p17, as compared with non-epileptic control. moreover, gfap-immunolabeling suggested that these astrocytes were reactive. we hypothesized that these modifications are linked with an alteration of astrocytic and/or neuronal physiological calcium excitability that could lead to the occurrence of epileptic seizures. to better evaluate the role of astrocytes and neurons networks during the development of ssc between p15 and p25, we used two-photon microscopy imaging coupled with eeg recording in animal under anesthesia and neuroleptanalgesia. we first analyzed neuropile, representing ascendant projection of deeper cortical layers, astrocytes and neurons calcium activities. preliminary results obtained from animals under isoflurane anesthesia showed similar neuropile activities. astrocytic and neuronal activities were too weak to highlight differences between gaers and non-epileptic control. this is likely due to the mode of anesthesia known to strongly decrease astrocyte calcium signaling and neuronal synchronization. current experiments are therefore performed using neuroleptanalgesia know to allow the occurrence of swd. the expected data should lead to a better understanding of neuron-astrocyte interactions during brain development and the mechanism underlying epileptogenesis in a genetic model of epilepsy. purpose: to study the effects of laser-induced ocular hypertension (oht) in the astrocytes of contralateral eyes two weeks after lasering. methods: adult swiss mice were divided into two groups: na€ ıve (n 5 6) and lasered (n 5 6). retinal whole-mounts were immunostained with antibodies against gfap. the gfap-labelled retinal area (gfap-ra) and the number of astrocytes were quantified. results: in comparison with na€ ıve: i) astrocytes were more robust in contralateral eyes. in oht-eyes, the astrocyte population was not homogeneous, given that astrocytes displaying only primary processes coexisted with astrocytes in which primary and secondary processes could be recognized, the former having less intense gfap-ir (p<0.001). the mean percentage of astrocytes in which only primary processes could be detected was 37.8%; ii) gfap-ra was increased in contralateral (p<0.05) and decreased in oht-eyes (p <0.001); iii) the mean intensity of gfap-ir was higher in oht-eyes (p<0.01), and the percentage of the retinal area occupied by gfap1 cells with higher intensity levels was increased in contralateral (p 5 0.05) and in oht-eyes (p<0.01); iv) the astrocyte number did not differ significantly among the eyes analyzed. however, in oht-eyes the number of astrocytes in which primary and secondary processes could be observed were decreased (p<0.01). conclusions: two weeks of laser-induced oht caused changes in the gfap-labelled retinal area but not in astrocyte number in both, contralateral and oht-eyes. on the basis of the astroglial changes detected in the present work, the use of the contralateral eye as an internal control in experimental model unilateral oht should be reconsidered. astrocytes show a high level of functional and morphological heterogeneity and are involved in many aspects of neural function, e.g., formation of the blood-brain barrier, regulation of ion homeostasis, synaptogenesis, and synaptic plasticity. despite the diversity of this cell type, the various astrocyte subpopulations are not well characterized, which is mainly due to the lack of markers that would allow for classification of astrocyte subsets. our study aimed at phenotyping of astrocyte subpopulations based on the expression of cell surface markers, which are associated with certain functions. we used two novel monoclonal antibodies, acsa-1 and acsa-2 (acsa: astrocyte cell surface antigen), directed against extracellular epitopes of astrocyte-specific cell surface markers to identify astrocyte subtypes. the acsa-1 antibody was generated by immunization of glast1 knockout mice and specifically detects the astrocyte-specific l-glutamate/l-aspartate transporter glast (eaat1, slc1a3), whereas the acsa-2 antibody results from an immunization of rats with astrocytes isolated from gfap-egfp transgenic mice. a mass spectrometric approach for the ligand-based identification of the acsa-2 antigen pointed to a number of candidates, which have to be validated by further experiments. the antibodies acsa-1 and acsa-2 were carefully analyzed by costaining experiments with commonly used intracellular astrocyte markers. we found that both antibodies specifically detect astrocytes in the developing and adult central nervous system. in contrast to antibodies against intracellular markers, such as gfap, s100ß, or glutamine synthetase, acsa-1 and acsa-2 can be used to detect and isolate living astrocytes, which enables further analysis and culture of the separated cells. flow cytometric analysis of acsa-1 and acsa-2 antigen expression on cells from different brain regions, as well as immunohistochemical analysis, revealed differences in the expression patterns. this allowed us to define different astrocyte subtypes especially in the cerebellum and olfactory bulb of the neonatal mouse brain. future work will focus on the identification of additional astrocytespecific cell surface proteins for a comprehensive classification of astrocyte subtypes based on cell surface marker expression or expression patterns. the reciprocal communication between neuronal and glial cells represents the key component of the immunosurveillance system of the brain. it has been proposed that the neuro-glial interaction is impaired in human alzheimer's disease. in this study we focus on neuronal "on" and "off" signalling molecules in the transgenic rat model for alzheimer's disease. using enzyme-linked immunosorbent assay we determined the level of cd47, cx3cl1 ("off" signalling molecules) and mmp3, trem2 ("on" signalling molecules) in brain homogenates. our results demonstrated significantly up-regulated level of cd47 (p < 0,001) in our ad rat transgenic animal model. on the other hand, quantification of mmp3 level revealed significant decrease of mmp3 expression (p < 0,001) in tested transgenic animals. previous studies showed that cd47 and mmp3 are predominantly expressed on neuronal cells in cns where cd47 functions normally as a marker of "self" to protect intact body component or "don't eat me" molecule which protect cd47-expresing cells from phagocytosis. our results indicate that overexpression of cd47 on neuronal cells expressing pathologically modified truncated tau protein can be used as a neuroprotective mechanism potentiating spread and accumulation of pathological modified tau protein in neurons affected by ad pathology which in final stage support progression of the developing disease. in conclusion, we showed, that pathologically modified tau protein can modulate specific "on and off" signalling molecules and thus affects neuron-glia interaction in ad. field-excitatory post-synaptic potentials (fepsp) were recorded from the ca1 area of hippocampal slices prepared from wistar rats (3-5 weeks old) as before (fontinha et al., 2008) . after obtaining a stable value of the fepsp slope for at least 30 min, ltp was induced by delivery of 3 trains of 3 pulses at 100hz, each train being separated by 200 ms. ltp magnitude (% increase of fepsp) was evaluated 50-60 min after induction. ltp magnitude in control conditions was 16 6 5.9%, whereas in a second independent pathway of the same slices but in the presence of bdnf (20ng/ml) it was 39 6 4.7% (n 5 7; p <0.05). ltp was completely abolished when hippocampal slices were superfused with the gliotoxin fluorocitrate (fc, 200 mm), which selectively reduces the astrocytic metabolism decreasing intracellular ca 21 signalling and the release of gliotransmitters. interestingly, in presence of fc, the facilitatory action bdnf (20 ng/ml) upon ltp was completely prevented. noteworthy, when fc (200 mm) treated slices were superfused with the selective adenosine a 2a receptor agonist, cgs21680 (30nm), the facilitatory action of bdnf (20 ng/ml) upon ltp was rescued (n 5 5; p < 0.05), so that dltp caused by bdnf (ltp in the presence of bdnf -ltp in the absence of bdnf in two independent pathways of the same slices) in fc 1 cgs 21680 treated slices (19%) was similar to that observed in control slices (23% in the absence of fc and cgs 21680). the results suggest that the facilitatory action of bdnf upon ltp is controlled by astrocytes, and that this role of astrocytes results from their contribution to the extracellular accumulation of adenosine allowing a 2a receptor activation to gate plasticity actions of bdnf. several studies demonstrated the ability of astrocytes to sense, respond to and regulate neuronal function. among the many functions of glial proteins, glutamate (glu) and gaba transporters play important roles in balancing excitatory and inhibitory signals in the brain. here we show that astrocytes regulate the tonic inhibition of neurons by the concerted action of glu and gaba transporters, thereby protecting both neurons and glial cells from overactivation. we demonstrate that the uptake of glutamate triggers the reverse function of glial gat-2/3 transporters by elevating the intracellular na 1 concentration in astrocytes. the released gaba significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. we also describe the source of the releasing gaba that is synthesized by an alternative pathway from polyamines. moreover, in the low-[mg 21 ] model of epilepsy, we show that blockade of the glial glu/gaba exchange mechanism increases the duration of seizure-like events and also results in increased activity of astrocytes, demonstrating the neuroprotective impact of the mechanism. finally, we show that the released glial gaba modulates the power of gamma range oscillation in vivo, suggesting that the glu/gaba exchange mechanism is also functioning in the intact hippocampus under physiological conditions. revealing this novel molecular mechanism by which astrocytes provide an adjustable, in situ negative feedback on the excitability of neurons is expected to broaden our understanding about the regulation of neuronal activity by astrocytes and may open up new targets for the treatments of pathological conditions, such as epilepsy or ischemia. chronic stress is well recognised to decrease the number of gfap1 astrocytes within the prefrontal cortex (pfc). recent research, however, has suggested that our understanding of how stress alters astrocytes may be incomplete. specifically, chronic stress has been shown to induce a unique form of microglial remodelling, but it is not yet clear whether astrocytes also undergo similar structural modifications. such alterations may be significant given the role of astrocytes in modulating synaptic function. accordingly, in the current study we have examined changes in astrocyte morphology following exposure to chronic stress in adult rats, using three-dimensional digital reconstructions of astrocytes. our analysis indicated that chronic stress produced profound atrophy of astrocyte process length, branching and volume. we additionally examined changes in astrocyte-specific s100b, which is both a putative astrocyte marker, and a protein whose expression is associated with astrocyte distress. while we found that s100b levels were increased by stress, this increase was not correlated with atrophy. we further established that while chronic stress was associated with a decrease in astrocyte numbers when gfap labelling was used as a marker, we could find no evidence of a decrease in the total number of cells, based on nissl staining, or in the number of s100b1 cells. this finding suggests that chronic stress may not actually reduce astrocyte numbers and may instead selectively decrease gfap expression. together, these results provide a significantly more elaborate picture of how chronic stress alters the pfc. the drosophila nervous system is ensheathed by several types of glial cells, one of which, namely the subperineurial glia, forms pleated septate junctions (psj) to build a tight blood-brain barrier (bbb). hence, nutrients from the surrounding hemolymph must cross these glial cells to reach the neurons. the amount of nutrients entering the nervous system is likely to be tightly controlled through neuron-glia communication to ensure optimal metabolic supply while avoiding disturbance of the extracellular homeostasis. we aim to elucidate signals involved in this regulatory interaction. to this end, an rnai-based screen in glia of adult flies is performed using the gal4/gal80 ts system. we analyze the intake of blue-dyed food by photometric measurement. since impairment of the metabolic supply of the brain should severely affect the organism, compensatory changes in feeding behavior are expected. for instance, knockdown of nutrient transporters specifically in glia would then induce excessive feeding in normally nurtured individuals. the first set of rnai lines screened includes genes that have previously been found to be essential in glia for survival or locomotion of the animal. furthermore it comprises metabolic enzymes, all carbohydrate transporters, neuropeptides and their corresponding receptors. candidate genes will be further characterized regarding their role in controlling the energy homeostasis of the nervous system. the discovery that activation of non-neuronal cns microglia plays a causal role in spinal processing of nociceptive signaling has shed new light on the processes underlying neuropathic pain facilitation. however, there remains much uncertainty as to the necessary contribution of microglia to enhanced pain states. we aim to define the particular role of microglia for the initiation of neuropathic pain and by answering this question also learn if the function of peripheral myeloid cells is distinct or redundant in this process. methods: to specifically investigate spinal microglia and peripheral macrophages in the pathogenesis of neuropathic pain, we model chronic pain by performing partial ligature of the sciatic nerve in cd11b-hsvtk mice engrafted with gfp bone marrow (gfp>cd11b-hsvtk). cd11b-hsvtk 1/mice allow the exchange with peripherally-derived, gfp 1 macrophages upon central depletion of endogenous cd11b 1 microglia. following this depletion/ repopulation paradigm, behavioral analyses of mechanical and thermal allodynia are conducted. results: we established a selective tool to exchange cns parenchymal microglia with peripheral gfp 1 myeloid cells. in chronic pain tests for mechanical and thermal hyperalgesia, gfp>cd11b-hsvtk 1/mice show considerable decreases in paw withdrawal thresholds in response to mechanical, but not thermal stimuli ipsilateral to the injury, indicating distinct roles of microglia and macrophages in the facilitation of thermal hyperalgesia. conclusions: we identified a differential contribution of resident spinal microglia vs. peripheral myeloid cells in the development of neuropathic pain in gfp>cd11b-hsvtk chimeras. future studies aim to examine the exact mechanisms underlying the distinction between these two populations. a deeper understanding of the processes involved in the compartmentation of brain energy metabolism is of major interest. not only to enwrap pathomechanisms of brain diseases associated with metabolic deficits but also for a more accurate interpretation of functional neuroimaging signals which often use metabolic signals as surrogate marker for brain activity (e.g. fdg pet). large amount of knowledge in the field has been obtained from small animal neuroimaging studies. but due to their invasiveness they often need anesthesia. anesthetics heavily alter physiological read-outs. the aim of this study was 1.) to test if metabolic imaging using two-photon microscopy (2pm) and genetically encoded glucose sensors is feasible in the awake, head-fixed mouse and 2.) to examine the effects of isoflurane anesthesia on the signals. in two mice, a recombinant adeno-associated virus as a carrier for the genetically encoded glucose sensor flii 12 pglu600md6 was injected into the whisker barrel cortex. by using an astrocyte-specific promoter (short gfap) astrocytic expression was ensured. in addition, a chronic window and a head post were implanted. behavioral training started a few days after surgery. animals had to learn to tolerate head fixation without spontaneous motor activity. within a single trial animals were trained not to move for 10 seconds (imaging period) to receive a water reward. animals were subjected to water restriction during the behavioral training. animals were trained for three weeks before 2pm imaging started. animals learned to tolerate head fixation up to 90 minutes allowing acquisition of up to 400 trials per session. upon oral glucose administration (per single water reward $1 mg glucose was administered) fret signal started to increase within 5 minutes and went up to a maximum of 8%. surprisingly, isoflurane anesthesia led to an even higher increase of the signal (mean increase of 12%) compared to the awake state. in some experiments the barrel cortex was activated by vibrotactile single whisker deflections generated by a piezo bending actuator. fret signal did not show a difference between baseline and stimulated condition (1.133 6 0.3 vs. 1.136 6 0.29). in conclusion, the presented experiments demonstrate for the first time the feasibility of awake 2pm imaging for metabolic measurements on a single cell level. the signal increase upon peroral glucose administration unambiguously supports the functionality of the sensor. isoflurane exhibits a large effect on intracellular astrocytic glucose concentration distinctly revealing the need for experiments in awake animals for unbiased results. future experiments will now be expanded to neuronal glucose sensors to enable comparisons between astrocytes and neurons. our previous studies indicate that neuronal electrical activity controls glial exosome release resulting in subsequent neuronal uptake and functional retrieval of the exosomal content. proteomic analysis of oligodendroglial exosomes revealed a list of candidates with potential neurotrophic action in neurons. to elucidate the impact of oligodendroglial exosomes on the neuronal metabolism, we analyzed the metabolic activity of neurons after co-culture with oligodendrocytes or direct treatment with exosomes. methods: neurons were subjected to different stress paradigms such as oxidative stress, hypoxia, and nutrient deprivation. neuronal vitality was assessed by mtt assay and the mitochondrial membrane potential was visualized by staining with mitocapture. results: neurons grown under optimal conditions were not affected by the presence of exosomes. intriguingly, when neurons were subjected to stress (oxidative stress, nutrient deprivation, oxygen-glucose deprivation) their metabolic activity was significantly increased in the presence of exosomes. when challenged with oxidative stress prior to exosome treatment, neurons were not able to recover. mitocapture staining demonstrated that oligodendroglial exosomes prevent the breakdown of the mitochondrial membrane in nutrient-deprived neurons. conclusions: our results indicate that exosome supply is protective for neurons but is unlikely to support their recovery. we suggest that oligodendroglial exosomes carry neuroprotective substances, which protect neurons from stress. interestingly, this effect of lactate on synaptic plasticity mechanisms was not fully mimicked by glucose, suggesting that the effect was not only related to supporting the potential increase in energy demands related to plasticity, but that l-lactate could act as a signaling molecule for the regulation of expression of plasticity-related genes. we have followed up on these observations and show that l-lactate significantly stimulates mrna expression of key immediate early genes (iegs), in a time-and concentration-dependent manner, in cultured neurons. following one hour of treatment with 20 mm l-lactate, arc, zif268 and c-fos mrna levels were increased by 5.2, 3.7 and 8.2 folds, respectively. in addition, the increased iegs mrna expression levels induced by l-lactate are correlated at the protein level with respectively 5.5, 4.0 and 3.2 fold increase compared to control values at the same time point. these effects are specific for llactate, since d-lactate (non-metabolized enantiomer of l-lactate), l-pyruvate and d-glucose (at equicaloric concentrations) have no effect on gene expression. interestingly, we observed that, in similar culture conditions, l-lactate-induced iegs expression is only observed in neurons but not in astrocytes, implying a cell specific effect. characterization of the underlying molecular mechanisms of l-lactate action on iegs expression demonstrate the involvement of nmda receptors. finally, we obtained evidence that such regulatory mechanisms of ieg expression also operate in vivo as direct intracortical injections of l-lactate (10 mm) into the somatosensory-motor cortex areas resulted, within 1 hour of application, in a significant stimulation of arc, zif268 and c-fos expression (by 61 6 13%, 46 6 8% and 60 6 12%, respectively) as compared to d-lactate (10 mm)-injected contralaterally areas. taken together these findings demonstrate that l-lactate acts as a direct signaling molecule which regulates neuronal plasticity-related iegs expression both in vitro and in vivo. interestingly, the underlying mechanism of action of l-lactate involves nmda receptors.this set of observations therefore reveals a novel signaling role of lactate which may represent a likely mechanism to account for the role of astrocyticderived l-lactate on ltm formation. neuronal activity in the brain is associated with a transient increase in the extracellular k 1 concentration. the excess k 1 is removed from the extracellular space, primarily by the surrounding glial cells, leading to an intracellular accumulation of k 1 via mechanisms not fully identified and/or quantified. post-stimulus recovery of [k 1 ] o has been proposed to be dependent on kir4.1-mediated spatial buffering and/or to na 1 /k 1 -atpase activity. to resolve the molecular mechanisms involved in k 1 clearance, we initially determined the contribution from the different k 1 -transporting mechanisms present in primary culture of rat astrocytes and their k 0.5 for k 1 . the na 1 /k 1 /2clcotransporter, nkcc1, increased its activity within a physiological concentration range of k 1 , while the na 1 / k 1 -atpase saturated at much lower concentrations. consequently, a concentration-dependent switch occurs between the two mechanisms of k 1 uptake in cultured astrocytes. in addition, nkcc1 was capable of mediating robust k 1 -induced astrocytic cell swelling. thus, nkcc1 could potentially act as a molecular mechanism responsible for clearance of the stimulus-evoked rise in [k 1 ] o and the associated shrinkage of the extracellular space. to determine the contribution of each of the three molecular mechanisms to k 1 -clearance in native brain tissue, we used rat hippocampal brain slices and employed ion-sensitive microelectrodes in association with high-frequency electrical stimulation as well as focal appliances of k 1 by ionophoresis. inhibition of kir4.1 (100 um bacl 2 ) or nkcc1 (10 um bumetanide) failed to show a significant effect on the rate of k 1 removal from the extracellular space. in contrast, inhibition of the a2 and a3 isoforms of the na 1 /k 1 -atpase significantly delayed post-stimulus recovery of [k 1 ] o and this delay was further potentiated by additional inhibition of the a1 isoform. the na 1 /k 1 atpase emerged as the primary factor responsible for stimulus-evoked k 1 -clearance with no evidence in favor of kir4.1 and nkcc1 involvement. further characterization of the different na 1 /k 1 -atpase isoforms, by heterologous expression in xenopus laevis oocytes, revealed that the a1 isoform reached its maximal activity already at resting k 1 concentrations, hinting at a "housekeeping" function. the a2 subunit displayed voltage-sensitivity and increased turnover rate along physiologically relevant increments in extracellular k 1 concentration. these a2-related features may render this astrocyte-specific subunit variant specifically geared for post-stimulus recovery of [ as a model of ischemic injury, and sham-operated mice were used as controls. we isolated gfp 1 cells from the dorsal part of the lv of sham-operated-and post-ischemic brains and employed a neurosphereforming assay in order to estimate their ability to proliferate and selfrenew. furthermore, we followed their immunocytochemical and electrophysiological properties during in vitro differentiation and compared the differentiation potential of gfp 1 cells isolated from the controls to those isolated from post-ischemic brains. the gfp 1 cells isolated from the dorsal part of the lv of controls formed neurospheres and differentiated only into a glial phenotype. the gfp 1 cells isolated from the dorsal part of the lv of post-ischemic brains were able to form neurospheres as well; however, besides their differentiation into a glial phenotype, they also gave rise to cells with the properties of neuronal precursors. we also performed in situ immunohistochemical/electrophysiological analyses of gfp 1 cells in the adult brain of controls and those after mcao. in situ analyses revealed that gfp 1 cells expressed the phenotype of adult nscs or neuroblasts in controls or following ischemia and that their number was increased in both hemispheres following mcao. compared to controls, we found that the number of gfp/doublecortin-positive cells significantly increased in the dorsal part of the lv as well as the number of gfp 1 cells in the olfactory bulb, where they probably differentiated into calretinin 1 interneurons. our results indicate that gfp 1 cells with an active mdach1 gene in the dorsal part of the lv play an important role in the increased production of neuroblasts after injury and that this process is also enhanced in the contralateral hemisphere. collectively, our results reveal the involvement of the mdach1 gene in adult neurogenesis. cells expressing this gene exhibit the properties of adult nscs or neuroblasts and respond to mcao by enhanced neurogenesis. experimental cerebral ischemia leads to activation of microglia and astrocytes, which subsequently release pro-and anti-inflammatory factors. furthermore, during the earliest periods of neuronal damage, large quantities of dopamine are released, which may exacerbate the vulnerability of the lesioned tissue. on the other hand, delayed treatment with levodopa has been proven beneficial in stroke patients, suggesting a more complex effect of dopamine. recent studies on astrocytic involvement in neuroinflammation have shown a remarkable modulation of the inflammatory response over the dopamine d2 receptor (d2r). after stroke, reactive astrocytes in the astroglial scar were found to be immunopositive for d2r. in this study, we report the expression of d2r in activated microglia, both after experimental stroke in vivo and after oxygen-glucose deprivation (ogd), an in vitro model of ischemia. using immunohistological stains, we analyzed the expression of d2r in iba1 positive cells 1, 3, 5, 7, and 14 days after middle cerebral artery occlusion (mcao; n 5 2-4 mice/time point). for each animal, >4 pictures were taken from the ischemic core as well as from the contralateral, non-lesioned cortex from 3 sections. a total of >300 (control) and >500 (ischemic core) iba11 cells were analyzed for each time point. in the ischemic core, $25% of cells immunopositive for iba1 expressed d2r in contrast to <1% in control areas. a strong depletion of d2r immunoreactivity was observed in the ischemic striatum, accompanied by an increase in d2r expression in the adjoining cortex and an elevated presence and activation of microglia. for in vitro experiments, we used fluorescence activated cell sorting to isolate cd45 low /cd111 microglial cells. no significant d2r expression at mrna level could be detected by rt-pcr in this population, while we were able to detect d2r in non-microglial cells. interestingly, cultured primary mouse microglia expressed low quantities of d2r, as revealed by western blotting and rt-pcr, suggesting that culturing is sufficient to induce d2r in microglial cells. upon in vitro activation of primary microglia through 60 minutes of ogd, we found a 1.5 fold increase in d2r expression after 24 hours (n 5 4). furthermore, treatment of primary microglia with the selective d2r agonist pramipexole showed a dose-dependent tendency to increase lipopolysaccharideinduced tnfa release (n 5 5). our studies indicate that activated microglia express a functional d2r, which may contribute to the pro-inflammatory reaction of microglia after ischemia. white matter (wm) is injured in most strokes and axonal injury and dysfunction contribute to disability associated with clinical deficits. in young wm, the damage from ischemic injury involves the sequence of energy depletion (ionic pathway), excessive glutamate release (excitotoxicity), generation of reactive oxygen species and oxidative stress (oxidative pathway). in older wm the injury is mediated by ca21-independent excitotoxicity due to an earlier and more robust glutamate release. because excitotoxicity leads to oxidative stress in wm we investigated whether blocking nitric oxide synthase (nos) activity before or after a period of oxygen glucose deprivation (ogd) promoted axon function in an age-dependent manner. acutely isolated optic nerves from young and old (1 and 12 month) swiss webster (sw) or c57bl/6 (bl6) mice were used to ascertain quantitative measurements of wm function and structure. to support a biological basis for nos inhibitor nitro-l-arginine methyl ester (l-name) action in themonpreparation, we evaluated the expression and localization of brain nos (bnos) using immunohistochemistry in conjunction with confocal imaging. the expression of bnos co-localized with gfap (1) astrocyte nuclei, cytoplasm, end-feet as well as nf-200 (1) axons. the pattern of bnos expression paralleled astrocyte morphology with age and became more punctate in appearance. evoked compound action potentials (caps) recovered to 21.8 6 2.8% (n 5 18) after 60 min of oxygen glucose deprivation (ogd) in young bl6 mons. pretreatment of mons with l-name (200 mm) promotedcaprecovery to 69.4 6 11.3% (n 5 8) compared to ogd while caps recovered to 39.9 6 4.4% (n 5 11) when l-name was applied after the end of ogd. pretreatment of mons from 1 or 12 month old sw with l-name improvedcaprecovery to 49.6 6 3.7% (n 5 8, vs ogd 21.3 6 3.7%, n 5 12) or to 51.1 6 9.3% (n 5 7, vs ogd 5.7 61.7%, n 5 8) respectively. l-name application after the end of ogd failed to promote aging axon function (8.8 6 3.5%, n 5 6). changes in nos activity help unveil agedependent oxidative injury mechanisms in ischemic white matter. question: cerebral endothelial cells have been reported to exert a protective effect against brain damage. does transplantation of healthy endothelial cells have a beneficial effect on the outcome of ischemic brain damage? methods: we injected endothelin-1 (et-1) into the rat internal capsule to induce lacunar infarction. seven days after et-1 injection, microvascular endothelial cells (mvecs) prepared from adult rat cerebral cortices were transplanted into the internal capsule. meningeal cells prepared from adult rat cerebra or 0.2% bovine serum albumin-hank's balanced salt solution were injected as controls. two weeks later, the footprint test and histochemical analysis were performed. results: we found that mvec transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (p <0.01) and induced remyelination (p<0.01) compared with the control groups. also the inflammatory response was repressed by mvec transplantation, judging from fewer ed-1-positive activated microglial cells in the mvec-transplanted group than in the other groups. conclusions: elucidation of the mechanisms by which mvecs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia. objective: though lactate is a known neuroprotective agent, its mechanism of action is not known. we hypothesized that lactate can provide neuroprotection by activating trek channels. the effect of lactate on the electrophysiology of trek channels was studied both in an in vitro brain slice model, after blocking the activity of other ion channels and in a heterologous expression system. results: whole cell patch clamp experiments, on ca1 stratum radiatum astrocytes, in acute hippocampal slices, significantly increased trek channel activity by 23.3 6 6.4% and hyperpolarized their resting membrane potential by 5 6 0.6 mv, on bath application of 30 mm lactate. comparison of rectification index at negative potentials between control and 30 mm lactate treatment showed no significant difference, suggesting that the conductance activated by lactate is outward rectifying. lactate-evoked increase in trek channel activity was reversibly inhibited by 200 mm quinine, a potent inhibitor of trek channels. further, lactate was unable to increase trek channel activity after disruption of intracellular lactate uptake with 2 mm a-cyano-4-hydroxy cinnamate, a blocker of monocarboxylate transporter. this was confirmed by inside-out patch clamp experiments on human trek1 channel expressed in hek293 cells. conclusion: the experimental observations suggest uptake of lactate by astrocytes to activate trek channels intracellularly. regenerative responses occurring after different types of postnatal brain injury often involve expansion of an endogenous neural progenitor cell pool, but the molecular mechanisms underlying this process are still poorly understood. we studied expansion of the glial progenitor cell pool in a mouse model of neonatal hypoxia (hx) that reproduces the hallmarks of perinatal brain injury in infants, including diffused white matter injury (dwmi). we have previously shown that hx causes proliferation of wm oligodendrocyte progenitor cells (opcs), and that this process is regulated by the cyclin-dependent kinase 2 (cdk2) pathway. our analysis in wm in vivo and in cultured cells demonstrated: i) higher expression of cdk2, cycline, prb806/811 and e2f1 in wm opcs after hx; ii) activation of the cdk2 pathway after hx, and iii) reduced hx-induced opc proliferation in cdk2-null mutant mice. here, we studied the upstream molecular mechanisms that regulate activity of the cdk2 signaling pathway resulting in opc proliferation after hx. we show that sirtuin 1 (sirt1) histone deacetylase activity is a major regulator of cdk2 signaling and of the regenerative response of opcs after hx. under hypoxic conditions, sirt1 is upregulated in wm, and interacts with both rb and cdk2. patterns of posttranslational modifications of cdk2 or sirt1 proteins after silencing either of these genes in cultured cells from normoxic and hypoxic wm indicate that cdk2 is a substrate for deacetylation by sirt1, and that sirt1 is phosphorylated by cdk2. also, sirt1 causes rb deacetylation resulting in dissociation of e2f1 from the rb/e2f1 complex, which ultimately leads to higher opc proliferation. knockdown of sirt1 in hypoxic wm cell cultures suppresses opc proliferation. finally, inhibition of sirt1 activity by sirtinol accelerates opc differentiation to mature oligodendrocytes, and reduces the number of opcs. we are currently analyzing the effects of hx on opc proliferation and differentiation in the wm of sirt1-null mice. our results provide evidence that sirt1 is an essential regulator of the cdk2-dependent regenerative response observed in wm opcs after hx, and that inhibition of sirt1 activity facilitates oligodendrocyte regeneration from opcs, which might ultimately promote wm recovery after hx. supported by nih r01ns045702, p01ns062686 and iddrc p30hd40677. gonadal steroid hormones reveal a potential neuroprotective role in acute brain ischemia. in rat in vivo studies using the transient occlusion of the middle cerebral artery as ischemic stroke model, we have shown that 17ß-estradiol and progesterone reduce the infarct area by more than 70% given 1h after the onset of stroke, prevent neuronal death and behavioral deficits. an intriguing aspect of this study was that the application of steroid hormones reduced the number of microglia and microglia-related markers in the penumbra during the first 24 h after the onset of stroke. besides microglia, penumbral astroglia coevally appeared as cellular target for steroid hormones by reducing the expression of proinflammatory-and molecules necessary for the attraction and activation of microglia and lymphocytes. this suggests that protective steroid hormones influence glia cell cross-talk and activity during early damaging conditions in the lesioned brain site. by using an in vitro hypoxic approach, we have now demonstrated that microglia express steroid hormone receptors and directly respond to ischemic conditions and steroid hormone treatment by changing expression and secretion of inflammatory compounds, trans-signaling factors and by modulating phagocytotic activity. this clearly shows that microglia is an important direct target for steroid hormones but also indirectly influenced via cross-talk by adjacent astrocytes at the damaged brain site. this generally points at an extensive bidirectional crosstalk between both glial cell types to convey steroid-mediated neuroprotection in ischemic brain tissue. in summary, the balance of inflammatory astrogliamicroglia interactions within the injured brain tissue during an early phase of ischemic destruction is a pivotal step for tissue repair. cannabinoids are considered as key regulators in the pathology of various diseases, including ischemic stroke. we previously demonstrated that post-ischemic treatment of two naturally occurring phenylpropanoids, trans-and cis-hinokiresinols (hrs) significantly reduced ischemic injury in rats subjected to middle cerebral artery occlusion (mcao) . in studies to screen their cellular targets, we found that hrs selectively bind to both type 1 and type 2 cannabinoid receptors (cb1r and cb2r). in the cb1r reporter gene assay, both hrs demonstrated antagonistic activity. in cb2r-overexpressing u 2 os cell lines, both hrs increased forskolininduced camp accumulation, suggesting their inverse agonism. since camp induces expression of heme oxyagenase-1 (ho-1), a well-known antioxidant stress protein, we further investigated the effect of hinokiresinols on ho-1 expression in mixed cortical neuron/glia cultures. trans-hr increased astroglial expression of ho-1 greater than cis-hr did. a selective ho-1 inhibitor, tin protoporphyrin ix significantly reduces the neuroprotective effect of trans-hr in cortical cultures exposed to oxygenglucose deprivation. in addition, both hrs reduced the number of ed-1immunopositive cells (i.e., active microglia and macrophages) in ischemic lesions. also, both hrs significantly reduced microglial migration induced by an endocannabinoid, 2-arachidonoylglycerol. the present study suggests that hrs reduce ischemic injury via regulation of glial cbrs. , the main anaplerotic enzyme in the brain, is predominantly located in astrocytes. in the adult brain, ischemia is known to reduce pyruvate carboxylation. moreover, reperfusion after ischemia leads to production of reactive oxygen species (ros). the pentose phosphate pathway (ppp) is, by making nadph necessary for the regeneration of reduced glutathione, a major pathway in the protection against ros. however, astrocytic and neuronal metabolic function in the neonatal brain after hypoxic-ischemic brain injury (hi) remains to be explored. methods: hi was induced in 7-day-old rats by unilateral severing of the carotid artery followed by 2 hours recovery and subsequent exposure to hypoxia (8%o 2 ) for 90 min. 30 min after end of hypoxia (the early reperfusion phase), rats were injected with [1,2-13 c]glucose and decapitated 30 min later. one group was sham-operated, and not exposed to hypoxia, thus reflecting normal metabolism in the neonate. extracts of ipsilateral hemispheres from hi and sham animals were analysed with 1 h-and 13 c-nmr spectroscopy. results: the sham animals had similar glutamine content, but lower amounts of 13 c labelled glutamine compared to corresponding values from adult rats, reflecting a generally lower rate of glucose metabolism in the neonatal astrocytes. however, approximately equal amounts of 13 c labelled isotopomers of glutamine were derived from pc and pyruvate dehydrogenase (pdh), resulting in a relatively high pc/pdh-ratio compared the same ratio in adults. following hi and reperfusion, a reduction in glucose metabolism and mitochondrial metabolism was seen by increased glucose and reduced incorporation of 13 c labelling in glutamate, glutamine and aspartate via pc and pdh. however, the pc/pdh-ratio in glutamine was similar in hi and sham. total mean values of glutamate, glutamine and aspartate were decreased, but only the latter to a significant degree. labelling in lactate via the ppp was reduced following hi. conclusion: the low labelling of lactate via the ppp indicates that the flux through the ppp was in fact reduced in the early reperfusion phase after hi. moreover, mitochondrial metabolism of pyruvate from glucose was reduced in both astrocytes and neurons. impaired anaplerosis in astrocytes was confirmed by lower amounts of aspartate. however, the proportionally similar decrease in 13 c labelling in glutamate and glutamine via pc, and the maintained pc/pdh-ratio implies that astrocytes continue to provide metabolic support for the neurons during the early reperfusion phase. ret receptor tyrosine kinase is the signaling component of the receptor complex for the family ligands of the glial cell line-derived neurotrophic factor (gdnf). ret is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons and it is necessary for the postnatal maintenance of dopaminergic neurons. ret expression has been as well demonstrated on microglia and several evidence indicate that gdnf regulates not only neuronal survival and maturation but also certain functions of microglia in the brain. here we demonstrated that isolectin ib4, commonly used as a microglial marker in the brain, binds to the glycosylated extracellular domain of ret both on the surface of living nih3t3 fibroblasts cells stably transfected with ret than in adult rat brain sections as revealed by immunoblotting. further, confocal immunofluorescence analysis demonstrated a clear overlap staining between pret and ib4 labeling in primary microglia cultures as well as in adult rat sections obtained from control or postischemic brain after permanent middle artery occlusion (pmcao). interestingly, ib4 staining identified activated or amoeboid retexpressing microglia under ischemic conditions. collectively, our data indicate ret receptor as one of the ib4-reactive glycoconjugate accounting for the ib4 stain in microglia under physiological and ischemic conditions. astrocytes can adapt to lower oxygen tensions by enhancing their glycolytic rate of energy production. in this case, adequate expression of monocarboxylate transporters (mcts) may be essential to sustain prominent lactate efflux and prevent glycolysis inhibition. here we show that oxygen level is an important factor controlling mct4 expression in primary cultures of mouse cortical astrocytes. indeed, while mct4 is clearly expressed by astrocytes in vivo, its basal in vitro expression in primary cultures of mouse cortical astrocytes is undetectable under classical culture conditions (95% air, 5%co 2 ). exposing astrocytes to more physiological brain oxygen tensions (hypoxic chamber flushed with 1%o 2 /5%co 2 /94%n 2 , leading to a concentration of dissolved oxygen in the culture medium of 40-60mmhg) restored mct4 expression. mct4 mrna expression became detectable after 12 hours under lower oxygen tension and was still present after 10 days with a maximum at 36 hours of incubation. this effect was paralleled by the appearance of the mct4 protein which reached a plateau between 48 hours and 96 hours, with a further enhancement at 7 and 10 days of incubation. this induction was specific for mct4 since mct1 expression was not altered. lactate release was significantly enhanced after 48 hours of incubation and further increased up to 10 days compared to astrocytes maintained under atmospheric conditions. treatment with dimethyloxalylglycine (dmog), a compound that mimics hypoxia by stabilizing the hypoxia-inducible factor 1 alpha (hif-1a) , produced a similar effect. both mct4 mrna and protein induction reached their maxima when astrocytes were treated with a concentration of dmog ranging between 1 to 2.5mm, which is also the range for greatest hif-1a expression. again, no effect was detectable on mct1 expression. transfecting astrocyte cultures with a sirna against hif-1a significantly reduced the effect of lower oxygen tension on mct4 expression. our results suggest that 1) under physiological conditions, changes in local oxygenation might regulate the capacity of astrocytes to supply lactate to neighboring cells via notably alteration in mct4 expression 2) under pathological conditions, mct4 may be upregulated as a consequence of hypoxia and contribute to neuroprotection by favouring the release and accumulation of lactate in the extracellular space. indeed, this process might give rise to the beneficial effect of lactate for neurons observed during the recovery from hypoxic/ischemic conditions in vitro and in vivo. early onset dementia and. the loss-of-function of trem2 or its coreceptor dap12 is responsible for the recessively inherited nasu-hakola disease (also known as plosl). the brains of plosl affected patients show strong microglial activation in the cerebral white matter. reports demonstrate that trem2-mediated phagocytic function of microglia is required for debris clearance and cns tissue homeostasis. perinatal brain injury is the underlying etiology for a host of developmental disabilities that includes spastic motor deficits and cognitive, behavioral and learning difficulties. hence, our aim is to characterize the expression of trem2 in the control of neuroinflammation following hypoxia/ ischemia (hi) in the newborn brain. we performed hi brain damage in postnatal day 7 (p7) c57/bl6 mice by permanent left carotid occlusion and litters were exposed to 8% of oxygen balanced with nitrogen for 50 minutes in a hypoxic chamber with controlled humidity and temperature maintained at 37 c. pups were then returned to their dam until sacrifice. the age-matched controls and samples from 3 hours to 7 days after hypoxia were collected and processed for immunohistochemistry. in control animals, trem2 expression was observed in the corpus callosum and in the subventricular zone at p7 that almost disappeared at p10. following hi, an increase in trem2 staining was observed in the corpus callosum, hippocampus, caudate-putamen, fimbria, cortex and thalamus in the ipsilateral (il) hemisphere, following a similar regional pattern of microglia activation. the increased expression of trem2 was observed from 24 hours to 7 days post-hypoxia. trem2 colocalized mainly with microglia markers, such as iba-1 and cd68. oligodendrocyte expression of trem2 was also analyzed. in conclusion, hi produced an increase in trem2 expression on the il damaged regions. these results suggest that the modulation of trem2 might be a possible target for damage control in neonatal brain. increasing evidence shows that astrocytes play a critical role in neuronal protection during an ischemic insult. in the vertebrate retina, most of astrocytic functions are executed by m€ uller glial cells, the major macroglial cell type in this tissue. the aim of this study was to evaluate the role of m€ uller glia in the onset and progression of retinal ganglion cell (rgc) degeneration after acute ischemic injury in the mouse eye in vivo. here, we used the selective gliotoxin fluorocitrate (fc) in order to transiently impair m€ uller glial metabolism at different time points during and after induction of an acute retinal ischemia/reperfusion by means of elevated intraocular pressure (iop). the effect of fc (3 mm) on metabolism was determined by measuring retinal glutamine levels, aconitase activity and mitochondrial membrane depolarization after intravitreal injection of the gliotoxin. retinal ischemia was unilaterally induced by elevating iop for 45 min. fc was intravitreally injected 4 h before onset of ischemia and after 6, 12 or 24h of reperfusion. saline was injected in the contralateral eye and served as control. surviving rgcs were quantified by means of confocal scanning microscopy and automated cell counting-based analysis on retinal wholemounts 7 days after lesion. fc treatment reduced glutamine levels to 35-38% as compared to control 4-6 h after injection. inhibition was completely reversed 24 h after injection. aconitase activity was reduced to $30% from control levels 4 h after fc injection. mitochondrial membrane potential was reduced by 50-70% as indicated by reduced intensity of the specific stain mitotracker red cmxros. one week after ischemia, only 40% of rgcs survived as compared to unlesioned controls. impairment of m€ uller glial metabolism during ischemia further reduced rgcs by 15% as compared to ischemia alone. fc treatment did not significantly modify rgcs survival 6 and 12 h after lesion. results support the notion for a critical neuroprotective role of m€ uller glial cells during ischemia. in the acute phase following ischemia, however, m€ uller glia inhibition does not further affect rgcs survival. further research is required to establish the time point for this switch in m€ uller glial function and the underlying mechanisms. extract has been widely investigated in animal models and clinical studies for various diseases including ischemic stroke. its major metabolite, cordycepin, has been reported to act as a selective a3 adenosine receptor agonist and to protect neurons against ischemic injury. however, their underlying mechanisms remain unclear. in the present study, we report that the standardized c. militaris extract, wib-801c, which contains cordycepin 8% of total dry weight of the extract, markedly reduced ischemic injury by inhibiting post-ischemic inflammatory responses. postischemic treatment with wib-801c (orally administered twice at 3 and 8 h after onset of mcao) significantly reduced infarct size and edema in rats subjected to transient middle cerebral artery occlusion (mcao, 1.5 h) and subsequent reperfusion (22 h). wib-801c also significantly improved integrity of glial cells in ischemic lesions, as evidenced by reduced white matter degeneration and loss of blood-brain barrier. importantly, wib-801c significantly ameliorated neurological deficits in not only transient but also in permanent stroke models. moreover, wib-801csignificantly improved long-term survival of mcao rats over 30 days. as we previously reported with selective a3 receptor agonists introduction: cd200 & cd200r are immune inhibitory molecules that have been shown to be involved in inducing immune tolerance and in contributing to immune privileged status of the cns. the developing brain exhibits distinct morphological as well as physiological characteristics determining a peculiar response to injury showing an aggravated susceptibility to excitotoxicity and pro-inflammatory cytokines, along with an exacerbated inflammatory response. previous studies from our group have described the expression of cd200-cd200r in brain during development showing a distinct pattern of expression in the cortex and the hippocampus. hence, the aim of this study is phenotypic characterization of cd200r1 microglia/macrophages and cd2001 neuronal cells following hypoxia/ischemia (h/i) in neonatal mice brain. wild-type c57/bl6 mice postnatal (p) day 1,3,5,7,10,14,21 and adult were used for developmental studies. p7 mice was used for h/i injury that was administered using vannucci model modified for neonatal mice (8% o 2 , 55 min) and samples were collected 3h, 12h, 24h, 48h, 72h & 7 days after hypoxia for immunofluorescence staining. results: phenotypic characterization of cd200r1 microglia/macrophages showed them to express markers of pro-or anti-inflammatory phenotype in a temporal fashion post lesion. they expressed m2 phenotype as observed by colocalization with cd2061 cells throughout the time points studied but a subpopulation of cd200r1 cells exhibited m1 phenotype as exhibited by mhcii1, cd861 cells from 24-48 hrs post lesion. calretinin (cr) used for the characterization of cd2001 neurons showed that most cr1 neurons were cd2001 especially the interneurons in the innermolecular layer of hippocampus, layer i of the neocortex and the hilus at most age groups studied. after h/i, cd200 immunolabeling was increased in the hippocampal fissure until 7 days post-lesion. this followed a change in cd200r1 microglial cells described above. to conclude, a balance between m1/m2 phenotype of cd200r1 microglia/macrophages and expression of cd200 by cr1 cells are involved in evolution of h/i induced brain injury in neonatal mice. objective: the cytokine interleukin-1 (il-1) and its naturally occurring receptor antagonist (il-1ra) play a key role in determining neuronal cell death and survival in focal cerebral ischemia. the objective of this study was to determine the cellular production of il-1/il-1ra, and to test the neuroprotective potential of post-surgically injected il-1ra overexpressing bone marrow (bm) cells in a mouse model of focal cerebral ischemia. materials and methods: c57bl/6 mice were injected i.v. with bm cells isolated from sil-1ra overexpressing mice, 30 min after permanent middle cerebral artery occlusion (pmcao). physiological parameters and behaviour were recorded post-surgically, infarct sizes were estimated, and microglial-leukocyte expression of il-1/il-1ra was analyzed by flowcytometry, in situ hybridization and immunohistochemistry. results: we identify microglia, and not recruited leukocytes as the major producers of il-1ra after pmcao in mice, and we show by using il-1ra knock out mice that microglial-produced il-1ra is neuroprotective. we report that the sil-1ra produced by the post-surgically injected bm cells, potentiates the neuroprotective effect of microglialderived il-1ra, at both 24 h and 5 days, which is consistent with behavioural improvement at 5 days, and detection of recruited bm cells in the ischemic area 1.5 h after pmcao. interestingly, we also find that the sil-1ra overexpressing bm cells stimulate microglial production of il-1ra 6 h after pmcao, at which time both il-1a and il-1b is upregulated. conclusion: our results provide proof of principle that increasing the production of il-1ra by recruited bm cells can counteract the effect of il-1a/b, increase neuronal survival and improve motor function alone or through induction of microglial-produced il-1ra after pmcao in mice. bogomoletz institute of physiology, kyiv, ukraine short-term oxygen-glucose deprivation (ogd) is known to activate excitatory glutamatergic synapses, induce long-term potentiation and result in neuronal and synaptical ultrastructural remodelling in organitypic cultured hippocampal slices (ochs). in this work we studied changes in glial synaptic coverage following 30 min ogd episode using 3d reconstruction of ochs confocal and electron microscopic images. propidium iodide (pi) and mitotracker orange cmtmros (mto) were used to estimate cell viability and their mitochondrial potential. astroglial and microglial cells were identified using immunohistochemical staining with anti-gfap and anti-iba-1 antibodies. the study demonstrated that astroglial and microglial cells retain viability (there was no significant increase in the amount of pi-labbelled cells). 3d reconstruction revealed significant increase in excitatory synapses glial coverage as early as 1 hour following ogd accompanied by a significant increase in number of astroglial and microglial processes. the deprivation also resulted in gradual increase of mitochondrial potential from 4 hr to 24 hr in both astroglial and microglial cells. it was previously shown that pyramidal neurons under the same conditions lose mitochondrial potential and became pi-positive after 4 and 24 reoxygenetion, indicating damage of cytoplasmic membrane, the effect prevented by application of nmda receptor antagonist d-ap5. thus, unlike neurons astroglial and microglial cells are ogd-resistant and demonstrate marked mitochondrial activation -a process that might be involved in maintaining neuronal function during oxygen-glucose deficiency. using a microfluidic high throughput qpcr platform, we measured the expression of 47 selected genes encoding astrocytic and polydendrocytic markers, ion channels, transporters and receptors that participate in maintaining k 1 and glutamate homeostasis in each of 292 individual gfap/egfp-positive glial cells. in this study we show how the expression of these selected genes changes during development (postnatal days 10, 20, 30 and 50) as well as 3, 7 and 14 days after middle cerebral artery occlusion (mcao). self-organizing maps and principal component analyses divided the cells according to their similarity in gene expression into three subpopulations of astrocytes present within the first 10-50 days of postnatal development (p10-p50). the first subpopulation comprises immature glia mainly from p10, characterized by the high transcriptional activity of all of the studied genes, including polydendrocytic markers. the second subpopulation is dominated by cells from p20 and displayed low transcript levels of all of the studied genes. the third subpopulation represents mature astrocytes mainly from p30 and p50. three, seven and fourteen days after ischemia (d3, d7, d14), additional astrocytic subpopulations appear: resting astroglia (mainly from p50 and d3), transcriptionally active early reactive astroglia (mainly from d7) expressing the mrna of polydendrocytic markers, and, presumably, permanent reactive astroglia (solely from d14). following focal cerebral ischemia, reactive astrocytes undergo pronounced changes in their expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and markers of reactive astrocytes and polydendrocytes. from the data obtained from single cell pcr analysis, we calculated the spearman correlation coefficients between pairs of genes, which revealed interesting positive gene expression correlations with polydendrocytic markers (gria2-4, grik1-5, grin3a, kcnj16, kcnk2 and kcnk10 genes). this study was further supplemented by an immunohistochemical analysis of nmda receptor subunits and pdgfar, which confirmed that changes in gene expressions correlate with immunodetected proteins. ga cr 13-02154s, astf 110-2011, gauk 604212. reactive astrogliosis is often regarded as universal type of astroglial response to various forms of injuries. among its most characteristic features one may include: glial fibrillary acidic protein (gfap) up-regulation, cellular hypertrophy and proliferation and gliotic scar formation. an important element of glial cells response is their ability of de-differentiation, which is related with their re-gaining of immunological and molecular properties characteristic for earlier developmental forms. the origin of cells proliferating in response to various types of brain injury is still a subject of debate. among others it is related to the pathomechanism of lesion, affected brain structure and developmental stage of the nervous system. aim: in our study we aimed to assess the differences of de-differentiation and proliferation potential of astroglia localized in the cerebral cortex and striatum to the transient focal brain ischemia. material and methods: transient focal brain ischemia was evoked in 20 adult male wistar rats by placement of the monofilament surgical thread into the internal carotid artery and occlusion of the middle cerebral artery for 1h. the postoperative survival period was up to 6 weeks. immunocytochemical double-staining for astrocytic marker (gfap), developmental (pax6) and proliferative (ki-67) markers was performed and subsequent qualitative and quantitative study was done by means of confocal microscopy. results: double-labeled gfap/pax6 and gfap/ki67 reactive astroglial cells were revealed in the cerebral cortex and striatum of the ischemic region, starting from 24h after initiating the ischemia. the apparent difference in the intensity of morphological changes, quantity of de-differentiating and proliferating astroglial cells was observed between ischemic cerebral cortex and striatum. intensity of astroglial response and proliferative potential were much higher in the striatum than in the cerebral cortex. summary and conclusion: apparent difference in proliferative response between the cerebral cortex and striatum may be consequence of metabolic and molecular differences between astroglial cells populating two mentioned above structures. it may be also consequence of uneven decrease of cerebral blood flow and resulting different metabolic conditions influencing astroglial function in various fragments of ischemic area. reassuming, the differentiated potential of astroglial proliferative response to ischemic changes in different brain regions must be taken into account while analyzing clinical consequences of cerebral infarcts of different localization. triggering receptor expressed on myeloid cells-2 (trem2) is a microglial surface receptor involved in phagocytosis. clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. the role of trem2 following stroke is currently unclear. as an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). quantitative pcr (qpcr) revealed a greatly increased transcription of trem2 after stroke ($10fold). we subsequently analyzed the expression of proinflammatory cytokines, chemokines and their receptors in trem2knockout (trem2-ko) mice via qpcr. microglial activation (cd68, iba1) and cd3-positive t-cell invasion were analyzed via qpcr and immunohistochemistry. functional consequences of trem2 knockout were assessed by infarct volumetry. the acute inflammatory response (12 h reperfusion) was very similar between trem2-ko mice and their littermate controls. however, in the sub-acute phase (7 d reperfusion) following stroke, trem2-ko mice showed a decreased transcription of pro-inflammatory cytokines tnfa, il-1a and il-1b, associated with a reduced microglial activity (cd68, iba1). furthermore, trem2-ko mice showed a reduced transcription of chemokines ccl2 (mcp1), ccl3 (mip1a) and the chemokine receptor cx3cr1, followed by a diminished invasion of cd3-positive t-cells. no effect on the lesion size was observed. conclusions although we initially expected an exaggerated pro-inflammatory response following ablation of trem2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in trem2-ko mice. we therefore conclude that trem2 appears to sustain a distinct inflammatory response after stroke. metabotropic glutamate receptors (mglurs) are seven transmembrane domain g-protein-coupled receptors for l-glutamate, the main excitatory neurotransmitter in the mammalian brain. in addition, glutamatemediated excitotoxicity plays a central role in mediating neuron and oligodendrocyte death during ischemic episodes. in vitro, developing oligodendrocytes (ols) in particular have been shown to be highly vulnerable to excitotoxicity and oxidative stress. notably, activation of group 1 mglurs has been shown to attenuate ol excitotoxicity in vitro and in brain slices has been shown to reduce ischemia-mediated impairment of astrocytes. here, we have examined the functional expression of mglurs and their role in acute ischemic injury in developing cns white matter of the mouse optic nerve. calcium imaging experiments were performed in optic nerves from p9-13 wild-type mice. optic nerves were isolated intact, loaded with fluo-4 am and visualized using a zeiss lsm 5 pascal confocal microscope; images were collected in optical z-sections and changes in fluorescence intensity were measured. the application of mglur agonists acpd and dhpg showed that group i mglur mediate a rise in glial [ca 21 ] i . the action of the group i mglur antagonist aida versus acpd indicated that the rise in [ca 21 ] i also involves group ii (and possibly group iii) mglur. immunohistochemistry confirmed glial expression of group i subunit mglur5 and group ii subunits mglur2 and mglur3 in the optic nerve, although further analysis of cellular localization is required. to examine the role of mglurs in white matter ischemia, optic nerves from p8-9 mice in which fluorescent reporters are driven by astrocyte (gfap-egfp) or oligodendrocyte (sox10-egfp) genes were isolated intact into artificial cerebrospinal fluid (acsf) and subjected to oxygen glucose deprivation (ogd) for 1 hour. activation of group i or group ii mglur with the agonists acpd, dhpg or ly379268 protected astrocytes and oligodendrocytes from ischemic cell death. further experiments are required to determine the mechanisms involved, but these findings demonstrate functional expression of mglur in optic nerve glia and indicate that activation of mglur is a possible therapeutic strategy to protect against glial damage in response to ischemia. the cerebral cortex is one of the most often affected areas after stroke, but there are no studies comparing the outcome in different cortical regions after focal ischemia. the aim of this investigation was to evaluate the patterns of glial activation, tissue degeneration and neuronal loss in different survival times following cortical ischemia. focal ischemia was induced by stereotaxic microinjections of endothelin-1 (et-1) into the somatosensory, motor and association cortices of adult rats (n 5 45). the control animals were injected with the same volume of sterile saline (n 5 27). the animals were perfused at 1, 3 and 7 days after ischemia. gross histopathology was evaluated using cresyl violet staining. 20 lm sections were submitted to immunohistochemistry for astrocytes (anti-gfap), activated microglia/macrophages (anti-ed1) and microglia (anti-iba1 and ed1). tissue loss and glial activation were more intense in the somatosensory cortex than in other cortical areas. the motor cortex was the second more affected area. the association cortex was the less damaged area, which was confirmed by quantitative analysis (anova-tukey). the results suggest that an ischemic lesion of the same intensity induces a differential pattern of tissue loss and neuroinflammation, depending on the cortical area, and that the primary sensory and motor areas are more susceptible to ischemia than association areas. tlr4 is required for ipc-induced neuroprotection. in order to elucidate the mechanisms by which microglial tlr4 contributes to ipc, we carried out cell-targeted genomic analyses specifically on microglia exposed to either ischemic conditions in vitro or ipc in vivo. for our in vitro model, we exposed wt and tlr4 -/mouse primary microglia to hypoxic/hypoglycemic conditions for 24 hours. for our in vivo model, we carried out transient middle cerebral artery occlusion (mcao) or sham surgery as our ipc pulse on wt and tlr4 -/adult male mice. three days later mice were sacrificed and microglia acutely isolated from ipsilateral cortex using magnetic affinity chromatography cell separation and ex vivo flow cytometry. microarray analysis was carried out on rna from both in vitro and in vivo microglia. results from both datasets identified robust expression of type 1 and/or type 3 interferon (ifn)-stimulated genes (isgs) as the predominant transcriptomal feature of ischemia-exposed microglia. in vitro, we found that 25 out of the 60 (40%) individual genes that were significantly up-regulated >2fold by hypoxia/hypoglycemia in wt, but not tlr4 -/-, microglia were isgs including mx1, oas2, irf7 and ccl5. promoter analysis demonstrated that the top four most active transcription factors were isgf3, irf2, irf1 and irf7; all ifn regulatory factors (irfs). a similar pattern emerged from the in vivo dataset with multiple irfs again among the most activated transcription factors in sorted microglia from ipcexposed mice. however, unlike the isg response in vitro, the isg response from the in vivo dataset was not tlr4-dependent. the ifn family of cytokines is recognized as a key component of the innate immune response to infection and other types of injury. the role of microglial ifn-signaling in ipc is unknown. these results suggest that broad activation of isgs may be a key feature of the microglial response to ischemic conditions. astrocytes respond to central nervous system (cns) injury by the formation of a glial scar that develops within few days after insult and is characterized by astrocyte proliferation and cellular hypertrophy. reactive astrocytes change their immunohistochemical profile, such as increasing expression of gfap, nestin and vimentin; however, they also markedly alter the expression of ion channels, receptors and transporters, which participate in the maintenance of ionic-, water-and neurotransmitter homeostasis, such as k 1 channels, aquaporins or glutamate transporters. here, we aimed to characterize the gene expression profile and functional properties of reactive astrocytes 5 weeks after global cerebral ischemia (gci) or focal cerebral ischemia (fci) with a specific focus on k 1 channels or non-specific cationic channels. gci was induced in adult rats by bilateral, 15-minute common carotid artery occlusion combined with low oxygen, while fci was induced in adult egfp/gfap mice by permanent middle cerebral artery occlusion. using the patch-clamp, we investigated the membrane properties of astrocytes in situ 5 weeks after gci in the rat hippocampal ca1 region and 5 weeks after fci in the mouse cortex. regardless of the type of ischemic injury, astrocytes from both cns regions depolarized their membrane potential by $14 mv 5 weeks after ischemia. when compared to astrocytes from the non-injured ca1 region or the cortex, the post-ischemic astrocytes displayed large hyperpolarizationactivated time-and voltage-dependent, non-inactivating inward currents, the current density of which increased 3-fold in response to gci or fci. in addition, these non-specific cationic channels were sensitive to zd7288 and to a low extracellular na 1 concentration, suggesting that they may belong to the family of hyperpolarization-activated cyclic nucleotide-gated (hcn) channels. we have taken advantage of fluorescently labeled astrocytes in egfp/gfap mice and isolated egfp 1 cells by facs from non-injured as well as ischemic regions and performed gene expression profiling using single-cell rt-qpcr. our data revealed that cortical astrocytes after fci express, besides the increased expression of gfap, gfapd, aqp1 and 9, extremely high levels of hcn1-3 transcripts that encode hcn 1-3 channels. moreover, our immunohistochemical analyses confirmed the presence of hcn1-3 in reactive astrocytes, especially hcn1, 2 and 3 channels. in summary, our results show that reactive astrocytes from post-ischemic tissue express hcn channels and that their activity might significantly contribute to na 1 influx, possibly maintaining atpase function and/or contributing to intracellular na 1 -dependent events, such as glutamate transporter or na 1 /ca 21 exchanger reversal. ga cr 13-02154s, p304/12/g069 and gauk 383711. the optimal operation of electrogenic astrocytic transporters and exchangers for some well-defined astrocyte brain homeostatic functions depends on the presence of k 1 channels in the cell membranes and the hyperpolarized membrane potential. our previous study showed that astrocytes functionally express two-pore domain k 1 channel trek-1, which helps to set the negative resting membrane potential. however, the roles of trek-1 on astrocytic function under normal and ischemic conditions remain unclear. in this study, we investigated the effects of trek-1 activity on astrocytic function and neuronal death under ischemic conditions. in an in vitro hypoxia model with astrocytes culture, trek-1 immunoreactivity was up-regulated after hypoxia. suppression of trek-1 activity inhibited the glutamate clearance capability, enhanced the inflammatory secretion of astrocytes derived s100b and led to increased neuronal apoptosis after ischemic insult. after middle cerebral artery occlusion induced focal ischemia in rats, the trek-1 expression was significantly increased which correlated with reactive astrogliosis. application of lineonic acid, a trek-1 agonist which could potentiate the astrocytic conductance and hyperpolarization membrane potential, up-regulated the expression of astrocytic glutamate transporter glt-1, inhibited the micro-glial inflammatory response and attenuated neuronal damage. our results suggest that trek-1 activity is involved in astrocytic function and neuronal survival. this would provide evidence showing astrocytic trek-1 involvement in ischemia pathology which may serve as a potential therapeutic target in stroke. oligodendrocytes are the myelinating cells of the central nervous system (cns). they are responsible for ensheathing myelin layers around axons, which contributes to improve conduction of neuronal impulses and additionally provides axonal support. oligodendrocytes are generated by oligodendrocyte precursor cells (opcs), a self-renewing and proliferating adult stem/precursor cell population in the cns. this process occurs not only during developmental myelination but also mediates cns remyelination, a regenerative process that restores myelin sheaths to demyelinated axons. although energy metabolism in the oligodendrocyte lineage is thought to be very active to support the lipid production specific to this cell type, it is still poorly characterized. as a first step towards a detailed characterization of the energy metabolism associated with specific lineage stages we investigated how glucose -the main cerebral energy substrate -is metabolized at an immature opc stage and in differentiated oligodendrocytes. primary cultures of opcs and oligodendrocytes were incubated with medium containing [1,6-13 c]glucose for 4h and metabolites in both cell extracts and culture medium were collected and analysed for excess % 13 c enrichment using gas-chromatography-mass spectrometry (gc-ms) as well as for total amounts using hplc. the significant 13 c-enrichment in citrate, malate, and aspartate in cell extracts indicate that oligodendrocytes oxidize glucose-derived metabolites extensively in the tricarboxylic acid (tca) cycle. moreover, they release 13 c-labelled aspartate and glutamate to the extracellular medium, although a significant consumption of aspartate, glutamate and glutamine from the medium was also observed. enrichment of extracellular lactate and increased alanine synthesis in oligodendrocytes as compared to opcs suggests that the rate of aerobic glycolysis is increased in immature as compared to differentiated cells. in conclusion, these data confirm that both opcs and oligodendrocytes are highly metabolically active and, in addition to extensively oxidizing glucose, synthesize and release distinct metabolites. we anticipate that further studies on this subject will contribute to a better understanding of the physiological role of oligodendrocytes in the cns and the role of glycolysis and mitochondrial metabolism during their maturation. schwann cells in the peripheral and oligodendrocytes in the central nervous system require a tremendous amount of lipids to be capable of producing all cell membranes forming the myelin structure. furthermore, when compared to other cells, myelin membrane presents an extremely high lipid content ($80% of dry weight) coupled to an unique lipid composition. the importance of myelin lipid composition towards myelin formation and maintenance is highlighted by the frequent appearance of myelin defects in lipid-metabolism human disorders. one of the main represented class of lipids in myelin are phospholipids, of which fatty acids (fas) represents the building blocks. most cells preferentially uptake circulating free fas. however, cells under increased membrane production, e.g. highly proliferating cells, can endogenously produce them through the enzyme fatty acid synthase (fasn), which synthesize palmitate. in these cells, fasn is transcriptionally augmented via the mtorc1 pathway. thus, we hypothesized that due to their increased lipid demand myelinating cells would require fasn activity towards myelin production. the functional role of fasn in the metabolic control of myelin membrane lipid composition and myelination is unidentified. thus, we addressed it experimentally by conditionally deleting fasn in myelinating cells in vivo. we show that endogenous fas biosynthesis in myelinating cells is essential to maintain myelin membrane lipid homeostasis. furthermore, maintaining a correct lipid homeostasis is critical for myelination. thus, we analyzed whether fasn is required to allow palmitoylation of myelin proteins and their targeting to the myelin membrane. in addition, we show that interfering with myelin fas homeostasis results in the activation of systemic compensating metabolic loops promoting fas uptake, although not sufficient to preserve efficient myelination. taken together our data demonstrate that in myelinating cells fas homeostasis is under the synergistic control of endogenous metabolic fas production via fasn and systemic functional regulation of free fas uptake. most importantly, maintaining a correct homeostasis of fa-derived lipids in myelin is critical for myelination. homeostasis and signaling, respectively (1). in mammals, two cytosolic and two mainly mitochondrial located glutaredoxins have been identified (2). we already described an essential role for the formation of a functional neuronal network during embryonic development of one of these proteins, the vertebrate specific glutaredoxin 2 (3). here, we will focus on the contribution of glutaredoxin 2 on basic cellular functions of oligodendrocytes, the myelinating cells of the central nervous system, under physiological and pathophysiological situations, e.g. multiple sclerosis, which is characterized by inflammatory axonal demyelination (4). oligodendrocyte progenitors and mature oligodendrocytes are very sensitive against oxidative stress induced by release of nitric oxide by activated microglia (5) . using a2b5 positive glia restricted progenitor cells as well as organotypic cerebellar slice cultures, we found that increased levels of glutaredoxin 2 protect against cell death induced by either activated microglia or s-nitrosoglutathione, a physiological nitric oxide donor. under physiological conditions elevated levels of glutaredoxin 2 increased migration of a2b5 positive cells nearly three fold. western blot analyses, quantitative real time pcr, as well as immunocytochemistry revealed that glutaredoxin 2 inhibited further differentiation of ng2 positive oligodendrocyte progenitor cells. in summary, our data indicate the importance of specific thiol redox signaling during cellular protection and function in this type of glia cells. current multiple sclerosis therapeutics act as immunomodulators and/ or immunosuppressors. this strategy is largely ineffective at preventing progression of the disease. significant benefits to multiple sclerosis patients might be seen with a therapeutic agent which induces remyelination, however, to date no such agents have been developed. using in vitro and in vivo models we have identified nefiracetam as a potential remyelinating therapeutic. to study remyelination in vitro, hippocampal organotypic cultures were exposed to lysophospatidylcholine (lpc) for 18hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for 24hrs of this recovery period. in all cases immunofluorescent labelling of myelin basic protein (mbp) was used as a measure of myelin. nefiracetam was shown to increase mbp expression, relative to untreated controls, accelerating recovery from lpc. two different models were used to investigate the efficacy of nefiracetam in vivo; an inflammatory model, experimental autoimmune encephalomyelitis (eae), and a toxin induced model of demyelination, achieved through the use of cuprizone chow. nefiracetam treatment did not alleviate the symptoms of eae. however, nefiracetam did restore the amount of myelin in cuprizone exposed animals, compared to saline-treated controls. in combination, these observations suggest that nefiracetam is capable of accelerating remyelination through a mechanism of action which is independent of immunomodulation. we conclude that nefiracetam encourages remyelination both in vitro and in vivo. these pro-myelin properties identify nefiracetam as a potential therapeutic for demyelinating disorders. university of naples "federico", neuroscience, naples, italy changes in intracellular [ca 21 ] i levels have been shown to influence the developmental processes that accompany the transition of human oligodendrocyte precursor cells (opcs) into mature myelinating oligodendrocytes and are required for the initiation of myelination and remyelination processes. in the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (ncx) family members ncx1, ncx2, and ncx3, play a role in oligodendrocyte maturation. functional studies, as well as mrna and protein expression analyses, revealed that ncx1 and ncx3, but not ncx2, were divergently modulated during opc differentiation into oligodendrocyte phenotype. in fact, while ncx1 was down-regulated, ncx3 was strongly up-regulated during the oligodendrocyte development. the importance of calcium signaling mediated by ncx3 during oligodendrocyte maturation was supported by several findings. indeed, whereas the knocking down of the ncx3 isoform in opcs prevented the up-regulation of the myelin protein markers cnpase and mbp, its overexpression induced an up-regulation of cnpase and mbp. furthermore, ncx3 knock-out mice exhibited not only a reduced size of spinal cord but also a marked hypomyelination, as revealed by the decrease in mbp expression and by the accompanying increase in opcs number. collectively, our findings indicate that calcium signaling mediated by ncx3 plays a crucial role in oligodendrocyte maturation and myelin formation. to explore the role of kif13b in myelination in vivo, we generated conditional knock-out mice with kif13b specific ablation in either schwann cells or oligodendrocytes. we found that kif13bfl/fl p0cre mouse nerves are hypomyelinated with reduced myelin thickness, thus confirming in vitro data. in the pns, axonal neuregulin (nrg) 1 type iii is one of the main signaling promoting myelination and remyelination after injury. nrg1 type iii binds to erbb2/erbb3 receptors in schwann cells, which signal through a plethora of downstream signaling pathways also including the pi3k/akt complex. the pi3k/akt signaling pathway is attenuated by the pten phosphatase, which dephosphorylates ptdins(3,4,5)p 3 (s. goebbels et al., 2010). dlg1 is believed to interact with pten and potentiate its ability to dephosphorylate ptdins(3,4,5)p 3 , thus negatively modulating the akt-mtor pathway (l. cotter et al, 2010). as kif13b interacts with dlg1 in schwann cells, we investigated the pi3k/akt pathway in kif13b-null nerves and surprisingly, we found that dlg1 expression levels are increased whereas akt phosphorylation is decreased in mutant nerves. our findings suggest that kif13b negatively regulates dlg1 activity and acts as a promoter of myelination in the pns. on the contrary, when kif13b is lost specifically in oligodendrocytes, myelin sheaths are thicker. at the molecular level, the hypermyelination is consistent with increased akt phosphorylation. we are currently investigating the molecular mechanisms at the basis of the kif13b/dlg1 interaction in both schwann cells and oligodendrocytes. we have identified the nootropic nefiracetam as a potential novel remyelinating therapeutic using in vivo and in vitro models of demyelination. in this study we investigate the mechanism of action of nefiracetam in the context of remyelination as regulated by wnt/b-catenin signalling and spontaneous activity. to investigate whether nefiracetam acts by modulating wnt signalling, organotypic hippocampal cultures were exposed to nefiracetam or the wnt/b-catenin inhibitor cardamonin. both increased immunofluorescent staining for the opc marker ng2. myelin formation, as assessed by mbp staining, was enhanced by nefiracetam but was not affected by cardamonin. thus, modifying wnt signalling appears to impact opc development without effecting differentiation, suggesting the pro-myelinating effects of nefiracetam involve an additional mechanism. we next investigated the effect of nefircatam on opc differentiation using purified opc cultures. nefiracetam increased the ol:opc ratio, as assessed by immunofluorescent staining for mbp and ng2 respectively, suggesting nefiracetam is capable of promoting ol maturation through a direct action on opcs. finally, we investigated the effect of nefiracetam on spontaneous activity. organotypic hippocampal cultures were exposed to lysophospatidylcholine (lpc) for 18hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for 24hrs of the recovery period. spontaneous calcium activity was then measured using fluo4-am. cultures not exposed to lpc displayed low levels of activity which were unaffected by nefiracetam. lpc exposure caused an increase in activity which was enhanced by nefiracetam. additionally, in these cultures, lpc decreased mbp staining compared to control while nefiracetam post-lpc caused a return to control levels. these data suggest increased activity may be an adaptive response related to myelin repair which is enhanced by nefiracetam. together, these findings suggest a complex mechanism of action for nefiracetam as a remyelinating agent involving modulation of wnt signalling, spontaneous activity and opc differentiation. in an assay in which cortical oligodendrocytes ensheath dorsal root ganglion cells 3 we found that nrg and nmda receptors interact to regulate myelination. without nrg, blocking nmda receptors had no effect on myelination. adding nrg increased myelination at all timepoints analysed (2-5 weeks), suggesting that nrg did not merely accelerate myelination. strikingly, nrg led to the majority of myelination becoming dependent on activation of nmda receptors and action potential activity. nrg's effect was associated with a 4-fold increase in nmda receptor current in oligodendrocyte lineage cells. thus, nrg switches myelination from a default programme, which is independent of neuronal activity, to a mechanism that is regulated by glutamate released from active axons. the effects of nrg were associated with an increase in the phosphorylation of akt and the transcription factor creb, but not associated with changes in the phosphorylation of erk. these data reveal a function for oligodendrocyte nmda receptors. the absence of nrg in multiple sclerosis lesions, and enhanced remyelination by added nrg, suggest a role for neuregulin/nmda receptor dependent remyelination after pathology. low-density lipoprotein receptor-related proteins (lrps) are endocytic receptors that internalize several ligands and are involved in lipoprotein homeostasis in adult brain. lrp-2, also known as megalin, is involved in the development of the central nervous system (cns) and in the transport of molecules across the blood brain barrier (bbb). in a previous work, our group revealed that megalin is selectively involved in sonic hedgehog-mediated effects on oligodendrocyte precursor cell migration and proliferation during the development of the cns. although there are some reports describing the presence of lrps in acute lesions of multiple sclerosis (ms) patients and pointing to an important role of shh in the pathogenesis of this disease, there are not data about the expression of megalin in ms tissue. in the present study, we analyse the distribution and the cellular characterisation of megalin in samples of human brain from ms patients to elucidate the role of this receptor in the pathogenesis of demyelination. changes in megalin distribution parallel the different ms histopathological lesions: we detected megalin in active demyelinating lesions and in the periplaque of chronic-active lesions, areas where remyelination spontaneously occurs. on the contrary, megalin was absent in the demyelinated region of chronic lesions. in addition, we observed the up-regulation of megalin in a subpopulation of perivascular astrocytes within the normal appearing grey matter (nagm) of ms patients. moreover, megalin was up-regulated in blood vessels within active lesions, in the periplaque of chronic-active lesions and in the nagm. in parallel, we also analysed the bbb profile to determine structural and functional alterations. altogether, we suggest that megalin is an important receptor expressed in diverse areas of the cns of ms patients representing different aspects of ms development: i) a potential target to improve remyelination; ii) an indicator of a functional rather than a structural disruption of the bbb. therapeutic strategies to modulate specific lrps would be useful to effectively treat ms. bdnf is known to promote central nervous system myelination both in vitro and in vivo. adopting in vitro myelination assays, we identified that bdnf acts through oligodendroglial-expressed trkb receptors to promote myelination. this was verified in vivo, as deletion of trkb in mature oligodendrocytes (in trkb fl/fl mbp-cre mice) resulted in a hypomyelinating phenotype during development. question: here we investigate the consequence of trkb deletion in oligodendrocyte progenitor cells (in trkb fl/fl cnpase-cre mice) and the signalling pathways downstream of trkb that promote myelination. methods and results: trkb fl/fl cnpase-cre mice were born in mendelian ratios and were phenotypically indistinguishable from littermate control mice. surprisingly, analysis of myelinated axonal tracts in the spinal cord and optic nerve revealed normal myelin development. in addition, the number of cells in the oligodendroglial lineage was the same as control mice during postnatal development. these data indicate that the timing of trkb deletion in the oligodendroglial lineage is critical, as deletion in opcs results in no significant phenotype, whereas deletion in mature oligodendrocytes results in a hypomyelinating phenotype. these data suggest that opcs are able to compensate for the loss of trkb and myelinate normally in the absence of bdnf signalling. our in vitro data have shown that the promyelinating influence of bdnf strongly correlates with erk1/2 activation. here we show that overexpression of erk2 in opcs exerts a more significant promyelinating effect than erk1. as erk1/2 are known to exert some of their effects through direct transcription factor phosphorylation, we have undertaken an in silico analysis of myelin-specific transcription factors and identified several that contain potential erk1/2 binding domains and phosphorylation sites. co-immunoprecipitation experiments show an interaction between erk1/2 and these transcription factors. investigation into the nature of these interactions and their functional consequences are ongoing. conclusions: this work suggests a novel role for erk1/2 signalling within oligodendrocytes that regulates cns myelination, possibly via activation of myelin-specific transcription factors. as erk1/2 is activated by multiple receptors, other receptors could potentially compensate for the loss of trkb in opcs, resulting in a normally myelinated cns in vivo. myelin is vulnerable to impairment by genetic, toxic, and immunological triggers, but the molecular basis for many aspects of myelin pathology has remained unknown. to identify molecules required for the structural integrity of central nervous system myelin we have systematically analyzed its protein composition by quantitative proteome analysis. based on the high abundance of filament-forming septins in myelin, we hypothesized that septin filaments may constitute a cytoskeletal membrane cortex in myelin. we find that septin filaments localize to the non-compact adaxonal myelin compartment, which is considered relevant for the metabolic maintenance and the turnover of myelin. for functional analysis, we generated mice lacking the most abundant septin of myelin, sept8, from myelinating glia. our results indicate that sept2, sept4, sept7 and sept9 multimerize with sept8 and that this interaction is essential for their incorporation into myelin. myelin lacking septin filaments display pathogenic outfoldings emerging from adaxonal myelin. we propose that this phenotype reflects that myelin septins provide long-term structural integrity to the adaxonal compartment of cns myelin. here, we used primary opc cultures to investigate the mechanisms underlying gpr17 endogenous regulation. this is quite important since gpr17 is upregulated at injury sites in both a demyelinating in vivo model (boda et al., glia, 2011) and in multiple sclerosis (ms) patients (chen et al., nat neurosci 2009), which, could, in turn, cause defective myelination. in line with these findings, we have recently shown that gpr17 forced over-expression at late differentiation stages, obtained by transfecting cultured opcs with a gfp-gpr17 fusion vector, indeed impairs terminal cell maturation. this suggests that the receptor needs to be down-regulated/ desensitized to allow opc terminal maturation. physiologically, gpr17 downregulation may occur through agonist-induced receptor phosphorylation via g-protein coupled receptor kinases (grks) (daniele et al., j pharm exp ther, 2011; frantangeli et al., j biol chem, 2013), which are, in turn, controlled by mtor kinases, and are altered in ms. our in vitro data show that rapamycin, an inhibitor of mtor kinase, reduces grk2 levels, with parallel increases in gpr17 expression and strong impairment of opc maturation. globally, these data suggest that dysregulation of these interconnected pathways leading to aberrant gpr17 overexpression may prematurely block opc maturation. analysis of gpr17 in vivo in ms models will confirm if the receptor is dysregulated during disease development and will help finding ways to re-establish myelin repair in demyelinating diseases. loss of central nervous system myelin, and the failure of remyelination by oligodendrocytes, contributes to the functional impairment that characterizes neurodegenerative disorders and demyelinating diseases such as multiple sclerosis (ms). since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting (re)myelination are pivotal in precluding progression of disease. although the reasons for remyelination failure are likely multifactorial, one of the major impediments to successful remyelination is the altered lesion microenvironment. our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure. here, we aim at elucidating whether exogenous gangliosides, i.e., cell surface lipids with a potential to modulate fibronectin-cell surface interactions, could overcome fibronectin-mediated inhibition of myelination. of the gangliosides tested, only addition of gd1a, perturbed adherence of oligodendrocyte progenitor cells to fibronectin, but not to poly-l-lysine, without affecting cell viability. gd1a slightly increased growth factor-mediated proliferation on fibronectin, but in the presence of the lipid migration of oligodendrocyte progenitor cells on fibronectin was reduced. furthermore, addition of gd1a increased myelin-like membrane formation on (aggregated) fibronectin, whereas gm1 reduced oligodendrocyte differentiation, as reflected by a decrease in the number of mbp positive cells. most interestingly, gd1a counteracted the myelination inhibiting effect of aggregated fibronectin in co-cultures of dorsal root ganglion neurons and oligodendrocytes. also, gd1a enhanced remyelination in demyelinated cerebellar slice cultures that contained fibronectin aggregates. taken together, gd1a is able to overcome the inhibiting effect of aggregated fibronectin in remyelination. given the persistent presence of these aggregates in ms lesions, gd1a might therefore act as a potentially novel molecular tool to selectively modulate the impeding signaling environment, apparently detrimental to remyelination. its mechanism of action and exploration of gd1a's potential in the treatment of ms, are currently investigated. in the peripheral nervous system (pns) it is secreted by schwann cells and fibroblasts and its expression is strongly induced upon injury. we are interested in the function of apod in the molecular events that take place after a pns injury. we have studied in vivo the process of wallerian degeneration following sciatic nerve crush and we have assayed ex vivo the phagocytosis of labelled myelin from wt and apod-ko mice by flow cytometry. the analysis of cellular processes and protein or mrna expression of a group of signalling molecules induced by injury shows that the lack of apod results in an exacerbated mcp-1 and tnfa-dependent macrophage recruitment. at the injury site, free aa decreases in the wt. lack of apod results in higher basal levels and a stronger injurytriggered depletion of aa. control by apod of the availability of aa to produce the lipid mediators involved in macrophage recruitment is therefore a key mechanism that conditions both wallerian degeneration and injury resolution later on. on the other hand, the phagocytosis of apod-ko cns myelin is less efficient than the phagocytosis of wt myelin. these results indicate that there are also genotype-dependent differences in myelin composition and/or in the interaction between myelin and macrophages. an electron microscopy analysis of crude myelin preparations reveals that the cns myelin from apod-ko mice has abnormal periodicity and shows defective myelin compaction. lipid analysis of apod-ko myelin shows altered phospholipid composition, particularly in phosphoinositid species. a study of the function of apod in the myelination process is currently underway, our results demonstrate that apod function is relevant for both, myelin membrane properties that influence myelin-macrophage interactions, and the control of the lipid-mediated signalling events controlling the extent of macrophage recruitment. adhesion g protein-coupled receptors (ad-gpcrs) are a subgroup of gpcrs that facilitate cell-cell and cell-matrix interaction. structurally, they differ from other gpcrs by the presence of an extremely larger extracellular n-terminal region that is separated from the 7-transmembrane region by a gpcr autoproteolysis-inducing (gain) domain that in most ad-gpcrs facilitates an auto-catalytic process to produce an nand c-terminal fragments during the maturation process. gpr56 is a member of the ad-gpcr family and its mutations are responsible for a human brain malformation known as bilateral frontoparietal polymicrogyria (bfpp). individuals with bfpp present with changes in the white matter tracts in the form of hypomyelination, in addition to cortical malformation. here, we show that gpr56 knockout mice and zebrafish exhibit a reduction of myelin-specific protein expression, fewer myelinated axons and decreased number of mature oligodendrocytes in the central nervous system (cns). during cortical development, gpr56 functions together with its ligand collagen iii to regulate neuronal migration and cortical lamination. however, it is not clear whether collagen iii is also the ligand of gpr56 during cns myelination. gpr56 has a very long and poorly characterized n-terminal fragment, allowing for the possibility of multiple binding partners that mediate cell-extracellular matrix interactions. indeed, gpr56 also binds to tissue transglutaminase (tg2) in melanoma cells. we have begun to study the possible role of collagen iii and tg2 in oligodendrocyte development and cns myelination. the likelihood of these two proteins as being the ligand(s) of gpr56 during oligodendroglial development will be discussed as will our ongoing work to define the role of gpr56 in cns myelination. proper control of cell cycle, proliferation and differentiation is fundamental to regulate oligodendrocyte (ol) development and recruitment of oligodendrocyte precursors (opc) after brain damage. failure in this process results in severe dysmyelinating disorders and defective brain repair. the molecular machinery that couples differentiation to proliferation withdrawal plays therefore a critical role in brain development and repair and is regulated by multiple extracellular cues including extracellular matrix (ecm) and growth factor derived signals. however, how these signals are integrated into ol to control cell cycle progression and differentiation is only partially understood. we are investigating the role of jun activating binding protein 1 (jab1), an intracellular signalling molecule, shuttling between nucleus and cytoplasm, involved in the control of cell proliferation, survival and gene transcription. recent evidence showed that jab1 function is regulated by ecm and growth factors. we demonstrated that jab1 is expressed in glial cells in peripheral and central nervous system (cns), and conditional inactivation of jab1 in schwann cells results in dysmyelinating neuropathy. here we present preliminary data showing that jab1 plays a role in cns development. mice with conditional inactivation of jab1 in ol, by cnp-cre tansgene, show cns hypomyelination, including corpus callosum, optic nerve and spinal cord. concomitant fiber degeneration was observed. our preliminary data suggest that jab1 expression in ol plays a role in cns myelination and possibility axonal survival. c. gonsior, j. trotter university of mainz, mainz, germany myelin basic protein (mbp), a major component of central nervous system (cns) myelin, is essential for oligodendroglial function and myelination in the cns and its expression is tightly regulated in time and space. the mrna of mbp is sorted to cytoplasmic rna granules and transported up to the distal processes of oligodendrocytes in a translationally silenced state. we and others could show that, dependent on axonal signals, activation of the non-receptor tyrosine kinase fyn leads to a release of translational inhibition allowing synthesis of mbp protein at the axon-glial contact site. a key factor in this pathway is the fyn target and rna-binding protein heterogeneous nuclear ribonucleoprotein (hnrnp) a2, that recognizes the a2 response element (a2re) in the mbp 3 0 utr and orchestrates the dynamics of the mbp mrna granule. moreover, we identified hnrnp f as an additional target of fyn kinase present in these ribonucleoprotein complexes and contributing to posttranscriptional regulation of mbp. cell stress conditions, as present in various disease states such as multiple sclerosis, may result in the formation of cytoplasmic rna-containing stress granules (sgs), potentially influencing the fate of myelin transcripts including their translation.mbp mrna was shown to be sorted to these cytoplasmic foci. we here identified the hnrnps a2 and f to be associated with oligodendroglial sgs. de-regulation of such factors involved in mbp mrna metabolism could affect stress dependent synthesis of mbp and thus (re-)myelination and myelin maintenance. we performed the experiments in biozzi abh mice known to develop a chronic relapsing-remitting form of eae following immunization with myelin oligodendrocyte glycoprotein. our findings suggest that in demyelinating lesions, myelin layers are pulled off from the myelin sheath and form bulb-like structures that we call "myelinosomes". we can detect the formation of such structures in acute and in chronic stages in the animal model, as well in biopsies from multiple sclerosis patients. we are currently investigating the molecular interactions that underlie the formation of myelinosomes to gather further insight into the mechanisms that underlie immue-mediated demyelination. adenosine is a potent neuromodulator which can be released from most cells and its extracellular concentration increases dramatically after brain injury. it acts on four g protein-coupled receptors, a 1 , a 2a , a 2b and a 3, and their activation is involved in myelination as well as in apoptosis linked to neurodegenerative diseases. in this study, we initially found that rat oligodendrocytes in vitro express all four adenosine receptor subtypes, adenosine transporters ent1 and ent2 and the adenosine degrading enzymes adenosine deaminase and adenosine kinase. activation of adenosine receptors caused mitochondrial dysfunction and oligodendroglial apoptosis. notably, selective activation of adenosine receptors revealed that a 3 receptors are the main subtype mediating oligodendrocyte death. thus, a 3 receptor agonist 2-ci-ib-meca caused a robust increase in ros levels, mitochondrial membrane depolarization and caspase-dependent apoptosis. in turn, selective a 3 receptor activation induced mapk modifications compatible with cellular damage, which was prevented by the selective antagonist mrs1220. the modulation of g protein and adenylate cyclase suggested that 2-ci-ib-meca acts via camp-pka. moreover, selective activation of a 3 receptor triggered intracellular calcium mobilization via plc pathway. finally, we used cerebellar organotypic slices and optic nerve ex vivo to investigate adenosinemediated oligodendroglial death in more integral preparations. consistent with data in dissociated cultures, a 3 receptor activation induced a significant damage in the optic nerve as well as in the cerebellum white matter, an effect that was prevented by caffeine, an adenosine receptor blocker. together, these results indicate that adenosine can trigger oligodendrocyte death via activation of a 3 receptors, and suggest that this mechanism may contribute to the etiology of demyelinating diseases. question-one of the commonest forms of inherited neuropathy, xlinked charcot marie-tooth disease (cmt1x) leads to progressive distal muscle weakness and sensory loss. the disease is caused by a large number of mutations in the gjb1 gene encoding the gap junction protein connexin32 (cx32). cx32 is expressed by schwann cells in peripheral nerves and forms important channels of communication between the layers of the non-compact myelin sheath and is necessary for the homeostasis and survival of the myelinating cells and the axon. transgenic disease models generated in our lab as well as clinical-genetic analysis of large number of patients have demonstrated that most cmt1x mutations result in loss of cx32 function and failure to form gap junctions. cx32 ko mice offer a well characterized and relevant model of the disease to study future therapies for cmt1x, for which gene replacement may be an effective treatment strategy. methods-for this purpose, we have generated a novel 3 rd generation lentiviral vector to allow gene delivery to peripheral nerves. in this vector, the cx32 open reading frame is placed under the control of cell-specific promoter for schwann cells, the rat mpz/p0 promoter. the expression cassette also includes a green fluorescent protein (egfp) as a marker. we have successfully produced lentiviral vector particles and delivered them to the sciatic nerves of 2-month old wild type (wt) and cx32 ko mice, immediately distal to the sciatic notch. the expression of egfp on wt background and of cx32 on ko background was determined by western blot analysis and immunohistochemistry of teased nerve fibers at different time points after injection. results-our initial studies in wt mice (n 5 15) show that more than 60% of schwann cells from different nerve fascicles expressed egfp as far as 5 mm distal to the injection site. expression started a week after the intraneural injection and remained stable for up to 3 months. when the lentiviral construct was injected into the sciatic nerves of cx32 ko mice (n 5 5), virally delivered cx32 was expressed and was properly localized at the non-compact myelin areas including the paranodal regions and incisures of myelinated fibers, where gap junctions are normally formed. poster abstracts s139 glia conclusions-thus, we have generated lentiviral vector that leads to efficient gene expression specifically in schwann cells. these results indicate that gene replacement therapy is feasible and may lead to correction of the gap junction loss in myelinating cells of the pns. perinatal inflammation is intensively discussed to have a long-term impact on various cell populations and on the development of autoimmune diseases such as multiple sclerosis (ms) in adulthood. in order to determine long-term effects of perinatal inflammation on the course of demyelination in adulthood we mimic a bacterial inflammation by exposure to lipopolysaccharide (lps) of either pregnant or newborn mice. demyelination as a "second hit" was induced by feeding adult mice with the oligodendrocyte toxin cuprizone (bis-cyclohexanone oxaldihydrazone). a single prenatal lps injection at e13.5 did not affect the course of demyelination in adulthood. in contrast, serial postnatal lps injections lead to a delayed demyelination and a reduced number of activated microglia in the corpus callosum, suggesting a long-lasting effect of perinatal inflammation on the function of microglia. surprisingly, mice exposed to lps after birth showed an enhancement of early remyelination accompanied by an increased number of newly differentiated oligodendrocytes. furthermore, independent of cuprizone administration the perinatal lps injections seem to induce a long-term impact on the blood-brain barrier (bbb) by decreasing the number of claudin-5 positive vessels. in summary, perinatal inflammation mimicked by the administration of lps has long-lasting effects on the bbb, microglia activation, and remyelination. question: epigenetic control is crucial for the differentiation of a variety of cells including oligodendrocytes, the myelinating glial cells of the central nervous system. however, studies about the implication of epigenetic factors in peripheral nervous system maturation are just emerging and we were wondering whether methylation of histones is involved in this process. methods: we describe the function of ezh2 in primary schwann cells and drg cocultures using immunohistochemistry, transfection and lentiviral transduction, shrna, qrt-pcr analysis, morphological analysis, western blotting and chromatin immunoprecipitation. results: here, we demonstrate for the first time the impact of a histone methyltransferase, encoded by the enhancer of zeste homolog 2 (ezh2) gene, on schwann cell differentiation. in sciatic nerves, ezh2 expression was found in schwann cells and to peak perinatally. suppression of ezh2 expression in cultured primary rat schwann cells reduced the length of cell processes. these morphological changes were accompanied by widespread alterations in the gene expression pattern, including downregulation of myelin genes and induction of p57kip2, which we have recently identified as an intrinsic inhibitory regulator of schwann cell maturation. in addition, we show that ezh2 suppression in dorsal root ganglion cocultures interferes with in vitro myelination. chromatin immunoprecipitation analysis revealed binding of ezh2 at the p57kip2 promoter and reduction of histone h3k27 trimethylation upon gene suppression. ezh2 suppression-dependent effects on morphology and myelin genes could be reversed by concomitant suppression of p57kip2, indicating that p57kip2 is a downstream effector of ezh2. furthermore, we describe hes5 as transcriptional repressor of myelin genes in schwann cells, which was induced upon ezh2 suppression and downregulated in p57kip2-suppressed schwann cells. conclusions: we have identified a molecular link between histone methylation and control of schwann cell differentiation and demonstrate that this epigenetic mechanism is crucial for glial differentiation to proceed. previous studies, performed primarily in cell culture, suggest that electrical activity may regulate multiple stages of oligodendrocyte development including proliferation, migration, initial wrapping and myelination. in this study, we have examined the requirement for electrical activity in oligodendrocyte development in vivo using zebrafish as a model system. results: we find that electrical activity is not required for proliferation, migration, or the initiation of axon wrapping. rather, our data indicate a specific role for electrical activity in oligodendrocyte differentiation by regulating a subset of myelin genes, including myelin basic protein (mbp). silencing electrical activity with tetrodotoxin reduced mrna expression of mbp, as assessed by rna in situ hybridization and quantitative rt-pcr. in contrast, myelin protein zero, claudin k, claudin 11a, ugt8, and 36k levels were not regulated by electrical activity. ongoing experiments are aimed to uncover the mechanism of activity-dependent mbp mrna expression during oligodendrocyte differentiation. conclusions: taken together, these data indicate that electrical activity can modulate myelin gene expression in vivo but do not support the notion that electrical activity drives key axon-glia messengers that are required for the initial stages of myelination. (fancy et al., 2011) . eight-week-old lgals3-/-and wild type (wt) mice were fed a diet containing 0.2% cpz w/w during 6 weeks, after which cpz was withdrawn in order to evaluate remyelination 2 weeks after. according to our results, cpz-induced demyelination in lgals3-/mice showed an exacerbated astrocytic and microglial response as compared to wt littermates. electron microscopy showed a significant lack of myelinated axons in lgals3-/-mice as compared to controls. furthermore, the few myelinated axons present were nearly 50% less myelinated than those of controls and were found to be collapsed. remarkably, the remyelination process seemed to be faster in lgals3-/-mice than in wt. remyelinated lgals3-/-mice showed a higher myelin basic protein (mbp) recovery rate as compared to their controls. flow cytometry assays showed a sharper microglial response in lgals3-/-mice, which was supported by an exacerbated number of cd11b1 and cd451 cells. however, electron microscopy images from remyelinated lgals3-/-animals showed, again, collapsed axons with a defective myelin wrap, as compared to wt mice showing normal axons without any relevant myelin wrap disruption. behavioral performance observed during cpz treatment recovery correlates with alterations in the morphological studies, which show that neither lgals3-/-nor wt mice reach basal myelination levels. we have shown that new oligodendrocytes (ols) continue to be generated in healthy adult mice after 8 months of age, even in the optic nerves, which are considered to be completely myelinated already [1] . this raises the question of whether the adult-born ols and myelin are engaged in remodeling existing myelin (e.g. intercalating among the existing myelin sheaths), or replacing ols that die in use. to ask whether ols die and are replaced during healthy adulthood we generated transgenic mice that express icreer t2 under the transcriptional control of the myelin basic protein promoter (mbp6-icreer t2 ). the $6kb mbp upstream fragment that we used is reported to direct transcription to ols but not to either ol precursors (ops) or schwann cells [2] . we confirmed that our mbp transgene targets cre recombination specifically to differentiated ols by crossing to ros26-yfp conditional reporters; when tamoxifen was administered to double-transgenic offspring, essentially all yfp 1 cells in the cns were also cc1 1 . when mbp6-icreer t2 is crossed to tau-mgfp conditional reporter mice (mgfp is membrane-tethered) we can visualize differentiated ols and their myelin internodes in white and grey matter, allowing us to follow the fates of myelinating ols for extended periods of time after tamoxifen labeling. following tamoxifen administration to mbp6-icre t2 : tau-mgfp mice on postnatal day 60 (p60) or p120, a large proportion of ols became gfp-labelled in the corpus callosum, optic nerve and spinal cord. examining these mice at increasing times post-tamoxifen will reveal if and how the number and fraction of mgfp-labeled ols/ internodes changes with age -whether there is net loss or gain of ols or whether new ol synthesis is balanced by the rate of ol death (i.e. there is ol turnover). our preliminary results indicate that new ol synthesis exceeds ol death, so the function of adult ol genesis is not simply to replace dying ols. this study aims to identify protein networks that participate in peripheral myelin formation and maintenance. we use a cell culture system, where c57bl/6j mouse embryos (e13.5) are used for the isolation of dorsal root ganglia neuron explants. explants are cultured in matrigel, and endogenous schwann cells appear in the culture within a few days. myelin formation is induced by adding myelination-promoting substances to the culture medium. immunocytochemistry is used to evaluate the myelination efficiency and the developmental timetable of myelination in culture. electron microscopy is used to study structural features of myelin in culture and to compare its ultrastructural features with those of myelin in situ. rna microarray analysis is performed to study gene expression changes during myelin development in culture. the rna expression data indicates the changes in the rna expression levels, but it does not give information about the amount of final protein product, their posttranslational modifications or stability. we combine the rna expression analysis with a proteomics approach. proteins are extracted for 2-dimensional electrophoresis analysis at the same time points, and protein spots exhibiting time-dependent changes in their expression levels are analyzed by mass spectrometry. we show that mouse dorsal root ganglion explant cultures produce substantial amounts of mature myelin in 3 weeks after the induction of myelination. ultrastructural analysis shows that myelin in our cell culture system has the same structural characteristics as those observed in peripheral nerves in situ. rna microarray analysis reveals changes in myelin-, nervous system-and schwann cell-related genes. the most significant changes in gene expression occur at the induction of myelination. at the protein level, peaks of specific proteins can be observed at the first and the last time points, which complement the results obtained using microarray data. we have developed a reproducible and efficient method to study molecular changes in myelination at the proteomic and genomic levels. this approach will be exploited further for the characterization of the molecular networks involved in the myelination process. results: our findings show that primary rat schwann cells respond to ivig stimulation with altered cell morphologies accompanied by an accelerated growth of cellular protrusions in early stages of the differentiation process. stimulation with ivig was also found to reduce cellular proliferation rates without affecting cell survival. furthermore, we observed that ivig treatment transiently boosts myelin gene expression of immature cells, whereas myelin gene expression of differentiating schwann cells is promoted on a long-term scale. importantly, myelin gene expression responses cannot be detected upon application of igg1 control antibodies (herceptin, synagis, avastin). in addition, we could demonstrate that primary rat schwann cells express the cd64 fc receptor and that in differentiating schwann cells cd64 levels were significantly increased. on the other hand, we could also reveal a specific binding of the human ivig on the schwann cell surface. conclusions: we therefore conclude that schwann cells are not only able to respond to but also specifically bind immunoglobulins and that ivig stimulation can promote their differentiation. here, we demonstrate that cnp deficient mice exhibit alterations in mechanical and thermal sensation. histological analyses of cnp deficient mutants reveal loss of sensory axons when quantified in the dorsal root and the predominantly sensory saphenous nerve. in contrast ventral roots remain unaltered. moreover, electron microscopical assessments of saphenous nerves display a hypermyelination of small to mid-sized caliber axons and an overrepresentation of the smallest fibers. the myelin pathology of sensory axons is reflected by alterations of sensory nerve conduction velocities. thus, our data demonstrate that, contrary to findings in the central nervous system, cnp has an impact on myelination and is especially important for the integrity of small to mid-sized caliber axons within the sensory peripheral nervous system. to unravel the pathogenetic mechanism of the s63del mutation, we generated transgenic mice that express p0s63del protein and manifest a demyelinating neuropathy similar to cmt1b. p0s63del is a misfolded protein retained in the er, where it activates a chronic unfolded protein response (upr). the upr is a cellular response to er stress, aimed to reduce the load of abnormal proteins by attenuating protein translation, increasing the er folding capacity and stimulating protein degradation. among the degradation strategies upregulated by the upr is the er-associated-degradation (erad) pathway. in erad, proteins destined for degradation are retrotranslocated from the er to the cytosol, ubiquitylated and degraded by the proteasome. microarray analysis of s63del nerves revealed that several erad genes, such as derlins, are strongly upregulated and derlin-3 expression returns towards normal in association with the amelioration of demyelination produced by ablation of chop, indicating a possible involvement of this pathway in cmt1b neuropathy. derlins elicit particular interest for exerting a protective role in several er stress-mediated diseases. in s63del nerves, derlins co-immunoprecipitate with p0s63del protein and, finally, derlin-1 overexpression reduces the level of er stress in cos7 cells expressing p0s63del protein, suggesting a positive role in the modulation of p0s63del protein retrotranslocation. these observations indicate that derlins may be part of the schwann cell adaptive response that attempts to cope with chronic er stress by modulating p0s63del clearance. we are now in the process of investigating the role of derlins, in the pathophysiology of pns myelination using in vitro, ex vivo and in vivo approaches. during the myelination process, the oligodendrocyte extends processes to and wraps around multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. this is one unique example of how cells in the central nervous system establish asymmetry. transport of mrna and local regulation of protein synthesis represent one mechanism by which such cellular asymmetry can be generated and the different requirements for myelin sheath at each axo-glia interaction can be controlled. the mrna of a key myelin sheath protein, myelin basic protein (mbp), is known to be transported into the oligodendrocyte processes, and a tight temporal and spatial control of mbp mrna translation is thought to be required for normal myelination. the molecular basis for the tight control of mrna transport and translation is the assembly of large mrna-protein complexes. prior work has identified a number of proteins that interact with the mbp mrna, including hnrnp-a2, hnrnp-k and hnrnp-e1. however, it is currently unknown how these protein functions together to prevent translation during transport. it is also unknown how targeting of the mbp mrna to the myelin sheets occurs. to delineate the precise role of the individual binding protein in regulating mbp mrna translation, we have identified regulatory elements within the 3 0 utr of the mbp mrna involved in translational inhibition, and we have identified binding sites in the mrna for hnrnp-k and hnrnp-e1. furthermore, we have analyzed the effect of sirna-mediated knockdown and overexpression of these proteins in primary oligodendrocytes. together, our results allow us to propose a model, in which the individual mrna binding proteins are assigned distinct roles for correct spatial and temporal expression of mbp. remarkably, this manipulation was sufficient to achieve both the expansion of oligodendrocyte precursor cells (opc) and the de novo myelination of a large fraction of enlarged parallel fiber axons in the cerebellar molecular layer, independent of axonal neuregulin-1 expression. by laser-guided microdissection and gene expression profiling of cerebellar gc, we identified neuronal factors that promote opc proliferation and myelin synthesis in vitro. these findings suggest that oligodendrocytes and their precursors respond to multiple 'instructive' signals expressed by cns neurons in vivo, but only on axons with diameters >0.2 lm that became 'permissive' for myelination. l. marziali, p. franco, j. pasquini university of buenos aires, caba, argentina promyelinating effects of both apo-transferrin (atf) and thyroid hormone (th) on rat brain are well documented. th effects are mediated by nuclear receptors which act as transcription factors and regulate different cell processes. previous results from our laboratory showed that th promotes the commitment of oligodendrocyte progenitors (opcs) to mature myelinating oligodendrocytes (olg). on the other hand, atf is able to promote the commitment of neural stem cells (nsc) to cells of the oligodendroglial linage and favors olg maturation after an intracranial injection. our previous demonstration that th administration is able to up-regulate tf mrna expression leads us to hypothesize that both factors converge in the control of oligodendrogenesis. to test if the combined effects of both atf and th are required for proper myelination in the rat brain, studies were done at p10 and p20 for each experimental condition. a hyperthyroid state was rendered by daily subcutaneous th administration from the day of birth (hyper). hypothyroidism was achieved by giving propylthiouracil to the mother from gestational day 18 to the end of the experiment (hypo). half of the hypo pups received an intracranial injection of atf at postnatal day 3 (hypo1atf). a set of euthyroid animals received an intracranial injection of atf at postnatal day 3 (atf). control groups received an intracranial injection or subcutaneous saline solution (c). at p10, hyper animals showed an up-regulation of 55% in tf mrna expression and 87% in protein levels as compared to controls. hypo showed a decrease of 25% in tf mrna levels. this effect was not affected by exogenous administration of atf. at p20, hyper showed no differences in the expression of tf mrna or protein levels as compared to controls. hypo showed a decrease of 25% in tf mrna and a 40% decrease in protein levels. this effect was not affected by exogenous administration of atf. no differences were observed in the expression of insulin growth factor i (igf-i) or igf-i receptor between groups at p10 or p20. immunohistochemical analyses were performed at p20 in all groups usingspecific markers anti caii, anti mbp, anti rip and pdgfra. our conclusion is that, in a hypothyroid state, tf is not able to produce olg and myelin maturation at the corpus callosum, which indicates that th is necessary for atf action. however, the finding that hyperthyroid animals showed a significant increase in tf mrna strongly suggests that tf could be involved in th effects. new experiments are carrying out in order to knock down the tf gen to probe this last piece of evidence. h. yigit, l. meyer, c. gonsior, p. hoch-kraft, j. trotter johannes gutenberg university, mainz, germany myelination is the prerequisite for fast saltatory conductance of action potentials. in the central nervous system, the myelin sheath is produced by oligodendrocytes which extend processes and wrap the axons. this structure is subsequently compacted by the help of certain proteins. of major importance here is the myelin basic protein (mbp) which interacts with the cytoplasmic leaflet of the plasma membrane. mbp is a highly basic protein and binds to the negatively charged plasma membrane with high affinity. it has 6 isoforms in mice produced by alternative splicing. the mostly studied 4 isoforms are 14 kd, 17 kd, 18,5 kd and 21,5 kd. interestingly, exon-ii containing 17 kd and 21,5 kd isoforms are actively transported into the nucleus while 14 kd and 18,5 kd isoforms are rather localized to the distal part of the plasma membrane. transport to the nucleus depends on temperature and energy. recently, it was reported that exon-ii contains a non-canonical py nuclear localization signal. as a cytoplasmic protein, mbp is essential for proper myelination. furthemore mbp is supposed to have multiple additional functions, e.g. in calcium homeostasis and cytoskeleton dynamics. it was also shown that the expression level of mbp exon-ii containing is changed in schizophrenia patients. selectively transport of exon-ii containing isoforms to the nucleus raises the question of its nuclear functions which have not been elucidated yet. this study aims to unravel the role of exon-ii containing mbps in the nucleus. gene expression profiling of the microdissected svz regions revealed that wnt-responsive genes were enriched in the postnatal dorsal svz compared to the lateral svz. we performed inhibition of gsk3b by intraventricular infusion of ara to initiate wnt-signalling. several approaches confirmed activation of the wnt-signalling pathway in the dorsal svz as well as within ops. thus, gene expression profiles of wnt-target genes of the dorsal svz indicated that axin2, lef1, fzdl1 and tcf4, but not those other pathways, were dramatically up-regulated. furthermore, nuclear b-catenin was transiently up-regulated in the dorsal svz including in a large population of sox10-egfp expressing cells following pharmacological wnt-activation. gene expression profiling and immunostainings revealed that the densities of nscs, cycling progenitors and subsequent op differentiation in the dorsal svz were augmented. in parallel, we genetically manipulated wnt-signalling in stem and progenitor cells of the dorsal wall of the lateral ventricle. targeted dorsal svz electroporation of cre-gfp plasmids in floxed stabilised b-catenin (gain-of-function) and floxed b-catenin (loss-of-function) transgenic mouse lines were performed and resulted in a phenocopy of the results observed following ara-intraventricular infusion. in summary, our findings illustrate that wnt-signalling endogenously persists in the dorsal svz after birth. pharmacological as well as genetic interventions reveal an important role of this signalling pathway in the regulation of op genesis during the period of myelination. in the cerebellum the generation of neurons and glia and the relationships between these two lineages are poorly understood. all the cerebellar neuronal phenotypes derive from two distinct embryonic germinal neuroepithelia that are restricted regarding the neuronal cell fate: the rombic lip (rl) gives rise to the glutamatergic neurons, whereas the ventricular zone (vz) generates all gabaergic neurons. in particular, different types of inhibitory interneurons are generated during the postnatal life from a common pool of progenitors expressing the transcription factor pax2. on the other hand, the glial development is relatively unexplored. part of the oligodendrocytes derives from extracerebellar sources, whereas some of them derive from endogenous precursors. astrocytes, instead, may derive from progenitors that delaminate from the vz and continue to divide in the prospective white matter (pwm) up to postnatal life. given the vn origin of both astrocytes and interneurons, we wondered whether a lineage relationship exists between these two cell populations and whether they share the same bipotent progenitor. in order to address these issues, we performed a genetic fate mapping study of glastcreert2 mice. we showed that both cerebellar gabaergic interneurons and astrocytes derive from proliferating glast 1 precursors. in particular, we found that these progenitors are able to produce interneurons at earlier developmental phases, whereas after p6 they appear restricted to the glial lineage. proliferating glast 1 cells that may produce interneurons comprise bergmann glia and pwm progenitors. however, when recombination was specifically induced in bergmann glia, we found that these cells were not neurogenic. therefore, we concluded that glast 1 progenitors reside in the pwm. to get further insight in the behaviour of these precursors, we injected in the pwm a gfp-containing lentiviral vector with a specific tropism for astroglial cells. some days after, infected cells generated pax2 1 -interneurons that were able to mature into interneurons of different cortical layers. moreover, in vitro and in vivo clonal analysis of pwm derivatives indicates that the pwm is neurogenic and both astrocytes and interneurons may derive from a common progenitor. evidence is accumulating that neurogenesis in the subventricular zone (svz) is boosted after trauma or ischemia, also through the interaction with surrounding parenchyma or niche cells. nevertheless, the number of newborn neurons that survive and integrate in the damaged areas is negligible, suggesting a non-permissive environment. thus, understanding the complex signaling network guiding neuroblast generation/survival could help identifying strategies to limit negative inputs and promote regeneration. extracellular nucleotides (ents) are among the hypothetical modulators of svz cell functions, especially under pathological conditions where their concentrations raise several folds and they contribute to reactive astrogliosis. few literature data have recently pointed for a role of the p2y 1 receptor subtype (one of the 8 known g protein-coupled nucleotide receptors) in controlling the proliferation and differentiative potential of svz cells. thus, we tested the ability of adpbs, a stable p2y 1 agonist, to modulate stem cell properties in the adult brain, with a focus on the possible modulatory effects exerted by reactive astrocytes. the administration of adpbs in the lateral ventricle of adult mice led to reactive astrogliosis in the surrounding brain parenchima, and to a massive reaction of gfapexpressing precursors and astrocytes in the svz. also proliferation was increased, paralleled by a significant expansion of the population of mash11 transit-amplifying cells and of doublecortin1 neuroblasts. thanks to the conditional glast::creert2 yfp mouse model, we also demonstrated that adpbs promoted the proliferation of glast-expressing progenitors in the neurogenic niche, and sustained their progression towards the generation of rapidly dividing transit-amplifying cells. in vitro the nucleotide analog increased the proliferation of svz cells grown as neurospheres, and their differentiation towards neurons, fully confirming in vivo data. interestingly, a significant enhancement in neurosphere generation was detected when svz cells were initially grown in the supernatant of astrocytes exposed to adpbs, and then shifted to normal medium. this suggests that adpbs stimulates the release of yet-to-be identified astrocytic mediator(s) whose removal from the culture medium boosted proliferation of svz cells. our results further strengthen the notion that the purinergic system is a key regulator of the neurogenic potential of svz cells, both directly and through the involvement of reactive astrocytes. cambridge stem cell institute, cambridge, united kingdom oligodendrocytes, one of the principal glial cell-types of the central nervous system, are critical for neuronal function and represent the primary target of many neurological diseases, including multiple sclerosis and leukodystrophies. despite the recent progress in stem cell research and the possibility to generate various neural cell types in vitro, the derivation of oligodendrocytes from human pluripotent stem cells remains inefficient and unreliable. we employed a new experimental approach termed "direct cellular reprogrammng" which was already succesfully applied for the generation of different types of neurons and neural stem cells. aided by bioinformatic filtering we have generated a pool of seventeen candidate reprogramming factors that could potentially convert fibroblasts directly into oligodendrocyte lineage cells. combinatorial testing of the candidate factors in overexpression experiments has revealed a combination of just two transcription factors that was sufficient to convert mouse embryonic fibroblasts directly into o4-expressing oligodendrocyte precursors. supplementation of additional factors and multicistronic gene delivery strategies render the reprogramming process more efficient and widen the applicability of this protocol. cellular reprogramming offers a new tool for the generation of oligodendrocyte precursors. this approach, that circumvents the protracted specification and differentiation process inherent to the oligodendrocyte lineage will foster the quest for a culture system of human oligodendrocyte lineage cells. transplanted are derived from progenitors within the intestine. to gain further insight in possible stem cell niches the compelling evidence that mouse enteric glia can also be neuronal precursors was related to human tissue. human colon from infants with hirschsprung's disease was investigated with a panel of glial and stem cell markers. tissue samples from infants with hirschsprung's disease were investigated with glial and stem cell markers along the gut axis. the tissue samples from ganglionic, aganglionic and transient segments were immuonstained either for s100/nestin, s100/p75, gfap/nestin and gfap/p75. in all samples investigated, nestin positive ganglia could be found, even in the distal parts with a severe hypo-or aganglionosis. beside nestin positive cells in all segments, there were also different expression pattern glial markers within the ganglia, indicating that distinct phenotypes of glia cells could be found. neural and glial precursor cells are present in the ganglionic as well as in the hypoganglionic segments of hirschsprung's colon, suggesting that these cells might be suitable for ncsc generation and further transplantation. the subventricular zone (svz) of the lateral ventricle, the largest adult stem cell niche, has a striking pinwheel organization specific to regions of adult neurogenesis. the pinwheel's core contains the apical endings of b1 cells and in its periphery two types of ependymal cells: multiciliated (e1) and biciliated (e2) cells. previous studies showed that svz cells are reactivated in response to demyelination. however, the mechanisms inducing svz reactivation in response to inflammatory demyelination such as in multiple sclerosis (ms) are poorly understood. we hypothesize that b1 and e, which are in direct contact with csf through cilia, may play a crucial role in initiation of svz reactivation. in the present study, we examined the svz reactivation in mice in which we targeted an ms-like pathology to the forebrain white matter (t-eae). animals were immunized with subclinical doses of myelin oligodendrocyte protein and after 20 days, received a stereotaxic injection of a cytokine cocktail consisting of tumour necrosis factor (tnfa) and interferon (ifn c) into the corpus callosum. to obtain an en face view of the ventricular surface, we dissected whole mounts of lateral ventricle of control and eae animals at 3, 7, 14 and 28 days post-eae induction (dpi). we investigated by immunohistochemistry whether and how the pinwheel architecture of the svz is modified and ependymal cells are reactivated during disease course of eae. in svz niche of t-eae animals we found that the number of b1 cells increased and peaked at 7 dpi compared to control. the number of e2 cells and pinwheels decreased and then reached control level at 7 dpi. while the number of e1 cells remained steady, their surface area increased at 3dpi and then returned to normal size over time. in addition, the number of their ciliary basal bodies increased during eae time courses, and e1 cells highly expressed gfap in eae animals compared to controls. thus, the findings of our ongoing research suggest that neuroinflammation induces some changes in cell number and morphology of svz niche as well as reactivation of ependymal cells, which altogether may contribute to svz reactivation mechanisms. further study will provide detailed information on the homotypic and heterotypic interactions between pinwheel cells in response to t-eae. to test whether hypothalamic tanycytes act as bona fide neural stem/ progenitor cells in vivo, we analysed their immumoprofile and proliferative potential, and lineage-traced them in vivo. for this, we exploited our earlier findings that in the adult hypothalamus, fibroblast growth factor 10 (fgf10) is expressed exclusively by tanycytes (hajihosseini et al. 2008 mcn 37: 857-68) , a radial glial-cell like population that lines the floor and ventral walls of the third ventricle. using a line of fgf10-lacz reporter mice, we found that fgf101 beta tanycytes express a panel of neural/stem progenitor markers such as nestin, musashi1, blbp and sox2. fgf101 tanycytes also incorporated brdu under cumulative brdu-labelling paradigms, and formed neurospheres in vitro. to directly test the potential of fgf101ve tanycytes, we lineage-traced them at p28 and p70 by activating the constitutive expression of the marker gene, tomato-dsred, in these cells and their descendants. this was achieved by applying tamoxifen to fgf10-creer-t2::rosa26r-tomato double transgenic mice. after short survival time-points, tomato was detected exclusively in tanycytes, whilst with longer survival time-point, tomato1 cells emerged in the neighbouring parenhcyma. the majority of these were neun1 neurons with fine arborizations. we noted that the newly-generated neurons become associated mainly with hypothalamic nuclei that regulate appetite and energy-balance. moreover, they respond to appetite/energy balance regulating signals such as acute food deprivation and leptin administration. our findings establish fgf101 beta-tanycytes as a putative population of neural stem/ progenitor cells that generate appetite/energy balance regulating neurons in the postnatal and adult hypothalamus. we used an inducible transgenic mouse line (hgfap-creert2) to conditionally label and fate map putative gfap(1) nscs populations in the adult svz and spinal cord (sc), and compare their self-renewal and multipotential properties. we evidence that a population of gfap 1 cells that share the morphology and antigenic properties of svz-nscs reside in the dorsal and ventral aspects of the central canal (cc) throughout the spinal cord. these cells are non-proliferative in the intact spinal cord, but incorporate edu, an s-phase marker following spinal cord injury. multipotent yfp-expressing neurospheres (i.e. which derive from recombined gfap-expressing cells) were successfully obtained from both the intact and injured spinal cord, confirming their early gfap origin. notably, only spheres isolated from the injured spinal cord revealed long-term self-renewal properties resembling those of svz neurospheres. altogether, this work highlights discrepancies in the identity of nscs that reside in distinct regions of the adult cns. our observations however reconcile divergent views on the location and identity of spinal cord-nscs by showing a population of multipotent gfap 1 progenitors with limited self-renewal properties co-existing alongside with multipotent, self-renewing ependymal cells within the spinal cord central canal. during development neural precursors (nps) both divide, to expand the cell population, and produce many different kinds of neurons and glia. this balance appears to be regulated by par complex proteins, which polarize neural precursors and can thereby direct daughter cells for different fates. how par complex proteins are regulated to appropriately polarize nps remains unknown. in recent years, regulation of gene function by micrornas has emerged as an important mechanism during development. using bioinformatics we identified the polarity gene pard3 as a candidate target for microrna219 (mir-219). mir-219 is specifically expressed in the developing central nervous system (cns) of vertebrates and mir-219-deficient zebrafish embryos have a deficit of oligodendrocytes, the myelinating glial cells of the cns. because a disruption in polarity could affect the types of cell divisions that nps undergo, thus altering the balance of cell types that arise, we hypothesize that neural precursor maintenance is regulated by modulation of polarity cues through mir-219. by using an in vitro reporter assay we found that mir-219 can downregulate expression of a luciferase gene fused to the pard3 3 0 utr. reduction of pard3 function in zebrafish embryos suppressed the oligodendrocyte phenotype resulting from loss of mir-219 function and injection of a morpholino oligonucleotide designed to block binding of mir-219 to its pard3 target sequence phenocopied mir-219 knock down. together, these data provide strong evidence that pard3 is a functionally relevant in vivo target of mir-219. to further investigate the role of mir-219 function we tested expression of np, radial glial and neuronal markers in mir-219-deficient embryos. the number of cells expressing sox2, a marker of nps and neural stem cells, was significantly increased upon mir-219 knockdown. concomitantly, mir-219-deficient embryos had a dramatic deficit of radial glia and late born neurons. these data provide evidence for a new mechanism of np regulation, in which mir-219 regulates pard3 levels, thereby regulating the transition of dividing neural precursors to differentiated neurons and glia. in demyelinating diseases such as multiple sclerosis myelin repair activities based on recruitment, activation and differentiation of resident progenitor and stem cells can be observed. however, the overall degree of successful remyelination is limited. it is therefore of considerable interest to understand oligodendroglial precursor cell (opc) homeostasis and maturation processes in order to develop remyelination therapies. mesenchymal stem cells (msc) were shown to exert positive immunomodulatory effects, to reduce demyelination, to increase neuroprotection and to promote adult neural stem cell differentiation towards the oligodendroglial lineage. we here addressed whether msc secreted factors can influence primary opcs in a myelin non-permissive environment. methods: to this end we analyzed cellular morphologies, expression and regulation of key genes/proteins involved in oligodendroglial cell fate and maturation upon incubation with mesenchymal stem cell conditioned medium. results: this demonstrated that msc derived soluble factors promote and accelerate oligodendroglial differentiation, even under astrocytic endorsing conditions. accelerated maturation featured elevated levels of 2 0 , 3 0 -cyclic nucleotide 3 0 -phosphodiesterase and myelin basic protein expression, reduced glial fibrillary acidic protein expression and was accompanied by downregulation of prominent inhibitory differentiation factors such as id2 and id4. conclusions: we thus conclude that besides the previously established immunomodulatory and neuroprotective roles of mscs these cells can also positively influence oligodendrogenesis in the adult central nervous system. in this study, we evaluated the effects of epo and the epo splicing variant (vepo) on murine neurogenesis ex vivo. for this purpose, we analyzed the survival of cultured neural stem cells (nscs) either exposed transiently to recombinant protein, or transduced with lentiviral vectors to achieve stable protein expression. in comparison to non-exposed controls, recombinant epo and vepo enhanced survival of nscs ex vivo (epo: 150 6 25%; vepo: 250 6 60% survival). nscs transduced with pcl20-mscv-epo-and pcl20-mscv-vepo showed also higher viability ex vivo compared to nontransduced nscs (epo: 182 6 4% vepo: 131 6 4% survival). furthermore, pcl20-mscv-epo, but not pcl20-mscv-vepo, increased the proportion of differentiated neurons when compared to non-transduced controls. thus, stable expressed vepo may have rather an effect on nsc survival than on nsc differentiation. our results demonstrate that vepos are neuroprotective in vitro. vepo may serve as therapeutic protein for treatment of neurodegenerative disease associated with impaired neurogenesis such as huntington disease. in the subependymal zone (sez) cytogenic niche of the adult mouse brain neural stem cells drive the continuous generation of new cells, mostly of olfactory bulb interneurons, but also of cells of the oligodendroglial lineage. previous reports have shown that newly-born cells exit the sez and migrate to the adjacent corpus callosum (cc) where they differentiate into oligodendroglial cells. however, the real contribution of these niche-derived oligodendrocyte progenitor cells and oligodendrocytes (nopcs and noligos) to the oligodendroglial population of the cc has not been assessed so far. in this study we used the hgfap-cre ert2 3 rosa26-eyfp double transgenic mice in order to label specifically the progeny of sez-located adult neural stem cells. we found that cells expressing markers of opcs, such as pdgfra and olig2, are constantly generated in the sez and migrate to the proximal fraction of the cc. interestingly though, these cells do not remain in the cc for more than 15 days with their contribution to the total population of oligodedroglial cells remaining stably below 2% even in the ageing brain. moreover, immunostaining for markers of cell cycle revealed that a higher fraction of nopcs undergoes mitosis, when compared with local opcs; hence, they constitute almost 25% of proliferating opcs of the cc. notably, during their transient presence in the cc, nopcs express markers of maturing oligodendrocytes and are incorporated in established local cell structures. when the cc is challenged with a focal demyelinating insult the local and the niche-derived oligodendroglial machineries exhibit differential cell kinetics, nopcs increase their contribution to the total pool of oligodendroglial cells; however, their presence remains transient. in the human and mouse retina m€ uller glia (mg) are well known to undergo gliosis in all major types of retinal diseases -which sometimes may even lead to scar formation due to proliferative gliosis. some studies suggest that in the mouse retina mg derived neuronal regeneration can be stimulated, but only to a very limited extent. here, we started to find out, if conditional immortalization might stimulate mg derived proliferative gliosis and /or neuronal regeneration. others and we reported that in the juvenile mouse retina, after retinogenesis is finished, some m€ uller glia shift from a quiescent differentiated state into a proliferative state upon damage of retinal explants ex vivo. we now observed that this process is differentially inducible by mitogens like egf (epidermal growth factor). edu (s-phase marker) pulse chase experiments revealed a tremendous increase in mg proliferation within the first two days, a peak of edu positive cells at day 4 (14 122 sem, n 5 4) and a massive decrease until day 6 (4 120.4 sem, n 5 4) per 400 mm of a central retina section. next, we used transgenic mice with tightly and temporally controlled expression of the proto-oncogene sv40 large t-antigen (csv40lt). it is well reported that csv40lt binds several proteins including the tumor suppressor p53 and retinoblastoma and bypasses cell cycle checkpoints. induction of the csv40lt for 6 days ex vivo led to an overall increase in proliferation compared to control. the number of edu1 cells was 8.5 fold increased (csv40lt: 23 126 sem, n 5 4; control: 3 120.16 sem, n 5 4; p our results so far suggest that induction of csv40lt not only overcomes the proliferative restriction of m€ uller glia but also maintains its progeny in the cell cycle over extended period of time. surprisingly, major parts of the generated cell progeny formed gliotic cell clusters, which were all located within the boundaries of the retina. further, we present data of successful m€ uller glia isolation at high purity by fac-sorting. in our current and future work we study the m€ uller glia and its derived progeny to find out the underlying mechanisms that enable neuronal regeneration and prevent gliotic scar formation. in mammals, regeneration capacity of the central nervous system (cns) is considered too low to recover the neurological functions after traumatic injury. in fishes and amphibians, however, the spontaneous regeneration is vigorous enough to complete the anatomical reconstruction and functional recovery even after severe damage of cns, in which the ependymal cells play the central role to exhibit proliferation, epithelial-mesodermal transition, cell migration, support of regenerating axons and neurogenesis to contribute to recovery from injury. these positive roles of the ependymal cells observed in fish and amphibian damaged cns may be good candidates for treating mammalian cns injury. in this study, we attempted to control the activity of the ependymal cells in the adult rodent spinal cord by virus-mediated gene transfer for imitating the reactions that are observed in the ependymal cells of fish and amphibian damaged cns. we introduced the gene known to be expressed in both the ependymal cells and neural stem cells (nscs) and found the proliferation of ependymal cells. in this case, the ependymal cells expressed glial fibrillary acific protein suggesting that this gene regulates proliferation and differentiation into astrocytes. in addition, introduction of another gene, also known to be expressed in the ependymal cells and nscs, caused cell proliferation without differentiation tendency. these findings indicate that the cell fate of the ependymal cells in the adult rodent spinal cord can be modulated by artificial intervention. future studies will clarify whether cell fate modification in the ependymal cells can contribute to anatomical reconstruction and functional recovery in the adult mammalian damaged spinal cord. methods neural stem cells are isolated from different parts of the human gastrointestinal tract, and also from different compartments (muscle, submucous and mucous layer) using enzymatical digestion approaches. generated neurospheres were screened for neuronal, glial, neural stem cell and pluripotency markers, prior and after differentiation. additional experiments were performed where neurospheres were either co-cultured with tissue blocks from different parts of the brain, or transplanted into brain slices from the rat. results neurospheres could be generated from all areas investigated and differentiated on glas coverslips. nestin and musashi-1 could be found in all neurospheres. moreover sox-10, nanog and oct-4 could be found in the differentiating cultures. after differentiation tubulin and pgp 9.5 positive neurons could be found. transplantation experiments showed that cells from the transplanted spheres migrated into the brain slices and differentiated into neurons or glial cells. neurospheres co-cultured with tissue blocks from cortex, cerebellum or hippocampus showed a significant higher outgrowth than neurospheres cultured without any brain tissue. conclusions in general, the ens is a suitable tissue for the isolation of neural stem cells in the individual patient and might so be an appropriate source for autologous neural stem cells. neurogenesis. at the end of embryogenesis, oligodendrocytes and astrocytes appear during the process of gliogenesis. although in the last decades diverse extrinsic and intrinsic factors involved in these developmental processes were identified, our understanding of detailed mechanisms is still comparatively low. in order to increase the knowledge about the cellular and molecular mechanisms that underlie central nervous system development, transfection of nscs showed to be a promising method to understand the function of specific genes and proteins. to overcome the well-known difficulties to transfect cells of the central nervous system, we improved the electroporation conditions to achieve high efficiency transfection and survival rates for embryonic and adult nscs isolated from mouse forebrain structures. one day after electroporation transfection and viability rates of around 80% were achieved in murine nscs (bertram et al. j neurosci meth 2012). due to our interest in differentiation and maturation of oligodendrocytes we are currently improving the transfection conditions of oligodendrocyte precursor cells (opcs), since a sufficient protocol for the transfection of opcs derived from neonatal rat cortices is currently not established. the opcs are isolated from rat cortical tissue by a shaking protocol and transfected with the 4d-nucleofector (lonza). when we used the same pulse protocol that successfully works on nscs, oligodendrocytes were sensitive to the transfection process, and many cells died or did not undergo normal differentiation. this demonstrates that other pulses are needed to electroporate opcs. in cooperation with lonza we tested several recommended pulses to increase the transfection and survival rates of opcs. a dramatic increase of the viability rate has already been achieved. in further experiments the pulse protocol will be improved to obtain transfection rates above 20%. by establishing this transfection protocol we want to accomplish transfection and viability rates comparable to murine nscs. this improved protocol for the transfection of sensitive opcs will allow for reliable studies of gene function by over-expression or knockdown experiments, and will enhance our understanding of cell biological mechanisms important for oligodendrocyte development. recent studies demonstrate that astroglial cells isolated from non-neurogenic brain regions have the potential to be reprogrammed into functional neurons through forced expression of transcription factors known to instruct neurogenesis. based on our previous studies on the potential of the neurogenic gene cend1 in directing neural stem/precursor cells (nsc) to exit the cell cycle and acquire a neuronal phenotype, in parallel with evidence demonstrating activation of cend1 expression by genes of the neurogenin family, we explored the combined effect of cend1 and neurogenin-2 on their reprogramming potential on postnatal cortical astrocytes. to this end, forced expression of either cend1, neurogenin-2 or both, resulted in an important increase of two morphologically distinct subpopulations of gfapcells with elongated morphology, that strongly expressed the radial glial marker glutamate transporter glt-1. further characterization revealed that a subpopulation of these cells differentiates towards the neuronal lineage, as they were exhibiting a differentiated neuronal morphology and expressed b-iii tubulin, as well as neuronal subtype-specific markers, including gaba and th. further analysis using long time live cell imaging and brdu cumulative labeling revealed that the majority of cend1-transduced astrocytes undergo 1-2 cell divisions before differentiating to gabaergic neurons, whereas most ngn2 1 astrocytes directly transdifferentiate giving rise to th 1 neurons. additionally, only in the doubletransduced cultures, cend1 1 /ngn2 1 astrocytes seemed to take a step back forming colonies of small round glast 1 /nestin 1 cells, detected 24h following transduction. a day later, these colonies grew as three-dimensional spheres of high proliferative potential attached to the culture dish. when 'astrospheres' were isolated and cultured under nsc conditions, they grew as neurospheres and were propagated for over ten passages. importantly, as soon as 'astrospheres' were cultured in the absence of growth factors, they differentiated into neurons, astrocytes and oligodendrocytes, implying that they possess neural stem cell properties. at the moment we are performing electrophysiology recordings to assess the functionality of cend1derived neurons in vitro, while studies are in progress to explore the potential role of cend1 and ngn2 on astrocytic reprogramming in vivo following cortical brain injury. average membrane potential (v m ) was 277 mv and input resistance (ir) was 92 mx, while dcx 1 or map2 1 cells expressing fast activating outwardly rectifying k 1 currents (ka), and delayed outwardly rectifying k 1 currents (kdr) were defined by a v m of 272 mv and high values of ir (1943 mx). ng2 1 cells with a complex current pattern expressed inwardly rectifying k 1 (kir) currents, in addition to kdr and ka currents, and their v m was 276 mv and ir was 383 mx. the electrophysiological analysis revealed that the inhibition of the wnt signaling pathway significantly increased the incidence of cells with a complex current pattern, while the number of cells with the marked expression of outwardly rectifying k 1 currents declined. wnt signaling inhibition also resulted in increased kir and decreased ka current amplitudes. 4oht-treated cells were also hyperpolarized when compared to controls. immunocytochemistry using antibodies against map2 and dcx showed that neuronal progenitors in 4oht-treated cultures were less developed, having fewer and less branched processes. in summary, our data imply that the canonical wnt pathway in the neonatal svz promotes nsc differentiation into cells with neuronal characteristics. gacr p303/12/0855; p304/12/g069. adult neurogenesis plays a critical role in the overall health of, and repair processes occurring within the nervous system. while a great deal of information has been gained about the elements of the neurogenic niche and the factors that mediate neurogenesis, many unanswered questions remain. specifically, studies suggest that microglia have a significant impact on adult neurogenesis, but no unifying theory exists to explain their exact role in this process. in order to determine if microglia play a pivotal role in adult hippocampal neurogenesis and to examine the mechanisms through which microglia exert their effects on the hippocampal neurogenic niche, we employed a transgenic mouse model that allows for the selective ablation of microglia in the central nervous system (cns), the cd11b-herpes simplex virus thymidine kinase (cd11b-hsvtk) mouse. using this system, we removed microglia from the adult hippocampal neurogenic niche and evaluated the effects of this manipulation on neurogenesis and hippocampal homeostasis, including immunohistochemical analyses of stem cell survival and proliferation, as well as analyses of neuroblast development, migration and electrophysiological activity. taken together, these studies help to provide a better understanding of the factors governing adult neurogenesis and further our knowledge about the increasingly diverse role of microglia in the cns. the current assumption is that ependymal damage, caused by viral infections may result in reactive gliosis in the subventricular zone (svz), leading to sgns. however, as the svz is currently recognized as one of the neurogenic niches in the adult human brain, and svz astrocytes have been identified as neural stem cells (nscs), the assumption of sgns being reactive astrocytes has to be reconsidered. therefore, we have immunophenotyped the cell types present in these structures in the brains of individuals of various ages. in addition, we assessed nodule incidence in the svz in cases infected with hiv, where cells in the svz are likely to be to be affected. we found sgns in almost 90% of all cases, independent of age or hiv infection. we determined that cells in the sgns express adult nsc markers, rather than markers for reactive astrocytes. we hypothesized that direct contact of the nscs with cerebrospinal fluid (csf) may induce nodule formation. indeed, we could show that human ventricular csf stimulated the proliferation of human nscs in culture. from our data, we conclude that sgns most likely are formed upon ependymal damage in response to exposure to csf and represent local expansions of the svz. . in experimental murine model of demyelination, the repair capacity is robust and sufficient, even if it might decrease with aging. one major therapeutic goal in ms is to favour endogenous myelin repair, to prevent neurodegeneration and disability progression. using a transcriptomic approach on pure populations of opcs, we try to identify mechanisms specifically involved in opcs activation. we set up a method to isolate a purified population of opcs, by fluorescent-activated cell sorting from pdgfar::gfp mouse brains. we created the gene expression profile of neonatal opcs, adult opcs and adult oligodendrocytes (ols) in control conditions and of adult opcs in cuprizone-induced demyelinating conditions. we analyzed the transcriptomic profile of adult opcs, neo-natal opcs and ols, and identified more than 1000 differentially expressed genes. after demyelination, we identified more than 2000 adult-opc-specific genes that are either up-or down-regulated compared to control conditions. preliminary analysis suggests that adult opcs are more "mature" than neonatal opcs and partially revert to a more immature profile after demyelination. bio-informatic analysis is ongoing, with the perspective of identifying key candidates for myelin repair capacity, then develop functional experiments to validate their involvement in remyelination. in parallel, we plan to compare the transcriptomic profile of adult opcs from aged versus young mice, to get insight into age-related decrease of repair efficacy. question: after damage, astrocytes are activated and exert specific roles for central nervous system (cns) repair. among these, astrocytes within the injury site may represent a novel source of cells with stem cell potential. in this study, we used sedimentation field-flow fractionation (sdfff) method to rapidly sort well preserved astrocyte subpopulations and we identified stem cell properties within one of these subpopulations. methods: cells were collected from newborn rat cortex, separated mechanically and subjected to sdfff. cell fractions obtained were analyzed by immunocytofluorescence using antibodies against specific epitopes: glial fibrillary acidic protein (gfap), o4, b iii tubulin, and cd68, labelling respectively astrocytes, oligodendrocytes, neurons, and microglial cells. then, proteomic analysis was performed on astrocyte subpopulations and their behaviour in culture was analyzed, particularly under culture conditions usually allowing neurosphere development. results: three main fractions (f1, f2, and f3) were isolated and compared with the total eluted population (tp). after one week in vitro, tp and f2 derived cell cultures contained respectively 74.5% and 11.9% of gfap expressing astrocytes while this percentage was extremely high in f1 and f3 derived cell cultures (95.6% and 98.0% respectively). f3 derived cells displayed higher migratory capacities than those of f1. proteomic analysis of f1 and f3 derived cells indicated a high expression of vimentin in f3 derived cells. in the presence of epidermal growth factor (egf), f3 derived cells formed neurospheres containing cells expressing gfap and nestin (figure) that, when placed in appropriate conditions, were able to generate the three major cell types of the cns (neurons, astrocytes and oligendrocytes). moreover, colony-forming cell assay in a collagen-containing semi-solid matrix allowed in the presence of egf the formation of large colonies. in addition, 7 days after cortical lesion in adult rats, cells presenting similar stem properties were obtained after sdfff cell sorting. conclusions. our study demonstrates that sdfff enables the isolation of almost pure astrocyte subpopulations after one week in vitro. in addition, after sdfff cell sorting, we isolated an astrocyte subpopulation expressing vimentin and displaying the self-renewal and multipotency properties of neural stem cells. our data support that sdfff is an efficient tool to explore the different astrocyte subpopulations of the cns, particularly those involved in cns repair. experimental studies have shown that loss of myelin results in axonal loss and disability. so, finding of an expandable and autologous source of myelin-forming cells to enhance remyelination is required. induced pluripotent stem cell-derived neural progenitor cells (ipsc-npcs) have been developed recently. the remyelination potential and safety of these cells still remained to be well-addressed. the main goal of this study is to characterize mouse ips-npcs in vitro and in vivo after transplantation. we used embryonic mouse neural progenitor cells (mnpcs) as control and characterized mouse ips-npcs for their expression of major markers of immature or mature cells by immunostaining. rt-pcr was performed to analyze expression of nestin, olig2, b3-tubulin, olig1, sox10, and nkx2.2 mrnas. furthermore, to investigate the fate of these cells in vivo, we injected the lysolecithin to induce focal demyelination in the spinal cord of adult nude or shiverer mice. we transplanted cells in the lesion site 2 days after demyelination. animals were sacrificed 2 days, 1, 2, 6 and 8 weeks post transplantation to assess survival and differentiation of the grafted cells. our preliminary results showed that mips-npcs similar to mnpcs were nestin1, ki671, olig21, b3-tubulin1 but o4-and map5-. mips-npcs, were more proliferative than mnpc. moreover, mips-npc expressed all mrna transcripts except sox10. a first line of mips-npcs, was transplanted in the newborn shiverer:rag forebrain or the demyelinated spinal cord of immunosuppressed shiverer mice with a constant failure in terms of cell survival beyond 2 days of transplantation, highlighting the fragility of some of the reprogrammed cell lines. cells of a second line of mips-npcs were transplanted in nude mice to be sacrificed at 1, 2 and 6 weeks. an additional serie of transplantation was performed in the demyelinated shi:rag spinal cord. data at 1 and 2 weeks indicate excellent survival and integration of the mips-npc at both time points. some of the grafted cells expressed gfap, olig2 and ki67 (few only) and so far, no tumors were observed at these timepoints. immunohistochemistry with other markers and analysis of 6 weeks post transplantation animals, are in progress. our preliminary results show that although mips-npcs have very similar immature phenotype of mnpcs in vitro, they may have different survival capacities in vivo. future studies will reveal whether transplanted cells which showed short-term survival after grafting are capable of differentiation into myelin-forming cells. . however these putative stem cells have only been partly characterized in vitro and their therapeutic potential for cns repair has not been addressed. we cultured adult mouse drg cells in sphere-forming conditions. in parallel, we derived spheres from adult spinal cord of the same animals and compared their properties. this last tissue contains well-described stem/progenitor cells whose repair capacities have already been proven for spinal cord injury but not for primary demyelination. in vitro, regardless of their origins, all tissues generated self-renewable spheres up to 10 passages. rt-pcr and immunocytochemistry revealed that sphere-forming cells actively proliferate and express neural stem cells markers. drg spheres were multipotent: as they differentiated into schwann cells, neurons and myofibroblasts, whereas spinal cord spheres differentiated into astrocytes, neurons and oligodendrocytes. we then compared the integration, differentiation potential and remyelination capacity of primary sphereforming cells isolated from actin-gfp transgenic mice after their engraftment in two distinct environments: the adult nude lpc-demyelinated spinal cord and the developing newborn shiverer forebrain. both cell types survived but with distinct integration and differentiation patterns. in the mbp deficient mutant, spinal cord cells exhibit a substantial tropism toward white matter tracts and differentiated mainly into myelinating oligodendrocytes while drg cells were largely distributed in the parenchyma in close association with blood vessels and differentiated in vascular smooth muscle cells. surprisingly, after focal demyelination, in the adult spinal cord, drg cells migrated extensively towards the lesion and differentiated into remyelinating schwann cells whereas adult spinal cord cells migrated less efficiently, and gave rise to oligodendrocytes and a large population of astrocytes. our study reveals that both cell types present different but promising potential for cns remyelination. funding by ela foudation, dim nerf, arsep foundation. ischemia-induced progenitor cell proliferation is a prominent example of the adult mammalian brain's ability to regenerate injured tissue resulting from pathophysiological processes. a key locus of regeneration is the neurogenic niche located in the subgranular zone of the dentate gyrus (sgz). within the sgz, radial glia-like progenitor cells generate neural precursor cells which migrate into the granule cell layer (gcl) of the dentate gyrus, mature, and integrate into the hippocampal synaptic network. in order to better understand and ultimately exploit the cell signaling mechanisms that regulate ischemia-induced proliferation in the sgz, we tested the role of the p42/44 mitogen-activated protein kinase (mapk) cascade effector ribosomal s6 kinase (rsk) in this process. using the endothelin-1 ischemia model in c57bl/6 mice, we report that intrahippocampal cerebral ischemia triggered rsk activation in sgz progenitor cells. further, microinjection of a rsk inhibitor significantly reduced ischemia-induced sgz progenitor cell proliferation. using the neurosphere assay, we also show that sgz-and subventricular zone (svz)-derived adult neural stem cells (nsc) exhibit a significant reduction in proliferation in the presence of rsk and mapk inhibitors. taken together, these data indicate that rsk functions as a cell-autonomous regulator of ischemia-induced progenitor cell proliferation. here we demonstrated that the inpcs not only possess npc-specific marker genes, but also have qualitites of primary brain-derived npcs (wt-npcs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. moreover, the mature neurons derived from inpcs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. importantly, besides survival, the transplanted inpcs exhibit the migratory property responding to inflammatory stimulation in vivo. these results suggest that directly reprogrammed inpcs closely resemble wt-npcs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (incs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders. the pathogenesis of neurodegenerative disorders, including human immunodeficiency virus (hiv)21 associated neurocognitive disorders (hand), is exacerbated by an imbalance between metalloproteinases (mmps) and their inhibitors, tissue inhibitors of metalloproteinases (timps). as the timps exhibit diverse non-classical functions including anti-apoptotic effects, the induction of timp-1 in neuroinflammatory conditions likely serves multiple roles in addition to modulating mmp activity. our work demonstrates differential timp-1 expression in acute versus chronic activation of astrocytes and hiv-1 associated dementia (had) brain tissues. the timp-1 promoter harbors five consensus ccaat boxes. ccaat enhancer binding protein (c/ebp)b levels were elevated in brain specimens from hiv-1 patients and this transcription factor regulated astrocyte timp-1 expression. hiv-relevant stimuli increased c/ebpb expression in human astrocytes and localized the factor to the nucleus. overexpressing c/ebpb in astrocytes increased timp-1 promoter activity, mrna and protein expression, while knockdown of c/ebpb decreased timp-1 mrna and protein expression. erk1/2 activation is critical for il-1b-mediated astrocyte timp-1 expression and p38k activation contributes to il-1b-induced astrocyte timp-1 and c/ebpb expression. these data suggest that erk1/2 signals downstream of c/ ebpb to facilitate il-1b-induced astrocyte timp-1 expression. the role of astrocyte-timp-1 as a neurotrophic factor was examined. interestingly, cotreatment with timp-1 protected neurons from apoptosis and reversed neuronal morphological changes induced by staurosporine (sts) and hiv-1 ada virus. further, the anti-apoptotic effect was specific to timp-1 and partially independent of mmp-inhibition. additionally, timp-1 modulated the bcl-2 family of proteins and inhibited opening of mitochondrial permeability transition pores induced by hiv-1 or sts. together, these findings describe a novel function, regulatory mechanism and direct role of astrocyte-timp-1 in neuroprotection suggesting its therapeutic potential in had and possibly in other neurodegenerative diseases. mounting evidence and our pervious study has also confirmed that the axon outgrowth inhibition receptor ngr was also expressed on microglia, and regulated cell adhesion and migration behavior in vitro. however, microglia has been proposed to play a pivotal role in the neuroinflammation through expressing a range of proinflammatory enzymes and proinflammatory cytokines under pathological stimulus. these findings initiate the possibility of nogo/ngr signal on mediating neural inflammation processes during cns injury beside neurite outgrowth inhibition. in the present study, we found that nogo-p4, a 25-aa core inhibitory peptide sequence of nogo-66, was able to induce microglia expressing cox-2, inos, as well as releasing proinflammatory cytokines including il-1b, no and pge2, which could been reversed by nep1-40 or pi-plc treatment. microglia stimulated with nogo-p4 increased the phosphorylation lever of signal transducer and activator of transcription 3 (stat3). the activation of stat3 further mediated the promotion of nogo-p4 on microglia expression proinflammatory factors. furthermore, administration nep1-40 was capable to attenuate the inflammation response in a mouse spinal cord compression injury model by reducing the expression of several proinflammatory mediators, as well as attenuating the activation of stat3. taken together, our results demonstrated, for the first time, that nogo-66 may directly take part in cns inflammation process by influencing microglia expressing proinflammatory factors, which was related to ngr /stat3 signal pathway. these results imply that the interaction of nogo-66 with ngr expressed on microglia may participate in diverse cns diseases related to neural inflammation, including trauma, stroke, and neurodegeneration diseases, etc. our results indicate that ccl-1 is one of the key molecules in pathogenesis and ccl-1/ccr-8 signaling system can be a potential target for drug development in the treatment for neuropathic pain. demyelination plays a central role in the pathophysiology of multiple sclerosis (ms) not only because it disrupts nerve conduction, but also because of effects that compromise axonal survival. there is a growing consensus that any effective treatment for ms must include a component that restores or enhances remyelination by endogenous oligodendrocyte progenitor cells (opc). however achieving this goal requires a far better understanding of why remyelination fails in the first place. we now report that fgf9 is selectively up regulated in demyelinating ms lesions and acts to inhibit (re)myelination in vitro. to explore the mechanism involved we compared the activity of fgf9 to that of fgf2, another member of the fgf family with well characterised effects on opc differentiation and myelination. fgf2 acts directly on opc to stimulate proliferation, while at the same restricting their differentiation into mature oligodendrocytes. in contrast, fgf9 is neither an opc mitogen, nor is it able to inhibit opc differentiation directly. however, opc differentiation and myelination in myelinating cultures were inhibited by supernatants harvested from fgf9 treated astrocytes, even in the presence of an excess of fgf9 neutralising antibody. microarray studies were performed to identify astrocyte derived factors responsible for this inhibitory effect. this approach identified several potential candidates that were significantly up regulated by fgf9 in astrocytes. these included lif, a member of the gp130/il6 cytokine family that is generally described as having pro-myelinating effects, although our own studies indicate when present in excess relative to other gp130/il6 family members it will inhibit myelination in vitro. our experiments demonstrate that fgf9-mediated signal transduction in astrocytes disrupts their ability to maintain a growth factor milieu that can support myelination. been shown that inflammation is primarily mediated by macrophages that are recruited to the tissue. similarly, a recent study demonstrated reactive microgliosis in the hypothalamus of mice fed a high-fat diet. in the cns, however, the relevance of microglial activation in response to a high fat diet remains unclear. we aim to determine if microglia activation occurring in response to overnutrition is a cause or consequence of dysregulation of energy homeostasis and obesity. to determine if the absence of microglia (and therefore microglia-mediated inflammation) may influence the metabolic response to a high fat diet, we used a transgenic mouse model, the cd11b-hsvtk mouse (tk), which allows for an inducible, specific ablation of microglia in response to treatment with the nucleoside analog, gancilovir (gcv). mice were treated with gcv for 4 weeks during which time they were maintained on a diet high in fat (60%) or a respective control diet. body weight, food intake, locomotor activity and energy expenditure were measured and compared to wildtype mice. analyses revealed alterations in the metabolic phenotype of mice fed both hfd and control diet in the absence of microglia cells. our findings point towards a physiological role of microglia in energy homeostasis, the study of which will be the focus of further experiments. interleukin-10 (il-10), a cytokine classically related with anti-inflammatory and protective functions in the central nervous system (cns), has been reported to be produced by astrocytes and microglia in different neurodegenerative and neuroinflammatory conditions. in order to study the specific role of il-10 in the cns, we have created a new transgenic mouse with astrocyte-targeted production of il-10 (gfap-il10tg). the aim of this study was to evaluate if production of this cytokine in the cns has any effect on the phenotype of glial cells. for this purpose, coronal cryostat free-floating sections from the brain of both adult transgenic mice and their corresponding wild-type (wt) littermates, were processed for the study of astrocytes using gfap immunohistochemistry and microglia using antibodies against iba1 and several markers commonly related to the activated phenotype of these microglial cells, such as cd16/32 (fc receptor), f4/80, cd11b, cd206, cd150 and mhc-ii. our results showed that astrocyte-targeted production of il-10 induces an increase in the area covered by gfap immunolabelling. microglial cells in these gfap-il10tg animals displayed also morphological changes in specific areas of the brain, including the hippocampus, cortex and cerebellum, characterized by coarser processes and a notable enlargement of the cell body. distinctively, in the hippocampus, microglial cells adopted an elongated morphology parallel to the dendrites of pyramidal neurons. in addition, in the aforementioned areas, microglia exhibited a statistically significant increase in iba1, cd16/32, f4/80 and cd11b markers and "de novo" expression of cd150, whereas no detectable levels of either cd206 or mhc-ii was found. in conclusion, these results indicate that in specific areas, glial cells are influenced by the presence of astrocyte-targeted il-10. further studies are necessary to evaluate whether after injury these transgenic animals will present changes in the microglial and astrocyte activation that can influence the evolution of the lesion. networks. brain-resident macrophages, microglial cells were considered ''resting,'' but it has recently become evident that they actively sense neuronal microenvironment with their motile and ramified processes. they develop specific activities (cytotoxic, neuroprotective or phagocytic) depending on brain molecular context. for example, they contribute directly to the development of neuronal networks by eliminating or maintaining synapses in an activity-dependent manner. morphofunctional plasticity of microglial cells is finely tuned in order to keep brain homeostasis and to ensure such developmental activity. like macrophages at the periphery, microglial cells indeed adopt m1 or m2 phenotypes, distinguishable through their pattern of cytokine expression and of specific cell surface markers, a m1 phenotype being more pro-inflammatory while a m2 phenotype is more related to phagocytosis. linoleic acid (la, 12:2n-6) and alpha-linolenic acid (ala, 18:3n-3) are essential fatty acids that cannot be synthesized de novo by mammals and have to be provided through the diet. they are precursors of arachidonic acid (aa; 20:4n-6) and docosahexaenoic acid (dha; 22:6n-3) respectively which are key structural components of brain cell membrane phospholipids and precursors of bioactive lipid messengers involved in the regulation of inflammation and microglial activity. most aa and dha accumulate in the brain during the perinatal period via placenta and milk. we previously demonstrated that depleting the diet in n-3 pufas from the first day of gestation alters some neuronal functions of the offspring at adulthood. we here hypothesized that this is due to an alteration of microglial mrophofunctional activity in the developing brain. to test this hypothesis, mice were submitted to a diet deficient or not in ala throughout gestation and lactation. microglial morphology, phenotypes and functions were determined in the brain of pups at post-natal day 21 (pnd21). we show that dha levels is decreased in membrane phospholipids of microglia. in addition, microglial cells from mice fed with an ala deficient diet present a drastic increase of the cd36 marker expression combined to a dysregulated cytokine/chemokine expression profile and an altered phagocytic activity. all together, our results show that dietary n-3 pufas deficiency is likely to impair brain innate immune system activity at pnd21. the outcome of such microglial impairment on brain function is discussed. in recent years, one of the most promising therapeutic means for ad was thought to be immunization with specific antibodies against ab. several therapeutic antibodies were developed, however, most of the clinical trials were canceled due to unexpected deaths and neuroinflammatory responses in some patients. the causes and mechanisms of such responses are currently only partially understood. we have recently raised monoclonal antibodies against oligomeric forms of ab and were investigating whether these antibodies would prevent the neurotoxicity of ab oligomers in primary neuronal-glial cerebellar granule cell (cgc) cultures. antibodies were not directly toxic to cgcs when applied at concentrations relevant to concentrations reported to be in blood serum. however, surprisingly, the antibodies when in complexes with ab oligomers or firbrils dramatically increased the neurotoxicity of ab. similar effects were also observed with antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein vp1, human metapneumovirus nucleocapsid protein and measles virus nucleocapsid protein strongly potentiated the neurotoxicity of their antigens. the neurotoxicity of antibody-oligomeric antigen complexes was abolished by removal of the fc region from the monoclonal antibodies or by the removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. in conclusion, we find that immune complexes formed by ab oligomers or other oligomeric antigens and their specific monoclonal antibodies can cause neuronal death in primary neuronal-glial cultures via fc-dependent microglial activation. the results suggest that therapies resulting in antibodies to oligomeric ab or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation. additionally, if endogenous antibodies contribute to neuronal loss in ad or viral encephalitis, suppression of microglial activation may be therapeutic. antidepressants have often been recommended for potential treatment of chronic pain, especially for neuropathic pain caused by nerve damage. it is known that nerve injury-induced activation of microglia and astroglia can be a cause for the development of neuropathic pain. recent data indicate that abnormal neuronal activity mediated by glia activation may influence the level of neuroimmune signaling molecules in chronic pain. however, it remains unclear whether the mechanism underlying antidepressant effects is related to glia activation. this study investigated the potential pain-relieving properties of milnacipran and venlafaxine, selective serotonin/noradrenaline reuptake inhibitors (snri), in neuropathic pain and attempted to resolve how these antidepressants affect the factors synthesized by the activated glia in neuropathy. chronic constriction injury (cci) was performed in wistar rats by loose ligation of the sciatic nerve. after a single intraperitoneal (i.p.) injection of antidepressants, the responses to mechanical and thermal stimuli in neuropathic rats were evaluated 7 days after cci by the von frey test and the cold plate test, respectively. to confirm the influence of antidepressants on microglia and astroglia, western blot analysis of iba-1 (microglia marker) and gfap (astroglia marker) in the spinal cord and dorsal root ganglions (drg) was performed. the experiments were carried out according to the iasp and local bioethics committee rules. our findings confirmed that the administration of milnacipran (20 mg/kg) and venlafaxine (10 mg/kg) elicited antiallodynic and antihyperalgesic effects in cci-exposed rats. western blot analysis showed that milnacipran elevated the level of iba-1 protein in the spinal cord (but not in drg) in cci-exposed rats. venlafaxine decreased the cci-induced iba-1 protein level in the spinal cord and in drg. we observed no changes in gfap in the spinal cord and drg after milnacipran and venlafaxine administration. these results provide a substantial evidence that both used antidepressants belonging to snri attenuated allodynia and hyperalgesia in an animal model of neuropathic pain, although their effect on microglial cells and astrocytes was different, namely, milnacipran increased the microglia activation, in contrast to venlafaxine. this effect can be explained by comparing the effects of these drugs during their repeated application, and relevant experiments are currently in progress. myeloid cells can respond to intra-and extracellular danger/stress signals by inflammasome-mediated activation. this process consists of two steps: toll-like receptor-induced production of pro-il-1b followed by inflammasome-mediated cleavage and secretion of bioactive il-1b. inflammasome-mediated activation is strictly regulated by expression of regulatory proteins and by inhibitory processes like autophagy. recent studies describe that microglia are markedly different from other myeloid cells. they originate from a separate progenitor, are chronically exposed to the neural microenvironment and their activation may be regulated differently. the objective of this study was to determine the expression profile of inflammasome components in primary microglia and to compare inflammasome-mediated microglial activation and its regulation with other myeloid cells. primary microglia, bone marrow-and blood-derived macrophages (bmdms) of adult rhesus macaques of the same donor were profiled for all nod-like receptors (nlrs), inflammasome-associated adaptor proteins, caspases, and regulatory proteins by qpcr. inflammasome function and kinetics were assessed by exposing lps-primed microglia to atp, silica or msu (danger-associated molecular patterns: damps) followed by measuring the transcription of pro-il-1b-encoding mrna and the secretion of il-1b. primary microglia expressed nlrs (nalp1-3, nod1/3/4, aim2, ipaf, naip), adaptor proteins (asc), caspases (1/3-5/7/8), and regulatory proteins (a20), and the expression profile closely resembled that of bmdms. priming of microglia with lps induced high levels of pro-il-1b-encoding mrna, but il-1b protein was only secreted in response to subsequent stimulation with various damps. interestingly, in lpsprimed bmdms the window for inflammasome-mediated activation was 4-8 hours, while lps-primed microglia remained sensitive for inflammasome-mediated activation for at least 20 hours. in conclusion, primary microglia express multiple inflammasome components closely resembling the expression profile of bmdms and they can be induced to form functional inflammasomes. importantly, primed microglia remain sensitive to inflammasome-mediated activation for much longer than other macrophages. we will present data on whether this reflects a difference in negative regulation of the inflammasome, or differences in autophagy or apoptosis sensitivity. objective: the susceptibility of the aging brain to neurodegenerative diseases may in part be attributed to the intrinsic senescence of the microglia. cellular aging or senescence is linked to telomere shortening/dysfunction. evidence is accumulating that aging microglia show morphological changes, referred to as microglial dystrophy, which may be accompanied by accumulation of the iron-binding protein, ferritin. here, we investigated the effect of telomere shortening on microglial morphology, numbers, and activation state in telomerase deficient (terc -/ -) mice, a model of premature aging due to shortened telomeres. methods: male terc 1/1 mice and 3 rd generation terc -/mice, which show a clear aging phenotype, were perfusion-fixed with 4% paraformaldehyde. brains were processed into 60 lm thick horizontal sections. every fourth section was used for stereological estimation of cd11b 1 microglia in the molecular layer of the dentate gyrus by the optical fractionator method. sections were also stained for the microglial markers iba-1 to monitor microglial morphology, cd45 and cd68 to study microglial activation, and ferritin to study microglial aging. further sections were stained for the apoptosis marker cleaved poly (adp-ribose) polymerase (cparp). results: unlike resting microglia in littermate terc 1/1 mice, which display thin, finely branched processes, microglia in terc -/mice exhibited partial retraction and hypertrophy of processes. microglial cd45 and cd68 immunoreactivity were similar in terc 1/1 and terc -/mice. absolute numbers of mac-1 1 microglia were significantly reduced in young and old terc -/versus terc 1/1 mice. an increased microglial density in old terc -/versus terc 1/1 mice could be attributed to a volume reduction of the dentate gyrus. we also observed an increased number of cparp 1 cells in old terc -/versus terc 1/1 mice. this increase was most prominent in the sub-granular zone, an active site of neurogenesis, but also occurred in the molecular layer of the dentate gyrus and might reflect microglial apoptosis. we observed no difference in ferritin staining in terc -/versus terc 1/1 mice. conclusion: our findings suggest that telomere shortening impairs microglial capacity for self-renewal. surprisingly, changes in microglial morphology in terc -/mice occurred without differences in cd45 and cd68 expression. ongoing studies will show if increased numbers of microglia in terc -/mice co-express cparp or ferritin as evidence of increased microglial aging. tetrahydrocannabivarin (d9thcv) and cannabidivarin (cbdv). we have developed a series of new cannabinoid quinones, among them the cbg quinone (vce-003) that shows pparg and cb2 receptors agonism. in addition we have found that vce-003 activates the nrf2/are pathway in neuronal cell lines. in the present study, we investigated the therapeutic potential of vce-003 in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis (ms) by immunization con mog 33-55 . vce-003 (5mg/kg ip, daily) was administered to susceptible c57/bl6 mice at the onset of symptomatology. clinical score and weights of mice were daily recorded until the day of sacrifice (28 days post-immunization). vce-003 treatment delayed the onset of disease and ameliorated the symptomatology. histological analysis of spinal cord of eae mice treated with vce-003 showed decreased microglia reactivity and reduced cellular infiltrates, in particular cd4 1 t lymphocytes. double labeling with neurofilament and the myelin protein, rip indicated that vce-003 diminished the axonal damage. demyelination was evaluated by luxol fast blue labeling. changes in the expression of several cytokines, adhesion molecules and nrf2-dependent genes were determined by qrt-pcr. the implication of pparg and cb2 receptors in the beneficial effects of vce-003 in the eae model of ms is being investigated by using specific receptors antagonists. taken together our results support the potential of vce-003 for the treatment of ms and other chronic inflammatory diseases. we found that the lipid molecule lactosylceramide (laccer), is upregulated in ms patients. to better understand the role of laccer in ms, we used an animal model of spms, and found that laccer synthase (b4galt6) is upregulated during disease progression by astrocytes, and that its inhibition halts the progressive phase of the disease, attenuating cytokine and chemokine production by the astrocytes, and reducing the recruitment of inflammatory ly6c high monocytes to the cns. moreover, it also skewed the microglia and monocytes towards a m2 phenotype, and shifted the astrocyte phenotype to support re-myelination and axonal growth. in vitro experiments, using primary cells demonstrated that the inhibition of laccer synthesis directly inhibits cytokines and chemokines production in astrocytes. importantly, similar results were also obtained with human astrocytes, reinforcing the biological importance of our findings. in summary, we identified a novel lipid-signaling pathway that promotes the activation of local cns immunity and neurodegeneration in experimental spms and is a potential therapeutic target for spms. converging clinical studies report an increased prevalence of comorbid neuropsychiatric symptoms, particularly major depressive disorders, in a number of conditions (e.g., aging, obesity) and diseases (e.g., atherosclerosis, congestive heart failure, rheumatoid arthritis) sharing inflammation as a common denominator. by using an experimental approach in mice exposed to innate immune system (iss) stimulation, we demonstrated that induction of depressive-like behavior is mediated by cytokine-induced brain activation of a tryptophan-catabolizing enzyme, the indoleamine 2,3-dioxygenase (ido). in the metabolic syndrome (mets), a widely accepted concept that identifies a cluster of individual risk factors for type 2 diabetes and cardiovascular disease (including obesity, metabolic dysregulations and basal low-grade inflammation), neuropsychiatric symptoms emerge as significant factors for aggravation of the disease and related outcomes. recently, we demonstrated in a model of mets, the diabetic and obese db/db mice that cognitive alterations and increased anxiety-like behavior are related to hippocampal inflammation. on the other hand, depressive-like behavior is not affected by basal inflammation displayed by db/db mice. given the data linking increased depressive-like behavior and ido activation by cytokines in conditions of iss stimulation, the question arises as to whether depressive-like behavior is increased in those conditions in db/db mice. to answer this question, we measured in db/db mice and in their healthy db/1 littermates the effect of a lipopolysaccharide (lps) challenge (5 mg/mouse, ip) on behavioral reactivity in a depression test (the forced swim test, fst), plasma levels and hippocampus expression of inflammatory cytokines and related neuronal targets, and brain ido activity. plasma levels and hippocampus expression of il-1b, il-6, tnfa and il-10 are similarly increased 2h after lps in both db/1 and db/db mice. as expected, brain ido activity and duration of immobility in the fst are increased in lps-treated db/1 mice 24h after lps. on the contrary, induction of brain ido activity is significantly blunted in db/ db mice compared to their db/1 counterparts and no increase of depressive-like behavior is observed. moreover, some data suggest an impairment of the neuroimmune interactions in db/db mice. we need now to understand how obesity and related neurobiological alterations impair ido activation by cytokines and their consequences in terms of vulnerability to infections in mets. in this study, we tested it in a collagenase-induced mouse intracerebral hemorrhage (ich) model using tlr2 knock-out (ko) mice. to induce ich, collagenase or blood was injected into the right caudate putamen in 8-10 week old male mice. tlr2 expression was upregulated in the ipsilateral hemorrhagic tissues of the collagenase-injected mice. brain injury volume and neurological deficits following ich were reduced in the tlr2 ko mice as compared to the wild type (wt) mice. heterologous blood-transfer experiments show that tlr2 signaling in the brain-resident cells, but not leukocytes, contributes to the injury. in our study to elucidate underlying mechanisms, we found that damage in the blood-brain barrier (bbb) integrity following the ich was attenuated in the tlr2 ko mice compared to the wt mice, which may be due to reduced mmp-9 activation in brain astrocyte. the reduced bbb damages accompanies with reduced neutrophil infiltration and proinflammatory gene expression the injured brain parenchyma, which may account for the attenuated brain damage in the tlr2 ko mice after ich. conclusively, these data demonstrate that tlr2 contributes to brain injury following ich by compromising bbb through activating mmp-9 in brain. the pathological relevance of this autoantibody response is unknown, but it is generally assumed that it exacerbates demyelination via activation of complement and/or cell meditated effector mechanisms. however in a majority of cases the mog-specific autoantibody titre is far lower than that required to induce widespread demyelination and exacerbate disease severity in animal models of ms. to explore the possibility mog-specific antibodies may play other more subtle roles in disease pathogenesis we investigated possible effects in myelinating cultures derived from embryonic rat spinal cord; a model system that allows us to explore antibody-dependent effects in the absence of exogenous complement and effector cells. myelinating cultures were treated continuously with monoclonal antibodies specific for either mog (clone z2, igg2a), sulphatide (clone o4, igm), proteolipid protein (plp) (clone o10, igm), or an appropriate isotype control from 18 days in vitro onwards. in the absence of an exogenous source of complement none of these antibodies induced demyelination, but by 24 div had all had a significant inhibitory effect on myelination compared to cultures treated with appropriate isotype control antibodies. to investigate possible mechanisms contributing to this inhibitory effect qpcr arrays were used to determine if this complement-independent effect on myelination was associated changes in expression of immune mediators. unexpectedly we found recognition of antigens exposed at the surface of the myelin sheath induced a rapid increase in expression of three chemokines known to be involved in recruitment of effector t cells into the cns. within 24 hours of adding antibody to the cultures expression of ccl5, ccl20 and cxcl11 increased by at least three orders of magnitude and then declined to baseline over the following five days despite continuous presence of antibody. this transient increase in mrna transcripts for these cytokines resulted in sustained protein synthesis and secretion of biological active products as demonstrated by analysis of culture supernatants. these results challenge the traditional view that myelin-specific autoantibodies contribute to the pathogenesis of diseases such as ms and adem by virtue of their ability to initiate immune-mediated demyelination. we now demonstrate that even in the absence of recruited immune effector mechanisms myelin-specific autoantibodies not only inhibit myelination but trigger secretion of chemokines predicted to trigger or exacerbate inflammation within the cns. the blood-brain barrier (bbb), comprising specialized brain endothelial cells, is crucial in maintaining a controlled environment within the central nervous system (cns) to safeguard normal neuronal function. in multiple sclerosis (ms) the function of the bbb is disturbed, leading to uncontrolled influx of metabolites and immune cells and neuroinflammation. therefore, restoring the function of the bbb may be a novel therapeutic tool to halt disease progression or new lesion formation. we previously showed the importance of the vitamin a metabolite retinoic acid (ra) in bbb function during cns development 1 , where fetal astrocyte-derived ra is needed to induce bbb function during cns embryogenesis. considering the possible involvement of developmental pathways that could regenerate the disrupted bbb, we investigated the possible role for ra in the protection or reinstatement of disrupted bbb function. we show that ra counteracts the deleterious effects of inflammatory cytokines such as tumour necrosis factor (tnf)-a and interferon (ifn)c on bbb function in vitro. moreover, ra diminishes the induction of inflammation-related genes in human brain endothelial cells, and induces general immune quiescence in resting brain endothelium. our preliminary data as well as a recently described study in the cuprizone animal model for demyelination suggest that ra synthesis in the cns is increased under inflammatory conditions. the impact of cns-derived ra on the inflamed bbb, as well as the cellular source is currently under investigation. insight into the ra-synthetic pathway and its protective effect on the bbb may lead to the development of novel therapies which are aimed to restore bbb function and reduce the inflammatory cascade in ms lesions. tissue transglutaminase (tg2) is a multifunctional enzyme whose expression and activity is enhanced during inflammatory processes. we previously observed that the expression of tg2 is increased in monocytes in lesions in post-mortem material of ms patients. moreover, tg2 activity and expression is enhanced in chronic-relapsing experimental autoimmune encephalomyelitis (cr-eae) in rats. in this same ms model, pharmacological inhibition of tg2 activity dramatically reduced clinical symptoms and attenuated the influx of monocytes. in the present study we question whether tg2 plays a role in mouse eae, as transgenic mice have to be used for subsequent in vivo imaging of leukocytes/monocytes during eae. methods and results: eae was induced in tg2 -/mice and littermate wildtype mice (c57bl/6) using mog 35-55 . during the early phase of disease (day 14-15), the clinical symptoms observed were significantly less severe in tg2 -/compared to the wildtype mice. moreover, the maximal clinical score was significantly lower in the knockout mice. there was no difference in the day of onset of disease symptoms between the two groups. secondly, we aimed at visualizing leukocytes/monocytes in the spinal cord of lysm-gfp and cd11c-yfp mice suffering from mog 35-55induced eae using intravital video microscopy. a permanent window was fixed on top of the spinal cord of mice to allow reimaging of the same field of view in the same mouse over the disease course. in a pilot experiment we observed numerous gfp positive cells in the white matter around the imaged blood vessel in the spinal cord of a lysm-gfp eae mouse. although equally present, this was less prominent in a cd11c-yfp mouse suffering from eae. subsequent immunohistochemical analysis revealed that various cell types had infiltrated the spinal of cord of both mice. outlook: to further address the role of tg2 in the infiltration of leukocytes/monocytes into the spinal cord of eae mice in vivo, specific inhibitors for tg2 activity will be administered to the transgenic mice and leukocytes/monocytes will be visualized using intravital video microscopy. z. muneer 1 , c. wiesinger 1 , g. regelsberger 2 , j. berger 1 , s. forss-petter 1 1 medical universty of vienna, center for brain research, vienna, austria 2 medical university of vienna, institute of neurology, vienna, austria x-linked adrenoleukodystrophy (x-ald) is the most frequently occurring peroxisomal neurodegenerative disorder and is often associated with cerebral demyelination and inflammation. x-ald is caused by mutations in the atp-binding cassette sub-family d member 1 (abcd1) gene encoding adrenoleukodystrophy protein (aldp), which is a transporter for coa esters of very long-chain fatty acids (vlcfa) across the peroxisomal membrane. adrenoleukodystrophy related protein (aldrp), another member of the abcd sub-family, encoded by abcd2, is the closest homologue of abcd1. upon over-expression, abcd2 has been shown to compensate for abcd1 deficiency in vitro and in vivo. several lines of evidence imply an important role of microglia/macrophages in the disease progression. here we used mouse peritoneal macrophages (mpmu) to correlate the gene expression levels of candidate modifiers with x-ald associated defects, like accumulation of vlcfa and decreased peroxisomal b-oxidation, in abcd1 and abcd2 single ko mutants as well as in abcd1/abcd2 double deficient (doko) mice. by quantitative rt-pcr analysis, in mpmu from wild type mice the abcd2 mrna was present at about half the level of the abcd1 mrna. abcd1 ko mpmu showed elevated accumulation of vlcfa (c26:0) compared to the mpmu from wild type mice as measured by gc-ms. the vlcfa levels of abcd2 ko mpmu were similar to wild type amounts. interestingly, there was much higher accumulation of vlcfa in the mpmu from doko mice. there were no differences in the mrna expression level of the elovl1 gene, encoding the enzyme catalyzing the elongation step of vlcfa biosynthesis. peroxisomal boxidation activity was decreased in abcd1 ko mpmu compared to wild type level. this defect was strongly enhanced in mpmu from abcd1/ abcd2 doko mice. these results show that the increased accumulation of vlcfa in mpmu from doko mice as compared to abcd1 ko mice was due to a more severe defect in peroxisomal b-oxidation in doko mpmu. we conclude that a substantial expression of abcd2 mrna prevents a more severe metabolic phenotype in abcd1 deficient mouse peritoneal macrophages. this study also supports the validity of abcd2 up-regulation as a potential therapeutic target in x-ald. j. claude, b. linnartz-gerlach, h. neumann reconstructive neurobiology, bonn, germany microglia have innate immune receptors recognizing pathogens and disease-associated molecular patterns but also molecules that could sense the intact tissue. a subfamily of these receptors is the inhibitory signaling sialic acid-binding immunoglobulin-like lectin (siglec) group including siglec-e that has an immunoreceptor tyrosine-based inhibitory motif (itim) in the cytoplasmic tail to suppress activatory microglial signals. in this study, we used primary and stem cell-derived microglia that were modified by lentiviral vectors. here we show that siglec-e is expressed on microglia and is up-regulated following interferon-c (ifnc) treatment. we performed lentiviral knock-down and overexpression of siglec-e. lentiviral overexpression of siglec-e decreased, while knock-down increased the phagocytosis of neural debris and its associated reactive oxygen burst. the extracellular domain of siglec-e linked to a fc-part of immunoglobulin bound to the sialic acid residues of the neuronal glycocalyx. therefore, we co-cultured these modified microglia with primary hippocampal neurons. overexpression and knock-down of siglec-e showed an increase and decrease in relative neurite-length, respectively. the neuroprotective effect of siglec-e was abrogated after removal of the sialic acid residues on the neuronal glycocalyx. treatment with the anti-oxidant trolox abolished the neurotoxic effect of the siglec-e knock-down on neurite length. in summary, our data suggests an immunomodulatory function of siglec-e on microglia which leads to a neuroprotective phenotype by decreasing the production of reactive oxygen species and a reduced phagocytosis rate of neural debris. a. nadjar, c. madore, a. sere, a. aubert, s. lay e university of bordeaux 2, bordeaux, france many epidemiological studies have linked maternal exposure to infections during pregnancy to later development of cognitive disorders in the descendants. these alterations are likely to originate from a generalized neuroinflammation in the fetal brain following activation of the maternal immune system. this neuroinflammatory response relies on microglial cells activation. these latter normally contribute to the development of neuronal networks especially by eliminating/maintaining synapses, a phenomenon also known as synaptic stripping, in optimal conditions of brain development. neonatal inflammation may alter the synaptic stripping capacity of microglial cells. thus, targeting this persistent inflammatory microglial activation could represent an original and promising strategy to improve cognitive performances in the descendants. omega-3 are known as immunomodulators and target microglial cells. we thus hypothesized that enriching the diet with omega-3 from the first day of gestation may impair the development of neurological deficits at adulthood, through limitation of microglial inflammatory response in favor of its synaptic stripping activity. to characterize the beneficial effects of omega-3 on microglial activity, pregnant mice were fed with an omega-3-deficient diet, or a balanced omega-3/ omega-6 diet or supplemented with omega-3 and received a peripheral injection of either lps or saline at e18 of gestation. using the golgi method, we first evaluated the morphology of dendrites and quantified the dendritic spines density as a measure of neuronal networks maturation in the hippocampus. we found that a prenatal exposure to lps increased the number of immature spines and this was reversed by an omega-3 supplemented diet. to correlate these data with alterations of microglia-neurons interactions, we took advantage of the cx3cr1-gfp mice and injected them with a neuronspecific lentivirus containing dsred protein, in the hippocampus, 7 days prior to two-photon experiments. we focused on microglia general dynamic and microglia-neuron physical interactions and found an alteration in microglial behavior and microglia-neurons interactions in pups from lps-treated females. these alterations were reversed by an omega-3 supplemented diet. overall, our data show that alterations of microglia-neurons interactions in the developing brain may explain the neurological disorders observed in the descendants of females with prenatal inflammation. these deleterious effects may be prevented by nutritional strategies. , which is characterized by a relapsing phase with inflammatory cell infiltrates and a remitting period, where patients partially recover. among the different cell types involved in the necessary immunomodulation to allow the relapsing-to-remitting transition, the role of myeloid-derived suppressor cells (mdscs) gains importance. mdscs form a heterogenic population of immature myeloid cells that is able to suppress the inflammatory response. this cells act, among other mechanisms, through arginase-i (arg-i) activity on tcells. a previous study of our group showed that arg-i 1 -mdscs transiently enter the spinal cord of eae mice and takes part in the immune response control by inducing t cell apoptosis around the peak of the clinical score. therefore, changes in the mdsc population during eae should modify the evolution of the disease. the retinoid acid family molecules are used for the treatment of different leukaemia due to their role as mdsc differentiation factors into diverse cell populations, abolishing t cell immunosuppression. am80, a synthetic analogue of the retinoic acid with a higher bioavailability and less side effects than the natural ones, has controversial actions on eae depending on its administration period. in this work, we administered am80 specifically in the critical moment for immune modulation (around the maximum clinical score). drug administration affected mdsc population and clearly worsened the eae course. our results point to endogenous strengthening of mdsc population as a new and promising therapeutic strategy to treat ms by speeding up the transition from the relapsing to the remitting period. sildenafil induces cgmp accumulation by pde5 inhibition. we have demonstrated that sildenafil decreases microgliosis and astrogliosis and proinflammatory cytokines expression in a demyelination model. however, little is known about mechanisms of sildenafil neuroprotection. since nfjb plays an important role in the regulation of glial activation, we examine the hypothesis that nfkb is part of the mechanism underlying the sildenafil neuroprotective effects. five male mice (c57bl/6), 6 weeks-old, were used per group. the groups received for four weeks: 0.2% cuprizone (cpz) mixed into the chow; cpz into the chow plus sildenafil (viagra v r , pfizer, 25 mg/kg) in the drinking water, starting concomitantly (sild-t0) or fifteen days (sild-t15) after initiation of cpz; controls received pure chow/water. cerebella were processed for western blotting and immunofluorescence (if). results showed that cpz increased gfap expression compared to control (astrocytes activation). sild-t0 (but not sild-t15) significantly decreased gfap compared to cpz group. in controls, microglia showed resting phenotype, with thin and branched processes positive to iba1/nfjbp65 (inactive fraction) double labeling. cpz treatment decreased inactive-nfjb expression, indicating that this transcription factor was activated. in agreement, ikba (nfjb inactivating protein) was also decreased. if showed increase of iba-1 expression, indicating microglia activation. sild-t0 strongly increased the nfjbp65 and its inhibitory protein, ikba, suggesting inactivation of nfjb. if showed decreasing in iba-1 labeling, suggesting inactive microglia. the delayed treatment (sild-t15) did not decrease iba-1 or increase inactive-nfjb and ikba expression. in conclusion, treatment with sildenafil concomitant with cpz exposure prevents micro-and astrogliosis in mice, possibly through ikba-nfjb signaling pathway. sildenafil may be suitable to improve neuroinflammatory/neurodegenerative diseases treatment. financial support: cnpq, capes, facepe, fapesp. mt-1&2 levels have been found to be increased in several human neurodegenerative diseases including alzheimer's disease (ad). moreover, mt112 are also upregulated in different ad mouse models, for example tg2576 mice, which show a significant up-regulation of these proteins in the vicinity of the amyloid plaques. in the present study we generated a double transgenic mouse line that develops ad-like pathology in addition to having an overexpresion of mt1, in order to determine the role of mts in different aspects of ad pathology. the results show that the overexpression of mt1 does affect the mortality rate of the tg2576 and control mice in a gender-dependent manner and partially reverses the behavioural phenotype of young (4-5 months) tg2576 mice, reducing the exploratory activity and improving the learning process; in contrast, mt1 does not cause relevant changes in deambulations and anxiety. on the other hand, the amyloid cascade and neuroinflammation is increased in the hippocampus of old (15-16 months) apptgmt mice, but the lower level of gliosis in the hippocampus of young male apptgmt mice suggests that the overexpression of mt1 is capable of reducing inflamatory response well before amyloid plaques are formed but not afterwards. further molecular and immunohistochemistry analyses are underway to give further insight into the role of mts in amyloidosis and neuroinflammation in this ad mouse line. exposure to prenatal inflammation is a risk factor for neurodevelopmental and neurobehavioural abnormalities that manifest in later life in disorders such as autism, schizophrenia and seizure development. the amygdaloid complex is composed of more than 10 nuclei with subdivisions that have different cytoarchitectonic, chemoarchitectonic, and projection characteristics and that are responsible for the processing of higher functions such as memory consolidation and emotional conditioning. it is also known that the basolateral nucleus of the amygdala is implicated in seizure propagation and initiation. seizure onset later in life may be linked to abnormal development of the amygdalaoid complex. neuropeptide y (npy) and its receptors are known to be involved in a number of important functions such as feeding behaviours, anxiety and seizure modulation. npy y1 and y2 receptors are the most abundant in the brain and are expressed at different stages of development. during foetal development different brain regions are populated by neurones and glia at different times and the interface between mother and foetus plays a crucial role in the development process. lipopolysaccharide (lps) induced maternal inflammation is a known animal model for studying the inflammatory effects on the developing foetus. in this study we are investigating the effects of an acute prenatal systemic insult using the lps model on amygdala morphology and structure. embryonic day 12 (e12) c57bl6j pregnant mice receive an intraperionteal injection of lps (50 mg/kg) or 0.9% sterile saline. embryos at e12, e16, e18 and pups at p1, p7 p14 and p40 are taken and used to study the developing hippocampus and amygdala. for each age and treatment group foetal brains, maternal brains, maternal serum and placentas are collected. incidence of preterm birth, resorption rate, litter size and weights are also assessed. immunohistochemistry, western blot and rt-pcr are then used to assess the expression of neuronal and glial markers in the amygdala and surrounding limbic structures. specifically alterations in expression, activation and distribution of npy and its receptors, microglia and astrocytes in the developing amygdala and hippocampus at different stages following lps treatment are being investigated. it is hoped that this study will further enhance our understanding of how the maternal environment influences brain development and, if disturbed at a critical time in the development of the amygdala, may predispose individuals to amygdala related disorders in later life. in vivo imaging of transgenic fluorescent mice in the cns by twophoton laser-scanning microscopy (2p-lsm) has become a powerful tool in neuroscience. for in vivo imaging of the cortex a craniotomy of the skull has to be made. in addition to anaesthesia and analgesia treatment, we occasionally apply anti-inflammatory drugs to prevent immunological activity that can obscure the images. as anti-inflammatory drug we used carprofen ((rs)22-(6-chloro-9h-carbazol-2-yl)propanoic acid), a known cyclooxygenase-2 (cox-2) inhibitor. adult cx3cr1egfp mice in which microglia are labeled by expression of the green fluorescent protein egfp were treated with a single dose of carprofen or with vehicle 12 hours before the craniotomy. in all mice we exposed the right somato-sensory cortex, and quantified the microglial response to a laser-induced micro-lesion. this micro-lesion was caused by increasing the power of the laser for 1 second in the middle of the region of interest. results -conclusions unexpectedly, we observed that carprofen reduced the microglial process motility significantly. the speed with which the processes approached the lesion dropped from 0.5 mm per minute in the untreated mice to 0.2 mm per minute in the treated mice. a molecular understanding of microglia response in inflammatory processes and how anti-inflammatory drugs modify normal microglia response will provide a strong impact in developing treatment strategies for diseases with strong inflammatory components. resting and activated (lipopolysaccharide, lps) cultured cstb-/-and control microglia were analyzed for cytokine production, chemotaxis and phagocytosis. microglia extracted directly from the mouse brains were studied for expression of mhc ii and m1-m2 polarization using flow cytometry. mouse brain cortex (p14) was analysed for m1-m2 microglial phenotypes and the presence of other immunological cells from the periphery. moreover, we have checked myeloid cell population in bone marrow and spleen of p14 animals as well as cytokines' level in blood serum. our results from cultured microglia show that secretion of proinflammatory chemokines is elevated by cstb-/-microglia. also, cstb-/microglia are chemotactically more active compared to the controls whereas their phagocytic activity is decreased. cstb-deficient microglia show decreased expression of mhc ii on the cell surface indicating reduced antigen presentation. activated cstb-/-microglia directly extracted from the brain has predominantly proinflammatory m1 phenotype. cstb-/-mice display inflammatory changes in the peripheral tissues at their early postnatal period. we have registered high concentrations of proinflammatory chemokines and cytokines in blood serum of p14 cstb-/-pups, but no difference in amount of neutrophils and lymphocytes between brains of wild-type and knockout mice. also, in bone marrow and spleen amount of granulocytes is enhanced in cstb-/mice during early postnatal period as well as level of granulocyte macrophage colony-stimulating factor in their blood serum. our results suggest a role for cystatin b in regulation of immune response and that cstb-deficiency is associated with early inflammatory processes both in the brain and the peripheral tissues. therefore, we consider that epm1 should be treated as a disorder combining neuropathological and immunological features. the contribution of glial cells in the pathophysiology of epilepsy is increasingly valued. furthermore, clinical and experimental evidence suggests a direct relationship between epileptic activity and cns inflammation, which is characterized by accumulation, activation and proliferation of microglia and astrocytes. concomitant glia-mediated mechanisms of action of several aeds have been proposed. however, their direct effects on glial cells, especially microglia, have jet been hardly investigated. we aimed to investigate the influence of commonly used aeds on the glial viability and microglial in-/ activation state in a physiological and inflammatory modified in-vitro astroglia/ microglia coculture model. methods:primary astrocytic cultures were prepared from brains of postnatal (p0-p2) wistar rats and cocultured with a physiological amount of 5% (m5), as well as 30% (m30) microglia in order to mimic inflammatory conditions. cocultures were treated for 24 hours with valproic acid (vpa), carbamazepine (cbz), phenytoin (phe) and gabapentine (gbt) with a concentration of 10, 25, 50 and 100 mg/ml. viability and proliferation was measured using the tetrazolium (mtt) assay. the microglial activation state was determined by immunocytochemical labeling using a monoclonal antibody to the ed1 marker. results:m5 and m30 cocultures showed a dose-dependent, significant reduction in glial viability after incubation with phe and cbz. furthermore, low doses of vpa led to highly significant microglial activation in m5 cocultures. however, cbz significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia in the inflammatory modified m30 cocultures. conclusion: cns inflammation is characterized by a disturbance of glial cell functions. strong microglial activation, a typical hallmark of inflammation, was induced by vpa in m5 and continued in m30 cocultures. with regard to the direct relation between cns inflammation and seizures, vpa seems to be unsuitable to reduce inflammatory condition. the reverse effect was achieved after cbz. we noticed significant microglial inactivation, after incubation of the m30 cocultures. as it has been demonstrated for levetiracetam before, we assume a beneficial therapeutic effect of anti-epileptic drugs (aed) with an antiinflammatory glial potential in epileptic patients with persistent inflammation. microglial cell activation due to homeostatic imbalances of external and/or internal etiology implies, among others, secretion of proinflammatory cytokines. the fact that microglial activation, inflammation and neurodegeneration often coexist, suggests that proinflammatory cytokines might induce and/or enhance neuronal damage. we investigated the neurotoxic impact of microglial activation in organotypic hippocampal slice cultures by exposing them to the toll-like receptor 4 ligand, lipopolysaccharide (lps), for 72 hours. microglial activation was characterized by unbiased estimation of their number (stereology) and quantification of soma and branching morphology (neurolucida v r -based cell reconstructions). moreover, proinflammatory cytokine and nitrite levels in the culture supernatant were estimated by elisa and griess. the neurotoxic impact was assessed by morphology (nissl staining, fluoro-jade v r b) and extracellular electrophysiological recordings of spontaneous and evoked neuronal activity in the hippocampal ca3 subregion. lps exposure induced microglial population expansion, morphological changes, such as process thickening and somatic enlargement, as well as substantial secretion of proinflammatory cytokines (tnfa, il6) and nitrite accumulation in the culture medium. however, these changes did not coincide with neurodegeneration and were associated with only minor effects on neuronal function, as demonstrated by the amplitude of evoked neuronal responses and short-term plasticity properties. we conclude that, in contrast to what has been observed in vivo, chronic microglial activation by lps is not sufficient to drive neuronal death and dysfunction in organotypic hippocampal slice cultures. the neonatal brain is particularly susceptible to oxidative stress. our group has previously shown that following hypoxic-ischemic injury, hydrogen peroxide (h 2 o 2 ) levels rise significantly particularly in the neonatal brain and are sustained for up to 24 hours. this rapidly accumulated h 2 o 2 is detrimental in the iron-rich immature brain as it can lead to the generation of dangerous free radicals that can cause extensive injury. to date, there is limited literature on the effects of increased h 2 o 2 levels, particularly on microglial cells, which have been extensively indicated in the mediation of ensuing injury. here we describe the effects of a continuous exposure of microglia to h 2 o 2 , as generated using the glucose oxidase-catalase (gox-cat) system. this system allows us to generate and continuously maintain for up to 24h pathophysiological levels of h 2 o 2 >1 um. microglial cultures were derived from the p1 mouse brain and exposed to either bolus concentrations of h 2 o 2 [1-100 mm] or varying concentrations of gox-cat for varying lengths of time. conditioned medium was collected from cells at 4, 18 and 24h of treatment and analyzed for secreted cytokine levels using the 25-plex cytokine microbead array kit. treated cell extracts were processed for protein and fixed cells were labeled with m1 and m2a phenotype markers. continuous exposure to very low levels of h 2 o 2 can produce 10-20-fold higher (over control) pro-inflammatory cytokine protein levels of il5, il6, il12p40, il12p70, il15, il17 and ifng and at least a 100-fold higher level of il1a, il1b, il7 and tnfa by 18h. anti-inflammatory cytokine (il4, il10 and il13) and chemokine (rantes, g-csf and gm-csf) protein levels were also increased by 18h. interestingly, no prominent cytokine responses were seen with bolus treatment at any of the time points studied. low, continuous h 2 o 2 promoted a predominantly m2a microglial phenotype by 24h. we conclude that studying continuous exposure effects will help delineate the various specific effects h 2 o 2 can have on microglial cells. these specific effects can then be used to clarify when and how microglial cell responses can be beneficial or detrimental following injury in the immature brain. we have previously shown that natural (15-deoxy-d 12,14 prostaglandin j 2 , 15d) and synthetic (pioglitazone) agonists of peroxisome proliferator activated receptor-c (ppar) potentiate intrinsic cellular mechanisms protecting oligodendrocyte (ol) progenitors (ops) from oxidative insults and promote their differentiation to ols. in addition, ppar-c agonists potentiate mitochondrial activities, as the mitochondrial respiratory chain activity and the regulation of cytoplasmic ca21 waves, which are known to be crucial for ol differentiation. in the present study we sought to investigate whether ppar-c agonists can protect ol cultures from conditions causing mitochondrial stress. first, to specifically induce a mitochondrial impairment, we used the complex i inhibitor rotenone. as expected rotenone, at concentration not affecting cell viability, significantly inhibited ol differentiation, as indicated by the reduced number of cells expressing specific markers of differentiation (o 4 and o 1 ). in ppar-c agonist-treated ols the inhibitory effects of rotenone were significantly attenuated, suggesting a protective effect of the agonists against the mitochondrial toxin. we next examined a condition mimicking inflammatory stress by challenging op cultures with tnf-a, an inflammatory cytokine known to retard the differentiating program of ops. in parallel with the expected reduction of the percentage of o 4 and o 1 positive cells, the cytokine induced a significant reduction of mitochondrial membrane potential (mmp), suggesting an impairment of the mitochondrial functions. the simultaneous treatment with tnf-a and ppar-c agonists (15d or pioglitazone) significantly reverted both tnf-a dependent reduction of ol differentiation and mmp. at the molecular level, we found that in op cultures, ppar-c agonists increased the expression of the uncoupling protein-2 (ucp-2), a mitochondrial protein known to contribute to the protection of mitochondria against oxidative stress. these findings suggest that ppar-c agonists protect ols and promote myelination through several mechanisms, including those involving mitochondrial functions. our studies support the therapeutic potential of ppar-c agonists in brain diseases in which mitochondrial alteration, oxidative stress and demyelination occur and point to the need to better understand the role of ppar-c and its agonists in ol biology. it is characterized by lesions of demyelination and inflammation, which can be classified according to the activation state of the microglia/macrophages. well before any myelin and blood-brain barrier damage clusters of activated microglia are noticeable. these clusters are considered to be the first stage of lesion formation and therefore called 'pre-active lesions. however, they do not always develop into demyelinating lesions. here we postulate that stressed oligodendrocytes play an crucial role in pre-active lesion formation, by producing factors that will attract and activate microglia to either a more pro-inflammatory (m1) or antiinflammatory (m2) activation status. this can contribute to the amount of damage to the environment and further lesion formation. this study sets out to identify the best method to mimic this early lesion formation in vitro. first, human primary isolated microglia are stimulated to either a m1 or m2 phenotype and characterized by marker expression and cytokine profile. oligodendrocytes are isolated out of the human brain. medium of (stressed) oligodendrocytes will be used as attractant as well as activator for microglia. by immunohistochemical staining the activation status of microglia in pre-active lesions will be determined. preliminary data reveal that human microglia are able to be skewed towards a pro-and anti-inflammatory phenotype. first experiments of primary human oligodendrocyte cultures, showed pure oligodendrocytes. thus far our data support our hypothesis, however further research is required to make our conclusions more robust. question: although cell transplantation is increasingly suggested to be beneficial for the treatment of various neurodegenerative diseases, the therapeutic application of such intervention is currently hindered by the limited knowledge regarding central nervous system (cns) transplantation immunology. in this study, we aimed to investigate the early post transplantation innate immune events following grafting of autologous mesenchymal stromal cells (msc) in the cns of immune competent mice. methods and results: first, the survival of grafted luciferase/egfpexpressing msc (msc-luc/egfp) was demonstrated to be stable from on day 3 post implantation using in vivo bioluminescence imaging (bli), which was further confirmed by quantitative histological analysis of msc-luc/egfp graft survival. additional histological analyses at week 1 and week 2 post grafting revealed the appearance of (i) graftsurrounding/-invading iba11 microglia and (ii) graft-surrounding gfap1 astrocytes, as compared to day 0 post grafting. while the density of graft-surrounding astrocytes and microglia did not change between week 1 and week 2 post grafting, the density of graft-invading microglia significantly decreased between week 1 and week 2 post implantation. however, despite the observed decrease in microglial density within the graft site, additional phenotypic analysis of graftinvading microglia, based on cd11b-and mhcii-expression, revealed > 50% of graft-invading microglia at week 2 post implantation to display an activated status. although microglial expression of cd11b and mhcii is already suggestive for a pro-inflammatory m1-oriented phenotype, the latter was further confirmed by: (i) the expression of nos2 by microglia within the graft site, and (ii) the absence of arginase 1-expression, an enzyme known to suppress no activity in m2oriented microglia, on graft-surrounding and -invading microglia. conclusions: in summary, we here provide a detailed phenotypic analysis of post transplantation innate immune events in the cns of mice, and warrant that such intervention is associated with an m1-oriented microglia response and severe astrogliosis. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). cd300f is a novel immunoreceptor that dampens inflammatory reactions in experimental autoimmune encephalitis (eae) and diverse allergy models. we have analyzed the function of cd300f immunoreceptor in an nmda excitotoxicity model and in a traumatic brain injury model (tbi). for this purpose, we determined the pattern of expression of both cd300f and its putative ligand(s) in the cns, as well as the neuroprotective role of cd300f after both acute brain injuries. first, we used a human and rat cd300f-ig soluble protein to show by confocal microscopy the presence of endogenous ligand(s) for this receptor mainly in cns white matter oligodendrocytes and on the surface of oligodendrocytes, fibrous astrocytes and neurons in vitro. interestingly, when we analyzed the in vitro expression pattern of rcd300f in brain cells by q-pcr and immunohistochemistry, in addition to the expected expression in microglial cells, we detected expression of cd300f in oligodendrocytes and neurons. in vivo, after a tbi, cd300f is expressed at early timepoints (3d) in small round macrophages, and at later time-points (14d) in neurons of the penumbra. q-pcr studies showed significant upregulation of the mrna for rcd300f at 7 and 14d after tbi. interestingly, a delayed (4 hours after the lesion) intraparenchymal injections of a non-viral modular recombinant gene therapy vector termed nlsct overexpressing hcd300f induced a decrease in the lesion volume 3d after nmda injection or tbi when compared to control gfp over-expression. in addition, the over-expression of hcd300f decreased the long-term sensitive functional deficits observed by the "sticky tape" test. in order to validate these results with the endogenous molecule, we cloned the rat ortholog of cd300f protein. on-going positron emission tomography (pet) studies using 2-[ 18 f]-fluoro-2-desoxiglucosa ( 18 f-fdg) are being used to evaluate the long-term functional recovery after tbi. the overexpression of rcd300f receptor had a comparable neuroprotective effect as the human molecule after nmda injection. these data suggest that cd300f may be important in neuron-glia inflammatory and trophic interactions. in addition, our results suggest that delayed cd300f overexpression by means of a modular recombinant gene therapy may constitute an interesting therapeutic strategy for acute brain injuries. depending on the type of signals produced after an inflammatory event, microglia develops specific activities, including cytotoxic, neuroprotective or phagocytic activities in order to come back to brain homeostasis. as described for macrophages, microglia can adopt m1 (proinflammatory) or m2 (phagocytic) phenotypes distinguishable through their pattern of factors expression and specific cell surface markers. interestingly, polyunsaturated fatty acids (pufas) are precursors of bioactive lipid messengers involved in the regulation of inflammation and in particular, docosahexanoic acid (dha, 22:6n-3) an n-3 pufa, presents anti-inflammatory properties. the aim of our study was thus to investigate the effect of an increase of n-3 pufas on the inflammatory response and cognitive abilities after a peripheral immune challenge. to increase n-3 pufas, we took advantage of transgenic mice carrying the fat-1 gene from the roundworm caenorhabditis elegans. this fat-1 transgenic mouse is capable of producing n-3 fatty acids from the n-6 type endogenously, eliminating confounding factors of the diet. this conversion leads to abundant n-3 fatty acids with reduced levels of n-6 fatty acids in tissues, including the brain. to induce a neuroinflammation, we injected mice intraperitoneally with lipopolysaccharide (lps), components of gram negative bacteria walls. we first studied the microglial phenotype 24 h after lps injection and found that microglia in fat-1 mice presented a significant increase of m2 phenotype markers. we then measured cytokine expression in the hippocampus after a lps challenge and found a significant decrease in il-1b mrna in fat-1mice compared to wildtype littermates. in terms of hippocampal memories abilities, only control mice presented a deficit in the y-maze task whereas fat-1 mice were able to perform the test correctly. our results indicate that 24 h after a lps injection, fat-1 mice presented a decreased of the inflammatory response and normal hippocampal memory abilities compared to wild-type littermates. glia integrity and homeostasis as they have prominent role in blood brain barrier formation and maintenance, as well as in active regulation of an immune response. however, astrocytes are affected in neuroinflammation, when their protective roles might be suppressed and when they might be shifted towards pro-inflammatory phenotype. experimental autoimmune encephalomyelitis (eae) is a widely used model of autoimmune neuroinflammation. chemokine cxcl12 produced by astrocytes and endothelial cells has profound anti-inflammatory and anti-encephalitogenic role in eae. nitric oxide (no) produced by inducible no synthase (inos) within the cns is considered as disease promoting, while interleukin (il)-10 as protective in eae. the aim of this work was to investigate effects of no and il-10 on cxcl12 gene expression in astrocytes. methods: astrocytes were stimulated with supernatant collected from concanavalin a-stimulated splenocytes (conasn) and simultaneously exposed to no donor, sodium-nitroprusside (snp) or anti-il-10 antibody. also, astrocytes were stimulated with ifn-c1il-17 in the absence or presence of il-10. peritoneal macrophages isolated from healthy rats were co-cultivated with astrocytes. after 24h incubation period cxcl12 gene expression in astrocytes was measured by rt-qpcr. phosphorylation of p38 mapk and nf-jb was assessed by immunoblot in astrocytes exposed to snp. results: we found that no released from snp or macrophages significantly reduced cxcl12 gene expression in astrocytes. this reduction was mediated by inhibition of p38 mapk activation, but not of nf-jb signaling. on the contrary, il-10 stimulated and anti-il-10 antibody suppressed cxcl12 gene expression in astrocytes. conclusions: these results imply that expression of cxcl12 in astrocytes is inversely regulated by no and il-10 in the central nervous system affected by inflammation. having in mind, profound regulatory role of cxcl12 in neuroinflammation, these data contribute to our understanding of pro-and anti-inflammatory nature of no and il-10, respectively. in the presence of pathological insults, they acquire reactive phenotypes aimed at re-establishing brain homeostasis and minimizing neuronal damage. however, reactive microglia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect. consequently, the progression and resolution of microglial activation have to be tightly controlled to avoid detrimental secondary effects. signals arising from neuronal cells play an important role in the activation state of microglial cells. among them, inhibitory mechanisms, such as neuronal cd200 and microglial cd200r1 interaction, keep the proinflammatory phenotype of microglia under control. alterations in the expression of cd200 and cd200r1 have been described in pathological conditions, and the modulation of cd200-cd200r signalling could be an interesting target to be considered in therapeutic approaches against neuroinflammation occurring in neurodegenerative diseases. little is known on the molecular mechanisms involved in the regulation of cd200 and cd200r1 expression. the aim of the present work is to study the possible modulation of cd200 and cd200r1 by ppar-c agonists, and the involvement of cd200-cd200r1 in the anti-inflammatory and neuroprotective effects of ppar-c activation. we have used mouse microglial, mixed glial and neuronal cell cultures, as well as neuron-microglia co-cultures. cd200r1 expression was detected in microglial cells, and it was decreased in response to pro-inflammatory stimuli such as lps/ifn-c. the ppar-c agonist 15-deoxy-d 12, 14 -prostaglandin j 2 (15d-pgj 2 ) abrogated the inflammatory response in lps/ ifn-c-treated microglial cells and prevented cd200r1 expression inhibition. cd200 expression was detected in neuronal cultures and, at a lesser extent, in astrocytes in mixed glial cultures. lps/ifn-c-treatment did not modify cd200 expression in neuronal cultures, but it induced an increase in mixed glial cultures. this increase was inhibited by 15d-pgj 2 pre-treatment. 15d-pgj2 also protected against lps/ifnc-induced neurotoxicity in neuron-microglia co-cultures, but this effect was abolished when cd200-cd200r1 interaction was interrupted using an anti-cd200r1 blocking antibody. these results show that ppar-c agonists modulate cd200 and cd200r1 expression in reactive glial cells, and that cd200-cd200r1 interaction is necessary for the neuroprotective effect of ppar-c agonists. to gain insight into the contribution of the cwbc pathway to remyelination in ms, we analyzed the expression pattern of tcf7l2, a downstream target of the cwbc pathway and coactivatior of b-catenin in ms lesions. tcf7l2 was expressed in ol and astrocytes in a subset of early ms lesions, but was also observed in tissue samples from patients with inflammatory, non-demyelinating diseases. in contrast, in chronic lesions, no expression of tcf7l2 was detected. by wb we found slightly increased b-catenin levels in ms lesions compared to normal appearing white matter. aspirin (ass), a non-steroidal anti-inflammatory drug, attenuates bcatenin signaling activity by inhibition of protein phosphatase 2a (pp2a) via increased phosphorylation. therefore, we determined the effect of ass on ol differentiation and myelin gene expression (mge). as a control for the cwbc pathway activation or inhibition the selective cwbc pathway inhibitor (icg-001) was chosen. icg-001 binds specifically to cbp and causes a disruption of the b-catenin/cbp interaction. exposure of ol to 0.3 mm icg-001 for 48 hours increased mge and had a positive effect on ol differentiation. in contrast, ass had no positive effect on process formation and did not promote mge in primary murine ol. our results suggest that icg-001, but not ass increases the expression of myelin genes. further experiments are required to determine the functional role of the cwbc pathway for ol differentiation and remyelination in ol and ms. (braak et al., 2003) . furthermore, neuroinflammation, i.e. activation of microglial cells in the substantia nigra (sn), has been widely implicated in pd progression (mcgeer et al., 1988) . in the present study, we question whether microglial activation occurs in brain regions beside the sn which are affected in pd patients. methods: to this end, we studied microglial activation and protein pathology in the ob and hc of clinically and neuropathologically verified pd patients (braak 4-6) and control subjects (braak 0). human post-mortem formaldehyde-fixed material of pd patients included sn (n 5 13), ob (n 5 9) and hc (n 5 12), and material from age-matched control subjects without neurological deficits included sn (n 5 10),ob(n 5 6), and hc (n 5 8). results: the presence of a-syn pathology concentrated in the anterior olfactory nucleus of the ob and in the ca1-2 region of the hc. furthermore, using cd68 as a microglial marker, we observed a significant increase in the number of immunopositive microglial cells, with an activated, amoeboid morphology, in the sn as well as in the ob and hc of pd patients compared to control subjects. co-localization studies indicated that these activated microglia were found in the proximity of a-syn inclusion bodies and neurites, but did not co-localize suggesting that the microglial cells do not actively phagocytose a-syn. conclusion: we conclude that microglial activation occurs in other brain regions beside the substantia nigra of pd patients. the functional role of the microglial cells in those brain regions and their contribution to pd pathology remains to be established. in the present study, we question whether ccl2 and cx3cl1 and its receptors are present in hippocampal lesions of ms patients. to this end, semi-quantitative rt-pcr was performed on cdna transcribed from rna isolated from hippocampi of ms patients and control subjects. moreover, immunohistochemical analysis of ccl2, cx3cl1 and its receptors ccr2 and cx3cr1 was performed on post-mortem, formalin-fixed hippocampal lesions (plp -) from ms patients and on hippocampal material (plp 1 ) from control subjects. ccl2 and ccr2 mrna was significantly enhanced in demyelinated ms hippocampal homogenates compared to non-demyelinated and control hippocampal homogenates. cx3cr1 mrna was significantly increased in demyelinated ms hippocampal homogenates compared tot non-demyelinated hippocampal homogenates, but cx3cl1 mrna levels showed no difference between groups. ccl2, ccr2, cx3cl1 and cx3cr1 immunoreactivity was present mainly in white matter of control and ms hippocampi and was clearly enhanced in hippocampal grey (cornu amonis) and white (stratum lacunosum, stratum radiatum, alveus) matter lesions. the intensity of the ccl2 and cx3cl1 immunoreactivity was most pronounced when active lesions were present. co-localization studies indicated that ccl2 and cx3cl1 are present in astrocytes, whereas cx3cr1 and ccr2 are present in or on microglial cells. we conclude that the chemokines ccl2 and cx3cl1 and their respective receptors are enhanced in hippocampal ms lesions. as no clear influx of leukocytes is observed in the grey matter lesions, we propose that astrocyte-derived ccl2 and cx3cl1, via interaction with their receptors, affect microglial activation and/or function thereby contributing to neuronal dysfunction in the hippocampus as seen in ms patients. , is a 17 kda cytokine-inducible protein, produced by activated macrophages during chronic transplant rejection and in inflammatory reactions. in central nervous system (cns), iba1 is a sensitive marker associated to activated microglia and is upregulated following neuronal death or brain lesions. iba1-like factors have been described in several metazoan and share a well conserved amino acid primary structure throughout evolution suggesting a common, functional role. the medicinal leech hirudo medicinalis is able to regenerate its cns after injury, leading to a complete functional repair. similarly to vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. we investigated the expression of hirudo iba1 to track the activation state of leech microglial cells involved in nerve repair events. results: we recently identified a gene, named hmiba1, coding a 17.5 kda protein showing high similarity with vertebrate iba1 factor. quantitative rt-pcr analyses showed that hmiba1 is constitutively expressed in cultured nerve chains. a weak down regulation was observed in the days following experimental injury. gene transcripts rise back to basal level one week later. cultured nerve chains stimulated with atp shows a significant increase of hmiba1 transcript 6 hours after treatment. immunoblot analysis, performed with anti-hmiba1 polyclonal antibodies, revealed an immunopositive band at the predicted size. the presence of hmiba1 protein in na€ ıve and experimentally challenged tissues was evaluated by immunohistochemistry. the protein is constitutively present in spread, stellar shaped microglial cells, distributed in connective fibers and in segmental ganglia. a few hours after experimental injury of cns, the amount of immunopositive microglial cells increases at the lesion site and at the cut end of nerve fibers until. the amount of hmiba11 cells in connectives rapidly increases in atp treated nerve chains. this augmentation is visible in ganglia microglia and in connective fibers, where cells located between axon fibers display an elongated and stretched shape. conclusion: hmiba1 is a good marker of activated microglia. like in vertebrates, the atp induces its expression in leech cns. also if the functional role of hmiba1 has to be further elucidated, this molecule appears as a good activation marker of microglia and an interesting tool to study and follow the activity of such cells during nerve repair in leech. hypertension is the single most important risk factor for cardiovascular disease. despite significant advancements, 20-30% of all hypertensives remain resistant to all current available pharmacotherapy. these patients exhibit elevated sympathetic drive, increased norepinephrine spillover, and dampened parasympathetic drive. the dysfunctional autonomic nervous system of these resistant patients indicates a neurogenic origin for their pharmacotherapy resistance. previous studies have proposed that neuroinflammation in the autonomic brain regions plays an important role in neurogenic hypertension. this coupled with the emerging interest in microglia led us to hypothesize that activation of microglial cells in the brain could be a critical event in the initiation and establishment of hypertension pathophysiology. we have previously established that chronic low dose angiotensin ii (ang ii) induced hypertension involves activation of microglia, and increase in proinflammatory cytokines and reactive oxygen species (ros) in cardioregulatory brain areas, such as the paraventricular nucleus of the hypothalamus (pvn). intracerebroventricular (icv) delivery of minocycline, an anti-inflammatory antibiotic that inhibits microglia activation, decreased activated microglia, inhibited increase in cytokines and ros, and attenuated hypertension. in addition, inhibition of brain mitochondrial ros attenuated hypertension, microglial activation, and the overexpression of brain pro-inflammatory cytokines. a time-course experiment demonstrated that there was a significant increase in activated microglia before the increase in blood pressure was detectable by radiotelemetry. furthermore, the origin of microglia in established hypertension appears to be both from resident and bone marrow derivation. these observations indicate that the activation of microglia is a critical player in the development of neurogenic hypertension. they suggest that activation of microglia in cardioregulatory regions of the brain is an early occurrence that may initiates a cascade of signaling events leading to increase sympathetic nerve activity and initiation of hypertension. this research is supported by nih grant (r37 hl 33610 to mkr). our results demonstrate significant inflammatory activation of the neurons and their sgc not only in drg associated but also non-associated with injured nerve. a distinct expression of cytokines and their receptors was identified in sgc surrounding largesized drg neurons. significant inflammatory reactions of sgc were found in all drg of pac-treated rats. moreover, inflammatory activation sgc was also observed in drg of sham-operated rats indicating other kinds of trigger than traumatic nerve injury. the nerve injury and pac-treatment also triggered socs3 expression in sgc to control stat3 activation. inflammatory activation of sgc significantly contributes to ectopic activation of the drg neurons not only associated with injured nerve, and is involved in npp induction. aging is associated with reduced function and degenerative changes of the central nervous system (cns). increasing evidence suggests that changes in microglia cells, i.e. the resident macrophages of the cns, contribute to the age-related deterioration of the cns. the most prominent age-related change of microglia concerns enhanced sensitivity to proinflammatory stimuli of microglia in mice, rats and primates, called priming. we have addressed this issue in ercc1 mutant mice, ercc1 d/mice, a progeroid, dna repair deficient mouse model that displays features of accelerated aging in multiple tissues including the cns. in aged ercc1 d/mice, microglia showed increased proliferation and enhanced immune function including expression of cytokines and antigen presentation molecules, increased phagocytosis and production of reactive oxygen species (ros). this microglial functionality was found to be surprisingly hyper-responsive to immune stimuli indicative of microglia priming. transcriptome analysis revealed an expression pattern that characterizes microglia priming, featuring genes associated with increased antigen pattern recognition and antigen presentation and phagocytic-, adhesion-and chemotactic ability. in mice where the ercc1related dna repair deficit was targeted to forebrain neurons, microglia priming was restricted to forebrain areas, suggesting that the dna damage-induced changes in neurons provided the signals leading to microglial immune priming. acidosis is a clinical consequence of many major diseases. non-infectious diseases are increasing in prevalence and many have been shown to have an inflammatory component, which in the absence of infection, will be sterile. the nlrp3 inflammasome, a multimeric protein complex which induces processing of il-1b into its active form, is a well established mediator of sterile inflammation and is known to drive the worsening of brain injury in numerous experimental disease paradigms. we sought to investigate whether acidic conditions, typical of a disease environment, affected il-1b processing in primary mouse glial cells. mixed glial cultures were grown from wild type and nlrp3 knockout mice. culture media was reduced to ph6.2 and il-1b release was measured following addition of activators of the nlrp3 inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) with or without pre-treatment with caspase-1 or cathepsin d inhibitors (yvad-cho or pepstatin a respectively). subsequently, il-1b release was measured following addition of lactic acid with or without pre-treatment with the above inhibitors. at ph6.2, activators of the nlrp3 inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) induced the release of il-1b from mixed glial cultures. the il-1b released at this low ph was 20kda in size in addition to the mature 17kda il-1b. this 20kda il-1b release was maintained in nlrp3 deficient cells and was not significantly altered by pre-treatment with the caspase-1 inhibitor yvad-cho. lactic acid, itself released during disease and a common cause of acidosis, also induced the release of 20kda il-1b from mouse glial cultures. as with the nlrp3 activators under acidic conditions, the lactic acid-induced 20kda il-1b release was maintained in nlrp3 knock-out cultures and with pre-treatment with yvad-cho. pre-treatment with the cathepsin d inhibitor pepstatin a, however, significantly reduced the 20kda il-1b released with the nlrp3 activators and lactic acid. here we show that under disease relevant conditions (low ph), 20kda il-1b is released from mouse glial cultures and this 20kda il-1b is independent of the classical nlrp3 inflammasome/caspase-1 pathway and likely mediated by cathepsin d. further investigation of this caspase-1-independent il-1b pathway may in future provide novel targets for the treatment of inflammatory disease. the phoneutria nigriventer (ctenidae-araneaeomorpha) spider venom (pnv) causes reactive gliosis, neuroinflammation and blood-brain barrier (bbb) impairment. nitric oxide(no)-soluble guanylate cyclase(sgc)-cgmp has been implicated in pnv-induced cavernosal relaxation. we investigate whether no-sgc-cgmp signaling acts on astrocytes/microglia activation and neuroinflammation induced by pnv. male wistar rats (6-8-week-old) were pre-treated (i.p.) with sgc inhibitor (odq, 10 mg/kg), nnos inhibitor (7-nitroindazole-7ni, 40 mg/kg), no donor (nitroprussiate-ntp, 3 mg/kg) or pde5 inhibitor (sildenafil, 10 mg/kg). after 30 minutes, the venom (0.85 mg/kg) was injected in the tail vein (i.v.). saline (i.v.), pnv alone or dmso (i.p) followed by pnv were used as controls. one hour after injection, cerebella were processed for immunofluorescence or western blotting. pnv increased gfap, iba-1, ifn-c and sgc, compared to saline control, indicating astrocytes and microglia activation, neuroinflammation and sgc-cgmp involvement. the sgc inhibition by odq intensified the pnv effects, increasing even more gfap, iba-1 and ifn-c levels. this indicates that sgc-cgmp production can be inhibited by venom. in agreement, the cgmp accumulation by sildenafil inhibited the pnv effects, decreasing gfap, iba-1, ifn-c and sgc. the increase of sgc by venom could result from a feedback mechanism, when sgc activity is inhibited and its expression is increased. 7ni and ntp pre-treatment did not show difference compared to venom alone, suggesting that no per se is not involved in the inflammatory effects of venom. however, it is not discarded no and sgs-cgmp coupling in the mediation of pnv effects. we suggest that pnv induces glial reaction and neuroinflammation by sgc-cgmp inhibition. the study gives evidence that sgc-cgmp, coupled or not to no signaling, mediates pnv cerebellar effects including the bbb impairment. pnv can be a useful tool for studies on the mechanisms involved in glial regulation. cnpq/fapesp/faepex support. j. engele, m. puchert, v. € odemis university of leipzig, leipzig, germany it is currently believed that cxcl12 predominantly signals through cxcr4. in addition, it is assumed that the alternate cxcl12 receptor, cxcr7, represents a non-classical g protein-coupled receptor which primarily acts as a modulator of the function of cxcr4. discrepant from this view, we demonstrated recently that in primary rodent astrocytes and human glioma cell lines, sdf-1 exclusively signals through cxcr7 by a g protein-dependent mechanism. we now provide evidence that cxcr4 is essential for cxcl11/i-tac signalling in primary rodent astrocytes and some human glioma cells. treatment of cultured rat astrocytes with cxcl11 for 10 min resulted in the dose-dependent activation (phosphorylation) of erk1/2 and akt with maximum activation in the presence of 100 ng/ml of the chemokine. cxcl-11-dependent activation of both signalling molecules persisted in astrocytes in which expression of the established receptors for cxcl11, cxcr3 and cxcr7, were inhibited by rnai. however, cxcl11-dependent activation of erk and akt was abrogated following sirna-mediated inhibition of cxcr4. likewise, cxcl11 failed to activate erk and akt in cortical astrocytes cultured from a cxcr4-/-transgenic mouse line. moreover, similar to primary astrocytes cxcl11 activated erk and akt in the human glioma cell line, a767. again activation of both signalling molecules was abrogated following rnai-mediated inhibition of cxcr4. cxcl11-dependent activation of erk and akt further remained undetectable in a non cxcr4-expressing subpopulation of a767 cells, previously isolated by flow cytometry. together, these findings unravel a unique processing of cxcl11 and cxcl12 signalling in primary astrocytes which is preserved at least in some malignant astroglial cells. long-lasting activation of gfap-positive astrocytes occurs in brain tissue exposed to injuries that promote epilepsy development. studies in experimental models of epilepsy showed that activated astrocytes release various molecules, such as proinflammatory cytokines and danger signals, that play a role in seizures generation and recurrence. these activated cells also loose their ability to buffer extracellular k 1 and glutamate. this set of evidence suggests, therefore, that astrocytes may contribute to epileptogenesis (i.e. the post-injury phase prodromal to generation of spontaneous seizures), thus representing a putative biomarker of epilepsy. to test this hypothesis, we set up an in vivo longitudinal study using 1 h-magnetic resonance spectroscopy (mrs) to measure the hippocampal levels of metabolites that could reflect the extent and the duration of astrocytes activation after an epileptogenic brain injury. status epilepticus (se), which provokes epilepsy, was induced by pilocarpine in adult male rats. 1 h-mrs measurements were done in the hippocampus every 24 h for 7 d post-se, thus encompassing the epileptogenesis phase, and in chronic epileptic rats using a 7 tesla bruker biospec. spectra were processed and analysed using jmrui and tarquin freeware softwares. we studied changes in myo-inositol (mins) and glutathione (gsh), which reflect astrocytes activation. we found a progressive (2-fold, pwhich was maintained in epileptic rats. immunohistochemical analysis (ihc) of s100ß and gfap confirmed the concomitant activation of astrocytes in separate timematched groups of rats. gsh levels during epileptogenesis showed a negative correlation with the frequency of spontaneous seizures which developed after se. a negative correlation was also found between gsh and mins levels during epileptogenesis and the extent of neurodegeneration in hippocampus of epileptic rats. ihc done in epileptic rats at the end of the mrs experiments, showed that hippocampal s100ß levels positively correlated with spontaneous seizure frequency. since this is a soluble protein, further investigations of its csf and blood levels are warrented. our mrs findings show that gsh levels during epileptogenesis could serve as a predictive biomarker of the ensuing seizure frequency (thus of epilepsy severity) and, together with mins levels, also predict the extent of neuronal cell loss which develops following se in epileptic tissue. notably, ihc-detected s100ß levels in the epileptic tissue also reflect seizure frequency. these findings highlight the potential use of serial 1 h-mrs analysis of astrocyte activation for predicting the severity of epilepsy and the extent of neuropathology in the clinical setting. interleukin-6 (il-6) is a highly plurifunctional cytokine, with many pleitropic actions, considered one of the main cytokines controlling the immune system and coordinating it with the nervous and endocrine systems. il-6 is produced in multiple cell types in the cns, and in turn many cells do respond to it. it is therefore important to ascertain which the contribution of each cell type is in the overall role of il-6 during both physiological and pathological conditions. astrocytes are major responders to il-6 as well as one of the main cns producers of il-6. for this work we used astrocytary il-6 ko (ast-il6 ko) mice, which we already proved to have an important role in physiological conditions (like body weight control and exploratory/ locomotion behavior), in order to test astrocytary il-6 role during a neuroinflammation situation. for this purpose, we induced either an extensively used animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (eae), or a traumatic brain injury (cryolesion) in our mice. regarding eae, results indicate that lack of astrocytary il-6 delays clinical course of eae and ameliorate eae symptomatology in ast-il6 ko respect littermate controls in a gender-dependent way. further immunohistochemistry analyses confirm a decreased number of cellular and lymphocytes infiltrates and lesser demyelination, angiogenesis and gliosis in spinal cord of ast-il6 ko animals. regarding traumatic injury, astrocytary lack of il-6 facilitates microgliosis, lymphocytes infiltration and a faster decrease of the injured area. all these results are likely to bring some answers to astrocytesecreted il-6 involvement in neuroinflammation pathways and eae pathogenesis. interleukin-10 (il-10) is a counterregulatory cytokine that plays an important role in controlling inflammatory and immune reactions. in the central nervous system (cns), production of il-10 has been demonstrated in activated astrocytes and microglia after different types of injuries. the specific role played by il-10 inmodulating glial responses is however still unclear. hence, the objective of this study was to evaluate the effects of local astrocyte-targeted production of il-10 on glial reactivity using the axonal anterograde degeneration paradigm. for this purpose, unilateral perforant pathway transection (ppt) was performed on adult gfap-il10 transgenic (tg) animals and their corresponding wild types (wt) littermates. at 3 and 7 days post-lesion (dpl), animals were intracardially perfused with 4% of paraformaldehyde, brains frozen and parallel free-floating sections processed for glial analysis using immunohistochemistry for iba-1 (microglia) and gfap (astrocytes). our results showed that in comparison with wt animals, microglial cells in the dennervated dentate gyrus molecular layer of gfap-il10tg showed differential reactivity changes. after lesion, astrocytic reactivity presented a higher hypertrophy in gfap-il10tg animals when compared with their corresponding wt littermates. in conclusion, this study demonstrated that local production of il-10 inthe cns can modify the glial response associated with ppt. further studies are warranted to evaluate if these alterations in microglial and astrocytic reactivity have any repercussions for the evolution of the lesion and the associated axonal sprouting. astrocyte and microglia become reactive under many brain pathological conditions, making this process of neuroinflammation a surrogate marker of neuronal dysfunction. several studies have reported that reactive microglia overexpress the translocator protein 18kda (tspo, formerly known as peripheral benzodiazepine receptor). positron emission tomography (pet) using radioligands of tspo is thus considered as a potent technique to detect reactive microglia in situ. however, it is still controversial whether tspo pet imaging is also able to monitor reactive astrocytes in situ. this is an important question as reactive astrocytes and reactive microglia play very different roles in brain physiology and could impact disease progression in opposite ways. to address this question, we used a model of selective astrocyte activation through unilateral lentiviral gene transfer of the cytokine ciliary neurotrophic factor (lenti-cntf) in the rat striatum. cntf induced an extensive activation of astrocytes, which overexpressed gfap and became hypertrophic. microglia, on the contrary, displayed a minimal increase in the expression of markers of reactivity. cntf-activated astrocytes overexpressed tspo at the mrna and protein levels. pet imaging experiments demonstrated a significant and specific binding of two tspo radioligands [ 18 f]dpa-714 and [ 11 c]ssr180575 in the lenti-cntf-injected striatum. we show that reactive astrocytes can be monitored by tspo-pet imaging in the rat brain. this technique is thus well suited to monitor reactive microglia as well as reactive astrocytes, the two cell types involved in neuroinflammation, but would not allow their discrimination in situ. chronicity might establish a neuroinflammatory ganglion profile with inflammatory cells contributing to the hypersensitive phenotype. we first investigated whether, in trigeminal sensory ganglia, cytokines such as tnfa might contribute to a local inflammatory phenotype of a transgenic mouse model of familial hemiplegic migraine type-1 (fhm-1, cacna1a r192q knock-in mice). with respect to wild-type, r192q ki trigeminal ganglia were enriched in activated macrophages and expressed higher mrna levels of il1b, il6, il10 and tnfa cytokines and the mcp-1. functional consequences of crosstalk between macrophages and sensory neurons were studied in primary ganglia cultures, where larger release of soluble factors and larger currents mediated by pain-transducing atp-gated p2x3 receptors were found. consistently, we observed that, following lps injection, tnfa expression and macrophage occurrence were significantly higher in r192q knock-in ganglia with respect to wild-type ganglia. our data suggest that, complex cellular and molecular environment of sensory ganglia could support a new tissue phenotype compatible with a neuroinflammatory profile. we propose that, in selected patients, this condition might contribute to pain pathophysiology through release of soluble mediators, including tnfa and atp that may modulate the crosstalk between sensory neurons and resident glia, underlying the sensitisation process. cultures of astrocyte/microglia are stimulated with ifn-gamma and then infected with tachyzoites of neospora caninum they release nitric oxide (no) that controls parasite proliferation. in order of elucidating the immune response of cns against this parasite, this study investigated the participation of inducible nitric oxide synthase (inos) in the control of its proliferation in co-cultures of neuron-glia obtained from rat brain. the cells were stimulated with ifn-gamma (100 iu/ml-24h) then supplemented with l-ng-nitroarginine methyl ester/l-name, an inhibitor of inos (1.5 mm/ml -60 min) before infection with tachyzoites of n. caninum (1:1 parasite:cell). after 72h, in the cultures supplemented with l-name, it was verified, a reduction of tachyzoites number in 55.7% (control cells) and 59.8% (ifn-y stimulated cells). however, in infected co-cultures, it was not observed any release of no, even when cells where modulated by ifn-g. additionally, in cultures infected and treated with l-name, the levels of il-10 were increased in six fold (non stimulated) and nine fold (ifn-g stimulated). these data suggest that no should not participate as a mediator of n.caninum control in neuron/glia co-culture but, the regulatory pattern of immune response must play a role in its down modulation regulating the inflammatory mediators released during the cns infection, perhaps to protect neurons. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n 5 6) and lasered (n 5 6). anti-iba1 was used in retinal whole-mounts immunolabelled to analyze the distribution, morphology, density and arbor area of iba11 microglial cells. results: in na€ ıve, contralateral and lasered eyes, iba11 microglias were distributed throughout the retina in the: subretinal space (ss), outer plexiform layer (opl), inner plexiform layer (ipl), nerve-fibre layer (nfl) and ganglion-cell layer (gcl). in na€ ıve eyes, iba11 microglias: i) had rounded bodies with fewer and shorter processes in the ss; ii) exhibited a branched morphology emanating from small cell bodies in the opl and ipl; iii) were less ramified and were related with the retinal vessels in the nfl and in the gcl. by contrast, the morphological features exhibited by iba11 cells in contralateral and oht-eyes differed from na€ ıve: i) somas were more robust; ii) processes were thicker and more branched in contralateral eyes and thicker and retracted in oht-eyes. in oht-eyes and contralateral untreated eyes the iba11 microglial number was increased in comparison with na€ ıve (p < 0.001 and p < 0.05 respectively, t-test). in comparison to na€ ıve retinas the iba-11 cell arbor in the opl and ipl decreased in both oht-eyes (p < 0.001 and p < 0.001 respectively, t-test) and in the contralateral untreated eyes (p < 0.05 and p < 0.001 respectively, t-test). conclusions: two weeks of laser induced-oht triggered microglial retinal changes suggestive of activation in the contralateral untreated and oht-eyes. the microglial activation in contralateral eyes could be related to the immune response. this behaviour in contralateral untreated eyes, lead us to suggest that the use of contralateral eyes as internal controls in experimental unilateral oht, should be reconsidered. interleukin-6 (il-6) is a pleiotropic cytokine involved in inflammatory and non-inflammatory responses. among the latter, it participates in the regulation of body weight and metabolism. il-6-deficient mice develop mature onset obesity and have higher blood glucose, as well as impaired glucose tolerance and elevated blood leptin levels, suggesting that il-6 participates in suppressing adiposity in mice. moreover, transgenic mice with astrocyte-targeted production of il-6 challenged with a high-fat diet are resistant to high-fat diet-induced obesity, highlighting the role of centrally produced il-6 in the regulation of body weight. in this context, we hypothesize that mice lacking il-6 produced by astrocytes (ast-il6ko) will be more prone to develop obesity. we therefore challenged ast-il6ko mice (obtained with the cre-lox technology) with a high fat diet (58% kcal from fat) for 17 weeks and compared their body weight and food intake with those of mice fed a standard diet (18% kcal from fat). on weeks 14 and 15, the metabolic status was evaluated by an insulin tolerance test (itt) and an oral glucose tolerance test (ogtt), respectively. regarding body weight gain, we see a clear increase in ast-il6ko females on a high-fat diet in comparison to their controls with no apparent difference in food intake, which is also already apparent in the control diet. in males a similar tendency is observed. part of this difference can be attributed to the heavier subcutaneous white adipose tissue depots (relative to body weight) in ast-il6ko mice (fig 1) . insulin and oral glucose tolerance are compromised in mice fed a high-fat diet, but no differences between genotypes are observed. taken together, these results indicate that centrally produced il-6 has indeed a major role in the regulation of body weight, affecting subcutaneous white adipose tissue, but without significantly altering peripheral glucose metabolism. we are currently working on assessing hypothalamic neuropeptides with in situ hybridization to study the effects of astrocytic il-6 deficiency at the central level. minocycline is an agent with pleiotropic properties that targets multiple proteins and cellular processes associated with development of neuropathic pain, including inhibition of the injury-induced glia activation. the aim of our study was to examine the effects of minocycline on the injury-induced changes in immune factors and on morphine effectiveness in a rat model of neuropathic pain. chronic constriction injury (cci) to the sciatic nerve in rats was performed according to bennett and xie (1988) and 2 behavioral tests were conducted to measure allodynia (von frey test) and hyperalgesia (cold plate test). minocycline was administered intraperitoneally 16h and 1h before cci and then twice daily for 7 days. the studies were performed using competitive rt-pcr in the spinal cord and drg from control, cci-exposed and minocycline-treated cciexposed rats. morphine was administrated i.t. (chronic catheterization according to yaksh and rudy, 1976) and ip 1h after the last minocycline injection. the experiments were carried out according to iasp rules (zimmermann, 1983) . repeated administration of minocycline attenuated allodynia and hyperalgesia when measured 7 days after cci. minocycline downregulated the spinal and drg level of several immune factors: c1q, mmp-9, mmp-2, il-1beta, il-6, il-18, nos1 and nos2 which were increased in consequence of the injury. moreover, chronic administration of minocycline improved the response to i.t. and i.p. morphine injections. our results demonstrate that minocycline reduces the level of pro-inflammatory factors and increases the effectiveness of morphine, which may have clinical significance for enhancing analgesic effects of opioids in neuropathic pain. to date, there is no standardized simple model available to investigate the biology of human microglia. the aim of this study was to establish a new in vitro microglia model using blood-derived precursor cells. for that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of a mixture of cytokines and chemokines (m-csf, gm-csf, ngf and ccl2) to generate monocyte-derived microglia (m-mg). monocyte-derived dendritic cells (m-dc) were also generated as a control population using gm-csf and il-4. the human microglia cell line hmc3 was used as control. m-mg were clearly different in morphology, phenotype and function from m-dc, but shared many properties with hmc3 cells. m-mg acquired a ramified morphology with primary and secondary processes, comparable to hmc3. they expressed very low levels of cd45, cd14 and hla-dr, cd11b and cd11c; but a distinct pattern of chemokine receptors, including ccr1, ccr2, ccr3, ccr4, ccr5, cxcr1, cxcr3, cx3cr1. similar to hmc3, under non-activated condition, the m-mg secreted of il-8 and il-6. in comparison with m-dc, m-mg displayed lower t-lymphocyte stimulatory capacity, as well as lower phagocytosis activity. in summary, we have established a new protocol for the generation of human monocyte-derived microglia, which is is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. this model will certainly be very helpful for future studies investigating the biology and pathology of human microglia. w. schaafsma university of groningen, neuroscience, section medical physiology, groningen, netherlands background: microglia are the innate immune cells of the cns. like other tissue macrophages, they can activate and commit to distinct reactive phenotypes in response to tissue damage and infections. however, the microglial response to tissue damage may change over age and by past experience. these changes in activation patterns may be caused by epigenetic modifications which in turn can result in changes in gene expression of, for example inflammatory cytokines. a well studied condition with an altered responsiveness of innate immune cells is 'endotoxin tolerance' (et). innate immune cells that are pre-exposed to endotoxins are dampened in their inflammatory response upon re-exposure to these endotoxins. while et is well described for peripheral macrophages/monocytes, very little is known about et in relation to microglia. objective: in our experiments we set out to investigate the presence of et in microglia and if an 'epigenetic' memory is involved. design/methods: in our in vitro experiments, microglia cells were stimulated with lps (100 ng/ml). naive microglia only received one lps stimulation at the end of culture, pre-stimulated microglia received an 24h pre-stimulation of lps (100 ng/ml) and a second stimulation 5 days later. in vivo, male c57bl/6 mice received an i.p. injection with either pbs or lps (1 mg/kg) and 4 weeks later again an i.p. injection with pbs or lps. transcription levels and secretion of il1-b and tnf-a were assessed by use of quantitative pcr and elisa. in order to answer if there is a long lasting epigenetic memory involved in this tolerance we used chromatin immune-precipitation (chip), to investigate changes in covalent histon tail modifications associated with either permissive or repressed chromatin. results : in vitro, we show a 'tolerant' phenotype in microglia which receive a second lps stimulation after 5 days. a reduced expression level of pro-inflammatory genes il1-b and tnf-a and reduced secretion of tnf-a were observed in response to a second lps challenge. furthermore, with in vivo experiments we showed that mice which received a second lps injection after 4 weeks, showed a dampened response in their microglia inflammatory reaction at the level of il1-b and tnf-a gene transcription. in both in vitro and in vivo experiments we showed a corresponding pattern for histon tail modifications in the enrichment of activation marks h3k4me3 and ach3 at the il1-b and tnf-a promotors. we are the first to show a long lasting 'tolerant' phenotype in mouse microglia in vitro and in vivo and the involvement of epigenetic changes at the level of histon modifications. morphological criteria and dual immunofluorescent labelling confirmed expression of er stress protein in neurons, astrocytes, microglia or oligodendrocytes. an intriguing finding was localisation of crt to the rim of oro-positive myelin fragments and the 'patchy' nature of crt staining seen when tissue was dual-labelled with crt and gfap or iba1. these results are the first demonstration of significantly higher levels of crt in rodent eae. chop and p-eif2a data has also not been reported in rat eae. this study highlights the potential importance of er stress in inflammatory demyelination. the authors acknowledge support from the irish research council and from the foundation office of nui, galway. (3). considering the responsible signaling pathways regulating adult neurogenesis (4), we observed differential regulation of wnt members in the hippocampus on transcriptional level, e.g. wnt dependent transcription factors (axin2, tcf4) and ligands (wnt3, 5a, 8b and 11), as well as on translational level represented by the wnt signaling cascade (active-bcatenin, phospo-glycogen synthase kinase 3b) during acute and chronic states of disease. by using in vitro studies in primary hippocampal cultures, we further show the role of transforming growth factorbb1 (tgfb1), a key cytokine early involved during eae (5), in regulation of wnt ligand expression and wnt signaling activity. furthermore we are able to visualize the connection between tgfb1 and wnt signaling by using the axin2-lacz mouse, a wnt reporter mouse, for functional and histological investigation of the hippocampus. taken together our results suggest a cross talk of inflammation and wnt signaling for dysregulated hippocampal neurogenesis. interleukin-6 (il-6) is a cytokine with major regulating effects of the inflammatory response. moreover, il-6 is a neuropoietin that has neurotrophic effects related to neuronal survival and protection. to establish the importance of il-6 produced only in the central nervous system, we have generated mice producing il-6 essentially only in the brain by crossing gfap-il6 mice (transgenic mice with astrocyte-targeted production of il-6) with il-6 ko mice (il-6-deficient). we studied the inflammatory response in a traumatic brain injury model, cryolesion, after 3 and 10 days post-lesion gfap-il6-il6 ko mice, comparing them with appropriate controls. in basal conditions wt and il-6 ko mice showed a similar phenotype. this was also the case for gfap-il6 and gfap-il6-il-6 ko mice, which showed prominent astrogliosis, microgliosis, increased recruitment of t lymphocytes and vascularisation compared with the other two groups. in response to cryolesion of the cortex an increased astrogliosis, microgliosis, recruitment of t lymphocytes and vascularisation was observed. il-6 deficiency produced an altered inflammatory response, in a time-and gender-dependent manner. this study with il-6 ko and gfap-il6 transgenic mice indicates that during an acute neuropathological insult such as traumatic brain injury il-6, from either the brain or the periphery, has an important role on the inflammatory response. increasing evidence suggests that iron accumulation in the brain might contribute to neurodegeneration. iron is a potential source of free radicals as it can catalyze the production of hydroxyl radicals under oxidative conditions. this can lead to amplification of tissue injury caused by oxidative damage. we thus characterized iron storage within the central nervous system (cns) of animal models of different neurodegenerative diseases. we examined animals with acute inflammation mediated by cd8 or cd4 positive t cells and animals suffering from t cell and antibody mediated chronic inflammation due to active immunization. further, we studied lps induced lesions which represent cns disease caused by the innate immune system. similarly, we characterized oxidative damage in the different models for inflammation. we did not find evidence for the presence of oxidized phospholipids or oxidized dna in the experimental lesions, which is in contrast to our results of ms lesions. none of these models showed iron accumulation in glial cells comparable to what is seen in ms tissue. however, some iron positive microglia and perivascular macrophages were observed in acute and chronic models. in preliminary experiments we found evidence that the presence of iron exacerbates h 2 o 2 induced cell death in purified glial cells in vitro. as a next step, we wanted to study a possible amplification of oxidative damage and neurodegeneration by iron in a more elaborate system. for this purpose we treated myelinating spinal cord cultures with iron chloride (fecl3) or ferritin. we observed iron loading in microglia in both experimental setups. moreover we found a selective decrease of microglia in iron chloride treated cultures but not after ferritin application. we did not find iron loaded oligodendrocytes in this in vitro model. iron accumulation is only minimal compared to the human brain in all the tested animal models. these observations necessitate the search for additional animal models mimicking the human situation more closely. objectives: microglia are a brain resident population of immune cells, involved in homeostatic surveillance of the brain parenchyma, with high importance in the regulation of inflammatory processes after injury. there are unresolved questions regarding microglia origin, and their similarities to peripheral macrophage populations. our objective was to compare the capacity of microglia and bone marrow derived macrophages (bmdms) to adopt different phenotypes, and how this influenced cell death after brain injury. methods: we compared the phenotype of bmdms and microglia (both bv2 microglial cell line and primary microglia) after treatment with well known polarising agents. lipopolysaccharide (lps) was used as an inducer of the classical m1 phenotype and il-4 was used to induce an alternative m2 phenotype. microglia or bmdms, of different phenotypes, were added to hippocampal organotypic slices (hosc) subjected to oxygen glucose deprivation (ogd) as an in vitro model of brain injury. results: bmdms and microglia (bv2 and primary microglia) both adopt m1 or m2 phenotypes after treatment with lps or il-4 respectively. however, addition of polarised microglia or bmdms onto hosc resulted in different outcomes. addition of activated bmdms, of either m1 or m2 phenotypes, led to cell death in control hosc and increased death when combined with ogd. on the other hand, addition of bv2-microglia did not induce any cell death in control hosc, and were protective after ogd, except for m1microglia which were toxic. endogenous microglia within the hosc were also able to adopt different phenotypes and exert neuroprotection after il-4 treatment. these ex vivo data correlated with the in vivo situation where we observed increases in microglial activation early after experimental stroke, although the vast majority of these activated cells did not express markers of the m1 phenotype. conclusions: this study highlights functional differences between macrophages and microglia, especially in response to brain injury. although microglia, like peripheral macrophages, can adopt different demyelination, thereby creating an unfavorable environment for remyelination. exploring efficient ways to prevent or overcome tlr-induced fibronectin aggregation by astrocytes might pave the way for new approaches in remyelination-targeted therapeutic strategies in ms. to study the tlr2 response in-vivo, sod1 g93a mice were crossed with the tlr2-luc-gfp reporter mice, a transgenic mouse bearing the dual reporter system luciferase and green fluorescent protein under the transcriptional control of the murine tlr2 promoter. double transgenic mice and littermates controls were monitored longitudinally using in-vivo biophotonic/bioluminescent imaging to measure microglial activation over the course of the disease. surprisingly, this analysis did not reveal an increase of activation in sod1 g93a mice as the disease progressed, with only weak signal coming from olfactory bulb and brain area. however, quantification of this signal suggested a lower level of tlr2 activation in the sod1 g93a mice compared to wild-type littermates. to further study the microglial impairment observed in the sod1 g93a mice, these transgenic mice were given lps with i.p. injections at 5 mg/kg and were followed for 48h by bioluminescence imaging, both at a pre-symptomatic age (60 days) and closer to paralysis (115 days). the level of tlr2 activation was lower in the sod1 g93a mice than the wild-type littermates, both at 60 days and 115 days. immunostaining of olfactory bulbs reveal the same phenomenon, which is less iba1 and less tlr2-positive cells in the sod1 g93a tissue. mrna analysis by in-situ hybridization confirms a similar pattern of microglial response. in a parallel study, primary microglial cell culture, derived from adult mice (60days) were similarly stimulated with lps to further understand the altered sod1 mutant microglia response. here again, sod1 cells appears less responsive to lps stimulation as observed in our mice studies. these combined results suggest that sod1 g93a microglial cells might have a pre-symptomatic suboptimal immune response. the role of reactive glia in the etiology and progress of neurological diseases is still unknown. reactive glia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect, highlighting the relevance of a strict control of the progression and resolution of glial activation. under some circumstances glial activation does not resolve, but it exacerbates or becomes chronic resulting in detrimental secondary effects. it is necessary to develop strategies to halt the negative outcome of glial activation in these situations, by controlling the pro-inflammatory phenotype of reactive glial cells and potentiating their beneficial effects. we studied the temporal pattern of inflammatory reaction in spinal cord and brain regions in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis. special attention was paid to the involvement of members of the c/ebp family of transcription factors, which regulate the expression of pro-inflammatory genes in reactive glia, and cd200, cd200r1 and trem-2, membrane-associated inhibitors of the pro-inflammatory response in resting/surveillant microglia. mice were scored for signs of eae for up to 28 days postimmunization (dpi). eae symptoms were present from 10 dpi, reaching score peak at 14 dpi. in the spinal cord, we observed a sustained increase in c/ebpa and a transient increase in c/ebpb and c/ebpd expression peaking at 14 dpi. cd200 expression decreased at 9 dpi, remaining below control values at 28 dpi. cd200r1 and trem-2 expression increased at 14 dpi, thereafter this effect progressively attenuated. no alterations were observed in the brain. an acute increase in the expression of pro-inflammatory genes (il-1b, il-6, tnf-a, inos, cox2) occurred in the spinal cord at 14 dpi. our results suggest that cd200 expression decreases before onset of eae symptoms resulting in downregulation of the microglia inhibitory signal mediated by cd200-cd200r1, which would facilitate the proinflammatory response observed after onset of the eae symptoms. the increase in cd200r1 expression observed after the onset of eae symptoms suggests a concomitant intend of microglia/macrophages to resolve the inflammatory response. the transient increase in c/ebpb and c/ebpd expression could be related to the acute increase in proinflammatory gene expression, while the sustained increases of c/ebpa expression and trem-2 could be involved in the resolution of the inflammatory response. recently, we showed a beneficial effect of myelin reactive t cells on oligodendrocyte precursor cell differentiation in zones of axonal degeneration in the hippocampal dentate gyrus, which mirrors grey matter injury in multiple sclerosis. this effect was associated with a marked expression of t-cell cytokines, such as interferon-c and interleukin-17, enhanced microglial clearance of myelin debris, and an enhanced sprouting of calretinergic axons. to gain better insight in which cytokines and signaling pathways are elevated in response to the t cell enhanced regenerative responses, we performed a mrna microarray study, in which the transcript profile in hippocampi from mice with adoptively transferred proteolipid protein specific t cells combined with an axonal lesion were compared to the transcript profile from mice with axonal lesion. thereby we identified 1446 transcripts, which were differently expressed. we identified the interleukin-1 (il-1) signaling pathway as a potential target by performing pathway analysis using david and amigo databases. we discovered that il-1a, il-1b and il-1 receptor antagonist (ra) mrna as well as the il-1 signaling pathway were highly upregulated in response to myelin reactive t cells. in order to determine if il-1b mrna was translated into protein, we performed immunohistochemical stainings on tissues from mice with 2 days post lesion survival. expression of il-1b was observed in the molecular layer of t cell infiltrated, but not in t cell na€ ıve mice with axonal lesion, suggesting that myelin reactive t cells stimulate il-1b protein expression. ongoing studies will determine the expression of il-1a and il-1ra protein and determine the cellular expression of the il-1 signaling pathway in t-cell infiltrated versus non-t cell infiltrated mice with axonal lesion. understanding the regulation of the expression of il-1a, il-1b and il-1ra in t cell compared to non-t cell infiltrated cns may lead to a better understanding of the functional consequences of inflammatory responses in the cns. when io were challenged with kainate in the presence of conditioned medium (cm) from astrocytes, susceptibility to kainate-induced cell death was reduced, but cm from l-ap4-treated astrocytes reverted kainate toxicity. in contrast, treatment with cm from control or lpsactivated microglia, either untreated or pre-exposed to l-ap4, was not able to affect kainate-induced io cell death. to establish whether mglur4 activation could modulate the antigen presenting activity of microglia, the bv2 microglia cell line was used. treatment with l-ap4 (50 lm/48 h) did not affect changes in morphology induced by activation with lps (0.1 lg/ml/48 h), but significantly reduced the percentage of cells expressing mhc class ii, as detected by flow cytometry. these data suggest that during neuroinflammation, activation of mglur4 directly on io or through astrocytes and microglia may contribute to a protective effect resulting in survival of the oligodendrocyte lineage. mirnas are small non-coding rna molecules that modulate gene expression at a post-transcriptional level and their role in the regulation of biological processes makes them an emerging class of therapeutic targets. dysregulation of mirna networks has been linked to neurodegeneration and immune dysfunctions and has become a research focus in the context of alzheimer's disease (ad). in this work, we demonstrate that mir-155 expression is increased in the brain of 33trg ad transgenic mice, prior to senile plaque formation, and co-localized with intraneuronal app accumulation in the cortex and hippocampus. in view of the pro-inflammatory functions of mir-155 and aiming at clarifying its role in ad, we evaluated the levels of mir-155 in ab-stressed astrocytes and microglia cultures, two brain-related cell types involved in neuroinflammation. we show that mir-155 expression levels are increased in astrocytes and microglia following activation with ab fibrils but not with ab oligomers. these findings correlate with the observed decrease in socs-1 expression, a mir-155 validated target, and with the increase in the production of tnf-a, il-1b and il-6 by these cells. importantly, we show that mir-155 expression is regulated by c-jun, since silencing of this transcription factor led to the reduction of mir-155 levels following astrocyte and microglia exposure to ab fibrils or lps. c-jun was also found to be upregulated in the 33trg ad transgenic model since early ages, which can help to explain the observed early increase in mir-155 expression. overall, our results demonstrate the important role of mir-155 in ad and show that silencing of c-jun in glial cells may constitute an interesting and promising anti-inflammatory therapeutic strategy towards this disease, by targeting mir-155-mediated inflammatory responses. here we demonstrate that tgfb is an important endogenous factor promoting quiescence of microglia in vitro. inhibition of microglial tgfb signalling resulted in a microglia polarisation towards the classical activation state. moreover, tgfb is able to significantly decrease ifnc-induced classical activation of primary microglia and to enhance il4-induced alternative microglia activation. we further provide evidence that il4-mediated alternative microglia activation is dependent on active tgfb signalling. these results demonstrate the essential functions of tgfb as an important regulator of microglia activation states and further suggest that tgfb might be an interesting therapeutical molecule to modulate microglial functions during development and progression of neurodegenerative diseases. we found that young mice lost weight only in the first 24h after infection, whereas aged mice exhibited a biphasic pattern of weight loss, with the second phase of weight loss beginning 1 week after infection and continuing for approximately 2 1 =2 weeks. aged mice also demonstrated a co-ordination and balance deficit in the static rod test 1 week after infection compared to uninfected or 3 week post infection aged mice, whereas young mice were unimpaired at any timepoint. s. typhimurium infection caused a significant, progressive reduction in open field rearing activity at 1 week and 3 weeks after infection in both young and aged mice. a similar but not significant trend was apparent in novel object performance. forced swim test data showed a trend towards depressive behaviour at 1 week after infection, but not at 3 weeks. analysis of pro-inflammatory mediators in the hippocampus did not indicate significantly upregulated il-1b, tnf-a, cox-1 or cox-2 transcript at any timepoint, suggesting that microglia might not play a significant role in mediating these behavioural effects, or that the changes in these molecules were too subtle to reliably detect using qpcr in the hippocampus. the effects of ageing on s. typhimurium induced weight and co-ordination changes may be a result of regional differences in the sensitivity of microglia to peripheral infection within the cns or increased sensitivity to activation of neuronal circuits involved in weight loss or co-ordination with age. we have studied microglial cells with anti-iba1 and anti-cd68 immunohistochemistry in the brains of 18 subjects with pretangle neuropathology ranging from 4 to 51 years of age. regions examined included the locus coeruleus and surrounding brainstem areas, entorhinal cortex, hippocampal formation, and other parts of the mesial temporal lobe. ten of the subjects (median age 34) showed severe neuroinflammation secondary to infectious disease (hiv, sepsis, toxoplasmosis) and cns trauma, while 8 subjects (median age 32.5) died from non-infectious conditions and lacked neuroinflammatory changes (controls). we also examined two 52-year-old subjects with down syndrome both of which showed braak stage vi neurofibrillary degeneration, where one had comorbid sepsis and the other one did not. pretangle neuropathology was staged 1a and 1b in all 18 subjects, i.e. they exhibited primarily subcortical tau pathology in the locus coeruleus and only sporadic lesions in the transentorhinal cortex. our findings show that rampant microglial activation in the ten subjects with infections and trauma was coincident with the same level of tau pathology as seen in the 8 control subjects, demonstrating that neuroinflammation does not initiate or exacerbate neurofibrillary degeneration. in addition, our findings show that the extent of neurofibrillary pathology seen in down syndrome is unrelated to neuroinflammation since the down subject without comorbid sepsis revealed a complete absence of microglial activation. overall we conclude that neuroinflammation is not causally involved in the development of neurofibrillary degeneration which is most characteristically associated with alzheimer's disease dementia. it has been suggested that peripheral infection/inflammation may play a role in the onset or development of not only neurodegenerative diseases but also depression, autism and chronic fatigue syndrome through inflammatory responses in the brain. to investigate the role of microglia in this hypothesis we used a model of neuroinflammation induced by an intraperitoneal (i.p.) injection of the toll-like receptor 3 (tlr3) agonist, polyinosinic:polycytidylic acid (poly i:c) in rats. as we reported previously an injection of poly i:c (i.p) decreased the daily amounts of spontaneous running wheel activity to $60% of the preinjection levels until day 7. microglia were morphologically activated in the prefrontal cortex (pfc) until 72 hrs after the injection of poly i:c. pretreatment with minocycline for the 3 consecutive days (40 mg/kg/ day) blocked the poly i:c-induced decrease in the running wheel activity as well as microglial activation. quantitative analysis of mrna levels demonstrated that interferon-a (ifn-a) increased in the pfc on both days 1 and 7. we found in the present study that poly i:c injection induced increases in mrnas relevant to tlr-signaling such as tlr3, interferon regulatory factor 3 (irf3) and irf7 in the pfc. furthermore, direct application of poly i:c to the primary cultured microglia also induced an enhanced expression of ifn-a, tlr3, irf3 and irf7. it has been reported that permeability of blood-brain barrier is increased after poly i:c injection (wang et al., 2004) . therefore, it is possible that the peripheral poly i:c enters into the brain to induce neuroinflammation by activating microglia. interestingly, intracerebroventricular (i.c.v.) injection of primary cultured microglia activated by poly i:c, but not by lipopolysaccharide, effectively produced a significant decrease in spontaneous activity in rats. taken together, these findings suggest that microglial activation is important for poly i:cinduced neuroinflammation through inducing tlr3-related genes. roles of each gene in the behavioral changes will be further studied. results: microglia produced nanogram levels of pgrn (10 fold higher in microglia than in astrocytes), and pgrn release from microglia was suppressed by the tlr ligands or il-1/ifnc, but increased by il-4 or il-13. unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of slpi, none were detected in microglial cultures. we also identified mmp-12 as a pgrn proteolytic enzyme, and slpi as an inhibitor of mmp-12-induced pgrn proteolysis in human microglia. conclusions: our results establish microglia as a significant source of pgrn. mmp-12 and slpi are modulators of microglial pgrn proteolysis. negative and positive regulation of microglial pgrn release by the proinflammatory/th1 and the th2 stimuli, respectively, suggests a fundamentally different aspect of pgrn regulation compared to other known microglial activation products. microglial pgrn appears to function as an endogenous modulator of innate immune responses. at pathological condition such as injury or ischemia to the central nervous system (cns), microglial cells and astrocytes become activate and inflammation occurs like proliferate, migrate and secrete some proinflammatory cytokines, il-1ß, tnf-a or il-6. however, it is unclear that the reason why inflammation is induced in the cns, and whether or not it is necessary for the brain during recovery from pathological situation. to know the molecular mechanisms for inflammation occurs in activated glial cells, we made a stab wound mouse model to the brain. we performed microarray analysis for rna extracted from the brain tissue around stab wound site compared among day after 0, 1, 3 and 7. it revealed that most of the genes from top 20 genes at high expression level around lesion site were concerned in immunological or inflammatory functions. we successfully identified and focused on to osteopontin (opn), which is an inducer of pro-inflammatory cytokine production expressed not only in reactive microglial cells but also in reactive astrocytes around the injured cns. furthermore, we also found receptors against opn functioning in the injured brain. however, functional role of opn in reactive astrocytes is not yet clarified. to analyze the central role of opn in reactive astrocytes, we examined primary culture of astrocytes from opn-deficient (opn/ko) mice and found that the morphology of these cells was unusual. by the stimulation with lipopolysaccharide to the primary culture of astrocytes from opn/ko mice, some of the pro-inflammatory cytokine expression was altered. moreover, reactivity of astrocytes caused by stab wound to the brain was examined using analogue of nucleic acid, bromo-deoxy-uridine (brdu), and clarified that it was decreased in opn/ko mice. from these results, we conclude that opn might play a major role in the activation of astrocytes under inflammation occurred to the cns. here we determined the influence of a hfd on eae. we show that feeding mice with a hfd exacerbates eae severity through the induction of the brain renin angiotensin system (ras), resulting in an increased immune cell infiltration into the cns, oxidative stress and th1 cytokines. moreover, peripheral inflammation was boosted upon a hfd due to a ras independent mechanism, indicating that a hfd exacerbates neuroinflammation in various manners. these data suggest that nutritional modulation may have an important influence on the course of ms and other neuroinflammatory conditions. the brain's immune privilege has been attributed to a lack of dendritic cells (dc) within its parenchyma and the adjacent meninges implying maintenance of antigens rather than their presentation in lymphoid organs (lo). using cd11c-gfp mice, we have recently reported the existence of a cd11c/iba-1/cd11b 1 -population in the juxtavascular parenchyma which extent their processes into the glia limitans, an ideal position for antigen-presentation. therefore, we phenotypically compared them to the cd11c 1 /cd45 1 population (all isolated with the same protocol used for brain cd11c 1 cells) from lung, liver and spleen in healthy mice using 7-color flow cytometry. we found unique, sitespecific expression patterns of f4/80, cd80, cd86, cx3cr1, ccr2, flt3 and mhc-ii. as described before, two different cd45 1 -populations (cd45 high and cd45 int ) in the brain can be separated, whereas liver, lung and spleen exhibit a more homogeneous cd45 high population. a higher percentage of the brain's cd45 1 /cd11c 1 -cells were f4/ 80-positive compared to spleen, liver and lung, but expressed it at lower levels. within the cd45 int /cd11c 1 -population most cells expressed the microglia marker cx3cr1 and ccr2 low (marker for inflammatory, motile cells). most importantly, compared to spleen and liver, cd45 int /cd11c 1 -cells from the brain almost completely lacked mhc-ii expression and cd45 high /cd11c 1 -cells from the brain have a lower percentage of mhc-ii 1 -cells. since the cd45 int cells are widely regarded as the resident, intraparenchymal microglial population and cd45 high as dcs and perivascular macrophages, our data confirm the view that a small subpopulation of microglia (cx3cr1 high , f4/ 80 1 ,ccr2 low ) share established immune phenotypical characteristics of dcs (cd11c 1 ). additionally we showed that intraparenchymal cd11c 1 microglia are unique in their low expression of mhc-ii. their weak expression of cd80 is in line with a tolerogenic phenotype. methods: we performed a series of experiments, where we examined the expression of pro-inflammatory factors in resident microglia and infiltrating macrophages after fluorescence-activated cell sorting (facs) at different eae stages. in order to properly discriminate the two cell types we used a set of markers where resident microglia were defined as cd11b 1 cd45 int ly6c -, and infiltrating macrophages cd11b 1 cd45 hi ly6c 1 . using this protocol we have analyzed the rna expression levels of mhcii (cd74, h2-aa), co-stimulatory molecules (cd80, cd86, cd40, cd274), pro-inflammatory genes (il-1b, tnf-a) and inflammasome-regulated cytokines in these immune cells with quantitative pcr. followed by analysis of mhcii, cd80 and cd40 at the protein level with flow cytometry. results: our results indicate that infiltrating macrophages have a pronounced activated (cd74 and h2-aa) and pro-inflammatory phenotype (il-1b and tnf-a) at rna level. this is supported with an increase of the expression of mhcii and the key co-stimulatory molecules cd80 and cd40 at rna as well as protein level. in contrast, microglia display a decrease in the expression of the co-stimulatory molecules cd80 and cd86 at the rna level and do not up-regulate expression of il-1b and tnf-a during the progression of eae. conclusion: our data suggest that upon eae, infiltrating macrophages display an inflammatory profile whereas microglia are only mildly immune activated. background: age-related hearing loss (presbycusis) affects half of people by the age of 75. it has a large impact on quality of life and is associated with accelerated cognitive decline. however, presbycusis is not an inevitable process of aging, suggesting that its progression could be minimised. hearing relies on sound vibrations from the middle ear to elicit movement of the basilar membrane in the cochlea, resulting in excitation of hair cells. spiral ganglion neurons relay the inner hair cell signals to the central auditory system via the vestibulocochlear nerve. sounds are integrated in the central auditory pathway and then processed by cognitive areas of the brain. degeneration of cochlear structures, including hair cells and spiral ganglion cells occurs in presbycusis. in the central auditory system there are alterations in neuronal plasticity and neurodegeneration may occur. chronic inflammation has previously been correlated with agerelated hearing loss in humans, implicating immune cells in the progression of presbycusis. microglia within the central nervous system are susceptible to priming by neurodegeneration or aging, both factors in presbycusis. it is feasible that microglia in the cochlea, an organ with similar immune privilege as the brain, could also become primed. priming increases the chance of microglia becoming activated by subsequent inflammatory events, such as 'flu, which may cause them to contribute to neurodegeneration in the cochlea and central auditory system. hypothesis: our working hypothesis is that as age-related hearing loss progresses, microglia in the cochlea and central auditory pathway will become primed. we predict that systemic inflammation will cause primed microglia to be activated to a pro-inflammatory damaging phenotype, which will increase the rate of cochlea and auditory pathway degeneration and hence hearing loss progression. methods: c57bl/6j mice are genetically predisposed to develop age-related hearing loss. young and middle-aged mice will be challenged with lps or saline (control) and microglial phenotype assessed in the cochlea and central auditory system. neurodegeneration will be evaluated at corresponding points in the cochlea and auditory pathway using molecular techniques. conclusion: if our hypothesis is proved this would suggest that rigorous control of infection in older individuals would reduce progression of hearing loss by minimising degeneration in the auditory system. this receptor triggers the response via both myd88-and trif-dependent signalling pathways, leading to production of cyto-and chemokines and thereby alarming and attracting the immune cells of the periphery to invade the brain. however, tlr4 is only fully functional in complex with several co/receptors, such as cd14. even though cd14 has been traditionally considered simply as a lps affinity provider, recent data indicate that cd14 is also necessary for the endocytosis of tlr4, which links tlr4 to intracellular signalling via the adapter protein trif. our latest data reveal the critical role of this receptor for the profile as well as the magnitude of microglial cyto/chemokine production in response to diverse lps variants. cd14 increases the sensitivity towards lps in a cell type-specific manner, making microglia far more sensitive to lps than bone marrow and peritoneal macrophages. while striatal applications of low lps doses reveal less incoming neutrophils into a brain of cd14ko mice, as compared to wildtypes, high doses lps lead to an excessive neutrophil infiltration. this phenomenon is well correlating with the observations in vitro, where cd14 absence in microglia stimulated with high doses lps causes an excessive production of selected chemokines, with the highest impact on the neutrophil chemoatractant cxcl1. these regulatory activities of cd14 require its membrane insertion and prolonged functionality, pointing to an involvement of signalling. in order to reveal candidates for such signalling mechanisms, we tested the role of syk and plc. even though these enzymes were described to play a role in cd14-dependent signalling in dendritic cells, they have no contribution in microglia, further pointing to a cell typespecific organization of the cd14/tlr4 complex in microglia. importantly, we identified receptor systems that impose influences on cd14 expression itself, which would thereby determine the extent and impact of a cd14-mediated regulation of tlr4 functions. supported by the dfg (for1336). in neurodegenerative disease misfolded proteins and cues released by dying cells can attract immune cells by chemotaxis and alter their phenotype. to better understand and differentiate between the effect of these cues, we aim to elucidate the spatiotemporal immunological events in the brain in vivo. zebrafish are an excellent model system to study leukocytes in vivo, and we previously showed by live imaging how engulfment occurs in the developing zebrafish brain (van ham et al., curr biol 2012). here we use genetically targeted cell ablation to specifically induce cell death of target brain cells. we subsequently track dying cells and immune cells by single and multiphoton 4d imaging using fluorescent reporter genes. to analyse immunological infiltrate and resident cells in an unbiased fashion, we use large-scale electron microscopy (em) of complete zebrafish cross-sections in parallel as a complementary approach. we find that within a day after onset of cell death multiple types of phagocytic cells, including microglia, accumulate in areas where cell death occurs, engulfing dying cells. granulocytes, do not migrate towards these dying cells and are not found inside the brain at these stages. we also find total numbers of immune cells, microglia in particular, are increased. in addition to microglia, we find phagocytic peripheral cells to be involved in clearance of dying cells. our findings provide an initial in vivo characterization of the immune response to programmed cell death in the brain, indicating involvement of resident and peripheral immune cells. we show spatiotemporal recruitment of these different immune cell types, and proliferation of microglia. the latter together with the recruitment of peripheral leukocytes are likely triggered to increase capacity to dispose of dying cells efficiently. our results indicate zebrafish provides a model to tease apart basic dynamic properties of immune maintenance of the diseased brain in vivo. our current studies focus on identifying the sequence of immunological events in resolving brain injury in vivo and how this is controlled, to ultimately help identify which of these aspects are harmful during brain damage or promote tissue recovery. in humans, lif and osm both signal through the lif receptor (lifr), however osm can also activate its specific receptor, osmr. lifr signaling promotes the survival of glial cells and neurons, and is thought to limit the development of pathogenic t helper 17 cells while promoting differentiation of protective regulatory t cells. osmr signaling is also neuroprotective, however the effects on immune cells are not yet elucidated. in this study, the immunomodulatory effects of lifr and osmr signaling in humans are investigated. we determined which immune cells express the receptors for lif and osm in healthy donors and compared this to the expression levels in ms patients. we found that in blood of healthy donors approximately half of the monocytes express the lifr and osmr, while 5-10% of the t cells and b cells express the receptors. more importantly, in untreated ms patients higher numbers of t cells and b cells express both the lifr and osmr as compared to healthy controls and treated ms patients. available treatments suppress the immune system, resulting in less activated t and b cells, which could explain the lower receptor expression. indeed, we measured the receptor expression on t cells and b cells after activation and demonstrated this strongly induces expression of both receptors. currently, we are investigating the effect of lif and osm on proliferation, cytokine production and antigen presentation of the different immune cell subsets. as blood circulating immune subsets of ms patients have a higher expression of lif and osm receptors, patients show increased susceptibility for modulation by these cytokines. thus, their immunoregulating properties together with the protection of glial cells and neurons indicates that these cytokines are promising candidates for the treatment of ms and other neuroinflammatory diseases. interleukin-6 (il-6) and interleukin-10 (il-10) are key cytokines with an important role in the regulation of the inflammatory and immune responses. in the central nervous system (cns), increased expression of both il-6 and il-10 occurs in a wide range of pathological conditions. meanwhile il-6 is recognized as a cytokine with a dual role acting as a pro-inflammatory or anti-inflammatory signal inducing glial activation, il-10 is well known by its ability to counteract the inflammatory and immune reactions. the objective of the present study was to evaluate the effects of local production of either il-6 or il-10 on the microglial response and the neuronal degeneration induced by facial nerve axotomy, a sterile neuronal injury model. to accomplish that, we used two transgenic mice, gfap-il6tg and gfap-il10tg, which express either il-6 or il-10 under the gfap promoter on astrocytes, i.e. a local production within the cns. unilateral facial nerve 1mm resection was performed, one set of transgenic and wild-type (wt) axotomized animals were sacrificed at 3, 7, 14, 21 and 28 days post injury (dpi) and cryostat free-floating sections were processed for immunohistochemical analysis. another set of animals were sacrificed at 21dpi to study neuronal survival. our results showed that, at 21 days after facial nerve axotomy, in comparison with the usual neuronal death observed in wt, selective cns il-6 production had a detrimental effect on neuronal survival, whereas, il-10 production was able to reduce neuronal death. these effects on neuronal survival correlated with changes in the expression of different molecules associated with microglial activation such as iba1, cd11b, cd16/32, mhc class ii, and some integrins like osteopontin and its receptors (cd44 and a5), along the different time points after injury. remarkably, when compared with their wt littermates, cd11b expression was higher on gfap-il10tg mice, whereas remains lower on gfap-il6tg animals along all the time-points analyzed. similarly, cd44 expression, which increases after axotomy in wt mice, was higher on gfap-il10tg mice at 14 dpi and 28 dpi, whereas no induction of this molecule was detected on the gfap-il6tg axotomized animals. in conclusion, our results indicate that astrocyte-targeted il-6 and il-10 production has a direct impact on neuronal survival, glial activation and integrin mediated signaling, producing a specific outcome of facial nerve axotomy. methods: we used bv-2 cells, a murine microglial cell line. in a first experiment aimed at determining the time course of the induction of expression of the lipoxygenases and receptors, they were incubated with lps. in a second experiment aimed at determining the effects of the resolvins and lipoxin on the production of il-10, il-1b, il-6 and tnfalpha, they were firstly incubated with lxa4, rvd1 and rve1 and then incubated with lps. results: our results indicated that lps only enhanced the expression of alx, the receptor for rve1 and lxa4 ( i2) (p < 0.05). the expression of the 15-lipoxygenase was also affected by lps (p < 0.01) and varied during the time course, reaching a maximum after 6h of incubation (p < 0.05). the production of the proinflammatory cytokines decreased after 18h of incubation: il-1b with rvd1 (100 nm, p < 0.001) and rve1 (10 nm, p < 0.01); il-6 with rve1 (1 and 10 nm, p < 0.05 and p < 0.001); tnfalpha with rve1 (1 and 10 nm, p < 0.001 and p < 0.01). the production of the anti-inflammatory cytokine il-10 increased only when cells were incubated with lxa4 after 6h (1 and 10 nm, p < 0.01). conclusions: these results suggested a potential role of pufa-derivates in the resolution of inflammation. methods: the expression of a panel of m1 and m2 markers on human monocyte derived m1 and m2 macrophages was analyzed using flow cytometry. this revealed that cd40 and mannose receptor (mr) were the most distinctive markers for m1 and m2 macrophages, respectively. we next examined the activation status of macrophages/microglia in ms and control tissue using a panel of m1 and m2 markers. results: our data show that m1 markers, were abundantly expressed by microglia in normal appearing white matter and by activated microglia/macrophages throughout active demyelinating ms lesions. m2 markers, mr and cd163, were expressed by myelin-laden macrophages in active lesions and perivascular macrophages. double stainings using anti-cd40 and anti-mr revealed that 70% of the results and conclusions: the aim of the present study was to evaluate the specific binding of [ 3 h]pk11195, a compound targeting tspo, to human tissue sections from patients with ms, and to correlate these findings with presence of tspo1 microglia in different types of lesions. specific binding of [ 3 h]pk11195 was evaluated using autoradiography, and histological analysis was performed on tissue sections to evaluate lesion type, tspo expression and presence of microglia. there was a clear correlation between level of specific binding of [ 3 h]pk11195 and lesion type, such that there was an increased binding to tissue with active or chronic-active lesions compared to control tissue or tissue with chronic lesions. this was concomitant with an increased tspo1 expression and increased presence of microglia in tissue sections with active or chronic-active lesions. in conclusion, specific binding of [ 3 h]pk11195 is correlated with lesion type, tspo expression and presence of microglia in tissue sections from ms patients. being astrocytes key regulators of microglial cell activation. during inflammatory response, glial cells secrete cytokines such as tnfa, il1b and tgfb, as well as nitric oxide (no), and activate phagocytosis, chemotaxis, increased expression of receptors that bind cytokines and inflammatory ligands. among ligand receptors are induced pattern recognition receptors such as scavenger receptor (srs), including class a receptor (sra), expressed by astrocytes and microglia. we will evaluate if sra has active roles in ab clearance and inflammatory activation of glia. methods: we evaluated the role played by sra in glial cell activation, and the regulatory capacity of astrocytes upon microglia in vitro, assessing production of no griess assay and cytokines by elisa; phagocytosis and activation of activity identity markers of signaling pathways by western blot. results: in primary glial cultures from wild type (wt) and knockout mice (ko) for sra, it was found that lps-stimulated sra ko astrocytes release 50% more no than sra wt astrocytes. these differences were associated with an extended activation of erk signaling pathway. in contrast, evaluation of lps-induced cytokines production, only wt astrocytes released il1b, whereas production of tnfa and tgfb reached similar levels in wt and sra ko animals. the abolition of il1b release by sra ko astrocytes appeared to be associated to the inhibition of activation of jnk and p38 signaling, and to the delayed activation of ijb/nfjb pathway in response to lps. microglia from sra ko mice also showed several differences in response to lps stimulation compared with their wt counterparts. they failed to show activation-associated morphological changes and were unable to secrete il1b, although they showed an increased release of no. in terms of activation pattern, sra ko microglia showed increased expression of mhc-ii, indicating that sra could be involved in a mechanism inhibiting mhc-ii expression that has not been previously documented. conclusions: our results suggest that sra participates in the activation of specific inflammatory responses by astrocytes and microglia, and the activation of signaling pathways involved in the production of soluble molecules, being also needed for astrocytes in order to be able to modulate microglia activation. microglia are the resident macrophages of the brain and the first responders to disturbances of brain homeostasis. they exist in one of two broadly defined states, a rather inaptly named "resting state" in which they exhibit a ramified morphology and, upon tissue disturbance, an "activated state" in which they exhibit an amoeboid morphology. the characterization as "resting" is in stark contrast with the frantic morphological changes that ramified microglia undergo in brain slices and in vivo, constantly extending and retracting processes. while the purinergic receptor p2y12 has been identified as a key receptor via which microglial processes are guided to a source of atp or to the site of an injury, the signals and pathways guiding microglial processes in the healthy tissue remain elusive. here, by imaging microglia in acute hippocampal brain slices, we assessed the baseline and targeted motility of microglial processes in the presence of different pharmacological agents targeting purinergic signaling. we found that while bath application of a p2y12 specific blocker alone (psb-0739 at 500 nm) or of a p2y1 specific blocker alone (mrs2179 at 25 mm) did not affect the baseline motility of microglial processes, bath application of a p2y1/12/13 blocker (mrs2211 at 25 mm) strongly reduced the baseline motility of microglia, leading them to retract most of their processes. moreover, a strong rebound of motility was observed after washing out mrs2211 that did not lead to microglial activation within the time window of the experiment (up to 90 minutes). in experiments measuring the chemotactic response of microglia to a pipette containing 1 mm of atp inserted into the slice, we found that chemotaxis was completely abolished by psb-0739 (bath applied at 2 mm) but not by mrs2211 (20 mm). finally, no chemotaxis was observed in response to a pipette containing udp-glucose (5 mm), an agonist of the p2y14 receptor. these results confirm that p2y12 alone controls the targeted chemotactic response of microglial processes to atp, with no discernable involvement of p2y1, 13 or 14, but suggest that p2y1, 12 and 13 might collectively play a role in controlling the baseline motility of microglial processes in the healthy tissue. how the different p2y receptors interact with each other to control microglial movements remains to be determined. supported by an eu marie curie fellowship, the wellcome trust and the erc. the ubiquitin-proteasome-system (ups) plays a central role in degradation and clearance of short lived regulatory as well as misfolded or damaged proteins. upon stimulation by interferons (ifn) the catalytic subunits b1, b2 and b5 of the 20s proteasomes (s-proteasomes) can be replaced with b1i/lmp2, b2i/mecl-1 and b5i/lmp7 subunits, giving rise to the catalytically active immuno-proteasomes (i-proteasomes), respectively. since i-proteasomes play an important role in clearance of oxidant-damaged proteins that preferentially accumulate upon inflammatory stimulation, and many neurodegenerative diseases, including alzheimer's disease (ad) and parkinson's disease (pd) are associated with a chronic inflammatory reaction, we tested the impact of i-proteasome deficiency on the pathogenesis and progression of ad and pd. while appps1 mice, a model for ad associated extracellular plaque pathology, exhibited increased levels of i-proteasome subunits in the brain, genetic ablation of the b5i/lmp7 subunit, which significantly impairs i-proteasome function, resulted in a reduction of cerebral plaque burden, indicating a disease exacerbating role of iproteasomes in ad. in contrast, transgenic alpha-synuclein (tgasn) mice, a mouse model for intracellular alpha-synuclein pathology associated with pd, crossed to b5i/lmp7 subunit deficient mice, highlight an increase of aggregated alpha-synuclein accompanied by worsening of pathology in tgasn mice lacking functional i-proteasomes, suggesting a protective role of i-proteasomes for intracellular amyloid pathologies. future studies will aim at resolving the mechanistic underpinnings of the seemingly opposing effects of i-proteasome deficiency on the pathogenesis and progression of extracellular and intracellular protein aggregation to aid better understanding and guide novel therapeutic opportunities for neurodegenerative diseases. in the most severe form x-linked adrenoleukodystrophy (x-ald) is a fatal neurodegenerative disorder with inflammatory demyelination of the brain caused by a deficiency of the adrenoleukodystrophy protein (aldp), encoded by the abcd1 gene. aldp, a member of the abc transporter subfamily d, is located in the peroxisomal membrane and transports very long-chain fatty acids (vlcfa) as coa-esters for degradation into the peroxisome. currently, the only curative therapies are allogeneic hematopoietic cell transplantation (hct) or genetically corrected autologous hematopoietic cd34 1 stem cell therapy (hsct). the success probably relies on the settlement of microglia and perivascular macrophages derived from the myelo-monocytic donor cells in the brain of the patient. therefore, we explored the phenotypes of the major immune cell types derived from the cd34 1 stem cell. immune cells from peripheral blood of controls and x-ald patients were isolated by magnetic activated cell sorting (macs). purity of cell isolations was verified by flow cytometry. in purified cells qrt-pcr analysis was used to determine mrna levels of the abcd transporters and gc-ms was used for the quantification of vlcfa levels, a diagnostic marker. the three peroxisomal abc transporters were differentially expressed in cd34 1 derived cell types of healthy controls: abcd1 and abcd2 mrna were inversely expressed in all cell types, whereas abcd3 was equally distributed. abcd2, the closest homolog of abcd1 could be expected to compensate for the loss of abcd1 function. however, the expression pattern of abcd2 was unchanged in x-ald patients; accordingly, cell types lacking abcd2 mrna (e.g. monocytes) displayed the most severe biochemical phenotype concerning vlcfa catabolism, whereas cells with substantial abcd2 expression (e.g. t-lymphocytes) were barely affected. thus, not all investigated immune cells present an intrinsic metabolic defect. therefore, we propose that the beneficial effect of hct and hsct may rely on the replacement of those cells lacking sufficient abcd2 expression in abcd1 deficiency. in addition, these findings support the concept that abcd2 is a target gene for pharmacological induction, as an alternative treatment strategy, to rescue vlcfa metabolism and possibly halt the inflammation in x-ald patients. interleukin-33 (il-33) is a cytokine that has important functions in inflammatory and autoimmune diseases. little is known, however, about il-33 in the brain. we analyzed expression of il-33 in the mouse brain during embryonic and postnatal development. being undetected until late embryogenesis, il-33 was highly expressed during the first two weeks of postnatal life, after which expression gradually decreased and was then absent from the normal adult brain. astrocytes and oligodendrocyte precursors expressed il-33, but not neural progenitors or neurons. the vast majority of il-33 positive cells displayed nuclear staining. apart from its function as a pro-inflammatory cytokine, a role for nuclear il-33, potentially in transcriptional regulation, is also emerging. the important role of neuro-inflammation in traumatic brain injury is being increasingly recognized, and local cytokine production can mediate the inflammatory response. because of its potential for attracting inflammatory cells we analyzed il-33 expression in a cortical contusion infarction model (cci). we found that il-33 expression was induced by injury, with a peak of expression three days after cci, and at six days the levels declined. il-33 positive cells showed an overlap with astrocytic and oligodendrocytic lineage markers, but not with neurons, neural progenitors, endothelial cells or microglia/macrophages. a similar time course for il-33 expression in human brain trauma was found, using cerebral microdialysis, which allows continuous sampling of the parenchymal concentrations of molecules over several days in vivo. the peak of released il-33 for the patient analyzed was 4 days post trauma. il-33 binds to a heterodimeric receptor composed of st2 and il-1racp. following injury of st2 knockout mice we found a general reduction of inflammatory cells at the site of injury, compared to wild type animals, and there were fewer microglia at the trauma site of mice lacking il-33 receptors. our data show that il-33 expression is under tight regulation in the normal brain, but is induced by traumatic injury where is important for attracting inflammatory cells. the small heat shock protein alpha b-crystallin (cryab) is expressed by oligodendrocytes (ol) and astrocytes at several stages of multiple sclerosis (ms) lesions. cryab has been shown to be protective and therapeutic in the eae model of ms, possibly due to its anti-apoptotic functions and regulation of inflammatory pathways. in this study, we further investigated the role of cryab in de-and remyelination in vivo, applying the cuprizone model of demyelination to cryab -/mice. surprisingly, we found that cuprizone-induced lesions are significantly smaller, less severe and less inflammatory in cryab -/mice, compared to wild type (wt) controls. staining for microglia and astrocytes revealed that astrocytes are the main inflammatory cell type that is affected by cryab expression: lesions in cryab -/mice display less reactive astrogliosis than wt controls. conversely, although less severe, lesions in cryab -/mice also contain fewer oligodendrocyte progenitor cells (opc) than in wt mice. in addition, our data suggest that remyelination is less efficient in cryab -/mice than in wt controls and that remyelinated areas in cryab -/mice contain fewer ol than remyelinated areas in wt mice. together, we hypothesize that cryab, due to its anti-apoptotic actions, mediates protection in both astrocytes and ol, enabling on one hand reactive astrogliosis, which contributes to demyelination, and on the other hand ol survival, facilitating remyelination. additional in vitro experiments support this hypothesis, revealing that cryab -/astrocytes are less reactive than wt astrocytes and that sirna-mediated knockdown of cryab affects ol survival. interestingly, analyzing phosphorylation patterns of cryab in cuprizone lesions revealed that cryab is differentially phosphorylated in astrocytes and ol. furthermore, we found that cryab is differentially phosphorylated in astrocytes in active demyelinating ms lesions, indicating that phosphorylation of the protein might underlie its (pathogenic) role in active demyelination. these paradoxical findings not only suggest a role for cryab as a potential target in a regenerative approach for ms treatment, but also point to a possible pivotal role for reactive astrogliosis in cuprizoneinduced demyelination and, more importantly, in early ms pathology. in a comprehensive immunohistochemical survey, we compared cortical ms lesions to those of inflammatory (tuberculous meningitis, rasmussen's encephalitis, b-cell lymphoma, and meningitis) and neurodegenerative (alzheimer's disease) diseases. although complex and fulminant immune responses were seen in many disease cases, primary demyelination was only detected in ms. in search of ms-specific molecular mechanisms, we performed whole-genome microarrays on micro-dissected archival formalin-fixed paraffin-embedded material. apart from cortical ms lesions, we also included brain tissue from patients suffering from tuberculous meningitis (inflammatory control) and alzheimer's disease (neurodegenerative control). additionally, control cases without brain pathology were included. using a restrictive cut-off, we identified 301 genes differentially expressed in ms lesions. more than 80% of these candidate genes could be assigned to t-cell mediated inflammation, microglia activation, oxidative stress, tissue injury, dna damage/repair as well as remyelination and regenerative processes. subsequent neuropathological analysis confirmed that significantly more oxidatively damaged neurons (stained positive for oxidized phospholipids) were present in cortical ms lesions than in any other examined disease. tunel stainings for the detection of dna strand breaks showed that neurons and oligodendrocytes with tunel-positive nuclei were most abundant in ms lesions containing zones of active demyelination and tissue injury. interestingly, immunohistochemical stainings for radical producing enzymes revealed that oxidative stress-mediated tissue damage in cortical ms lesions seems to be driven by nadph oxidases rather than by nitric oxide synthases. taken together, we were able to show that ms-specific primary demyelination is driven by mechanisms of tissue injury that differ from any other investigated inflammatory and neurodegenerative disease. among these mechanisms, oxidative tissue injury seems to play a major role. f. labombarda 1 , s. gonzalez 1 , i. jure 2 , a. de nicola 1 1 ibyme/conicet/uba, buenos aires, argentina 2 ibyme/coni-cet, buenos aires, argentina reactive gliosis and inflammatory mediators are implicated in demyelination and secondary damage after spinal cord injury (sci). we have previously reported that after sci, short-term progesterone treatment (3 days) stimulates oligodendrocyte precursor cells proliferation and decreases reactive gliosis, whereas chronic treatment (21 days) differenciates oligodendrocytes precursor cells into mature oligodendrocytes and enhances remyelination. presently, we further studied whether progesterone was able to modulate the inflammatory reaction and cytokine production by astrocytes and microglial cells. thus, the timecourse mrna expression of pro-inflammatory cytokines (il1b, tnfa and il-6) and pro-inflammatory enzymes (cox-2 and inos) was studied by pcr in real time. results showed that the highest increase in cytokine and pro-inflammatory enzymes mrna production occurred in rat spinal cord 6 h after sci. progesterone treatment significantly decreased the early rise of proinflammatory mediators mrnas at 6h. as progesterone action in spinal cord involves multiple mechanisms, the role played by the classical progesterone receptor (pr) on the progesterone inhibitory effects on cytokines, was assessed in prko mice. in agreement with data obtained in the rat model, sci strongly stimulated tnfa, il1b and il-6 mrna levels in spinal cord of both wild type and prko mice. however, whereas progesterone treatment inhibited the mrnas of cytokines in wild type mice 6h after sci, it was ineffective in prko mice, involving pr in the inhibition of cytokines production. finally, we measured astrocyte and microglial cells density by immunohistochemsitry after 6 h of progesterone treatment. the steroid administration significantly decreases gfap1 and ox-421 cells, which correlated with cytokine and pro-inflammatory enzymes inhibition. we conclude that progesterone attenuates reactive gliosis and inflammatory reaction, probably reducing the secondary spinal cord damage and favouring remyelination. in the steady state they are rarely observed in the cns parenchyma. however, during cns inflammation they cross the blood-brain barrier where together with microglia (cns-resident apc) they can present antigen to infiltrating t cells. the goal of this study was to compare two subpopulations of microglia: cd11c-and cd11c1 with brain dc in terms of promoting different t cell responses. two subpopulations of microglia: (cd11c-cd45dim cd11b1) and (cd11c1 cd45dim cd11b1) as well as (cd11c1 cd45hi) bdc have been sorted from the cns of c57bl/6 mice subjected to eae. sorted cells were used either for ex vivo proliferation and cytokine assays or for qrt-pcr. our results show that bdc and cd11c1 microglia are similar with regard to ability to induce proliferative t cell response from both primed and na€ ıve t cells. nevertheless, they differ in their cytokine profile and ability to promote cytokine release by t cells. our findings suggest that different subtypes of apc in the cns promote different immune responses. this work has been supported by lundbeckfonden. melanocortin 4 receptor (mc4r) is predominantly expressed in the brain and it is the only mcr expressed in astrocytes. our previous results showed that a-melanocyte-stimulating hormone (a-msh) the anti-inflammatory action is mediated by mc4r in astrocytes and in the hypothalamus of male rats and that these effects may lead to neuroprotection. we have already demonstrated that ndp-msh (an a-msh analogue) increased brain-derived neurotrophic factor (bdnf) expression through the camp-pka-creb pathway in astrocytes. in the present study we examined the participation of mitogen activated protein kinases (mapk) and phosphatidylinosotol-3 kinase (pi3k)-akt pathways in mc4r signaling in astrocytes. we also investigated the effect of a-msh on bdnf expression in vivo in brains of male rats.we preincubated cultured rat primary astrocytes with mapk (p38, jnk and erk) and pi3k-pdk1-akt inhibitors 15 min before addition of 1 mm ndp-msh for 1 h. we found that ndp-msh-stimulating effect on bdnf expression assayed by qrt-pcr was abolished only in the presence of erk and pi3k inhibitors. accordingly, levels of phospho-erk1/2 determined by western blot were increased by ndp-msh whereas phospho-akt levels were not modified at 30 min. ndp-mshinduced erk1/2 activation was decreased by adenylate cyclase and pi3k inhibitors but not by a pka inhibitor, suggesting a camp and pi3k involvement in this effect. we also investigated if a-msh was able to induced bdnf expression in vivo. for that purpose we injected male rats with a-msh (0.5 mg/kg, ip) or vehicle (saline) once and sacrificed them after 3 h. rats were also injected twice daily and for two days and killed 48 h after the first injection. we observed that bdnf mrna levels increased after a-msh treatment at 3h in the hypothalamus whereas in the cortex bdnf expression was not modified. at 48 h bdnf expression was not modified by a-msh. we showed by immunohistochemistry that mc4r and bdnf co-localizes with neurons and astrocytes in the brain. since melanocortins have anti-inflammatory and neuroprotective effects in the brain, the mechanisms described in astroglia help understand mc4r action. our results indicate that melanocortins induce bdnf expression in astrocytes through erk and pi3k, suggesting that this effect could be involved in the neuroprotective actions of melanocortins. the fact that bdnf expression also increases in vivo in the hypothalamus reinforce this idea. methods: we analyzed microglia activation and the role of nmda receptors in this process using the microglia cell line bv2 and cortical slice cultures. the in vivo analysis was performed in two models: first, in odtr mice that express the diphtheria toxin receptor specifically in oligodendrocytes (odcs) which allows induced killing of odcs (locatelli et al., 2012) and second in mice that were immunized with mog/ cfa to induce eae. results: the proinflammatory cytokine il-17a plays a pivotal role in the pathogenesis of ms and eae. we showed il-17a activates microglia in vitro. therefore, we activated the microglia cell line bv2 and cortical slice cultures with il-17a to and subsequently treated the cultures with the nmda receptor antagonists mk801 and ap5 to block nmda receptor signaling. this treatment inhibited il-17a-mediated activation of microglia and in particular ros production, proliferation and migration. additionally, we detected increased secretion of il-6 and g-csf. furthermore, il-17a enhanced activation of the nmda receptor through phosphorylation leading to higher influx of calcium upon ligand binding. to further analyze nmda receptor-depended microglia activation we used two different mouse models that provoke different mechanisms of microglia activation. whereas in eae a strong inflammatory process activates microglia, in odtr mice, activation occurs due to induced odc death. interestingly, in the context of eae microglia activation was clearly diminished when mice were treated with mk801 whereas inhibition of the nmda receptor had no effect on microglia activation in the odtr model. in line with the in vivo data we show that inhibition of microglia activation takes place only when the cells were stimulated with il-17a and not upon stimulation with lps. conclusions: our findings show that nmda receptors are not involved in general microglia activation but rather play a role in microglia activation in an inflammatory context with specific cytokines such as il-17a. aims of the study: to investigate whether cx3cl1-cx3cr1 signaling contributes to microglia migratio00ctivation and subsequent astrogliosis following msc transplantation in the cns. methods: first, in order to allow localization of grafted msc in vivo, wt c57bl/6 msc were transduced with a lentiviral vector encoding the blue fluorescent protein (bfp). next, bfp1 msc were injected into the cns (striatum) of cx3cr1 1/-(n 5 10) and cx3cr1 -/-(n 5 7) transgenic mice. these mice have respectively one or both copies of the cx3cr1 gene replaced by the egfp reporter gene. at 10 days posttransplantation, histological analyses were performed to determine iba11 microglia and s100b1 astrocytes within and surrounding the bfp1 msc graft site. astrogliosis was determined based on staining for gfap. cx3cr1 expression was evaluated based on egfp expression. all histological evaluations were quantified using tissuequest and/or imagej cell analysis software. results: (i) bfp1 msc graft survival is observed in both cx3cr1 -/and cx3cr1 1/mice; (ii) microglia migration towards grafted msc occurs independent of functional cx3cr1-cx3cl1 signaling; (iii) down-regulation of egfp gene-expression (and thus also cx3cr1 gene expression) is observed in both cx3cr1 -/and cx3cr1 1/mice on msc graft-invading microglia, but not on msc graft-surrounding microglia; (iv) the absence of functional cx3cr1-cx3cl1 signaling in cx3cr1 -/mice results in significantly less astrogliosis around the msc graft site, as compared to the msc graft site in cx3cr1 1/mice. conclusions: here we identified cx3cr1-cx3cl1 signaling as a potential target to modulate and/or control astroglial scarring following (stem) cell grafting in the cns. the latter is of significant importance as grafted cells can only functionally interact with (injured) brain tissue in the absence of a graft-surrounding astroglial scar. this accumulation is known to be essential for the usual sprouting of injured axons and leads to a functional nerve repair. because of the insignificant infiltration of macrophages in the injured leech cns, we investigate the chemotactic mechanisms of the recruitment of only resident microglial cells in order to explore in fine the crosstalk between damaged neurons and activated microglia leading to the leech cns repair. methods: in vitro and ex vivo chemotactic assays were performed by using recombinant form of hmc1q on microglial cells which were pre-incubated or not with anti-cc1qr or anti-gc1qr antibodies. then, affinity purification analyses using biotinylated c1q were performed from leech microglial cell protein extracts. finally, immunohistochemistry analyses were located the production of newly characterized c1q receptors in microglial cells. results: we demonstrated that rhmc1q contributes to the recruitment of some leech microglial cells. chemotaxis assays showed that the blocking of respective receptors, homologous to human gc1qr and cc1qr, inhibits the hmc1q-dependent recruitment. importantly, affinity purification analyses demonstrated the interaction between both receptors and hmc1q. finally, those receptors were located on distinct microglial subsets. conclusion: this work is used to specify the activation processes of only resident microglial cells. the results show the importance of hmc1q in the microglial accumulation leading to the nerve repair. in h. medicinalis, hmc1q activity is driven through two different receptors which are not present on the same cells suggesting that hmc1q activates two distinct microglial cell subpopulations. the question whether blood-borne immune cells are infiltrating brain areas afflicted with neurodegeneration in ad and other tauopathies yielded contradictory results. some authors showed that the chronic neuroinflammation in ad was provided almost exclusively by resident cns cells without any apparent influx of leukocytes from the blood while others reported that hematopoietic cells can enter the brain in alzheimer's disease and may contribute to an increased inflammatory burden. in order to address this issue we have used two independent transgenic lines expressing human misfolded truncated tau in two different genetic background (wistar and shr). we have shown that tau neurodegeneration can induce inflammatory responses mediated by reactive microglia in both transgenic lines, however the microglial responses showed a striking difference between the lines. we have identified two significantly different inflammatory responses to the same inducer. moreover, we found that the genetic background significantly modified the molecular cascade influencing leukocyte influx into the brain. in both transgenic lines, the majority of the blood cells entering the brain belonged to antigen presenting cell family -monocytes and dendritic cells. at the proteomic level we found striking differences between the lines. while in the wistar transgenic line we observed significant increase of cytokine-induced neutrophil chemoattractant-1, in shr transgenic line, tissue metalloproteinase 3 was significantly reduced. these findings suggest that blood cell infiltration of the brain affected by tau neurodegeneration is modified by genetic background. this inter-individual variability should be taken into the consideration in the development of novel drugs with anti-inflammatory properties. acknowledgement: this work was supported by axon neuroscience and research grants vega 2/0161/11, 2/0205/11, 2/0193/11, apvv 0200-11, apvv 0206-11 and structural fund 26240220046. charit e -universit€ atsmedizin, berlin, germany toll-like receptors (tlrs) are key molecules of the innate and adaptive immune response in vertebrates. the original protein toll in drosophila melanogaster regulates both host defense and morphogenesis during development. tlrs recognize host-and pathogen-derived stimuli. single-stranded rna is sensed by tlr7 localized to the endolysosomal compartment of immune cells. we found that both extracellular viral rna and host-derived micro-rnas induce cell-autonomous and microglia-mediated neuronal cell death through the endosomal receptor tlr7 in vitro and in vivo. however, the exact intracellular signalling cascade and the cellular mechanisms through which tlr7 leads to tissue injury in this context remained unclear. unc93b1 is a molecule specifically involved in trafficking of nucleotide-sensing tlrs, such as tlr7, in immune cells of both humans and mice. unc93b1 physically interacts with this receptor in the endoplasmic reticulum (er), and the function of the membrane protein unc93b1 is to deliver the nucleotidesensing receptors from the er to endolysosomes. making use of real-time pcr, immunocytochemistry, and histochemistry we systematically examined the expression of unc93b1 in the murine cns. whereas a distinct expression for this molecule was observed in microglia and astrocytes, expression of unc93b1 in cultured neurons was negligible. however, expression of this molecule was detected in cortical neurons of brain sections. moreover, unc93b1 was strongly regulated during different embryonic, postnatal, and adult stages of the developing mouse brain. neurons of various brain regions were identified as the main cell type expressing unc93b1 in the developing brain. taken together, our data reveal a specific expression pattern of unc93b1 in the cns, in particular in a developmental context, and lay foundation for further investigation of the pathophysiological significance of this molecule for both injurious and developmental processes in the central nervous system of vertebrates. has benefited from a number of studies that precisely depicted different types of plaques in white or grey matter areas. also, both inflammation and diffuse axonal loss in the normal appearing white matter (nawm) were extensively described. however, little attention was given to the so-called periplaque, which is usually defined as a partially demyelinated ribbon of tissue surrounding the plaque. whether periplaques correspond to expanding lesions, sites of ongoing remyelination or areas of tract degeneration remains uncertain. methods: in this context, our study aimed to bring quantitative insights to the neuropathology of ms periplaques in the spinal cord of primary or secondary progressive ms patients. a neuropathological quantitative assessment of inflammation, axonal loss and myelin loss was performed concurrently in the periplaques, plaques and normal-appearing white matter (nawm) of cervical spinal cord samples from 16 patients with progressive ms. results: periplaques formed large areas of incomplete myelin loss that extended distant away from the border of plaques. axonal loss was quantitatively similar in plaques and periplaques but signs of axonal dystrophy were predominantly observed in plaques. surprisingly, axons that remained myelinated in periplaques presented a thicker myelin sheath than myelinated axons of the nawm. inflammation in the periplaque was mainly characterized by an accumulation of macrophages/microglia that were closely apposed to myelin sheaths but exerted poor phagocytic activity. finally, we found that neuropathological features of periplaques were overall disconnected from that of plaques with regard to size, shape, inflammation and axonal integrity. conclusions: our work indicates that in ms spinal cords, periplaques correspond to demyelinating rather than remyelinating lesions. it further suggests that in progressive forms of ms, periplaques are likely to impact significantly on neurological disability. finally, we propose that tract degeneration might not be the only cause of periplaque extension and that slowly-expanding demyelination, temporally remote from plaque formation, might occur in periplaques. molecular results will be presented that support these hypotheses. cell-adhesion between endothelial cells at the blood-brain barrier (bbb) makes the endothelium impermeable to blood-derivatives and immune cells. to establish and maintain this barrier during development, adulthood, and during disease, brain endo-thelial cells must develop and sustain these strong adhesive contacts, through expression of tight junction molecules. however, we do not know whether netrin supports inter-endothelial cell adhesion at the blood-brain barrier. given this, we hypothesize that netrin tightens the bbb during development, adulthood, and protects it during disease. methods: to test this, we used both human adult primary brainderived endothelial cells and newborn netrin-1 knockout mice and evaluated netrin's effect on inter-endothelial cell adhesion and barrier permeability. we also assessed netrins' therapeutic potential to maintain the barrier and limit immune cell infiltration into the central nervous system (cns) during experimental autoimmune encephalomyelitis (eae). results: our results demonstrate that brain endothelial cells express netrins. they help to form a tighter bbb during development. they also maintain and protect the adult barrier by increasing the expression of endothelial junction molecules, thus promoting inter-endothelial adhesion and reducing protein leakage across the barrier. netrins also reduce bbb breakdown and diminish initial myeloid cell infiltration into the brain and spinal cord during eae, which delays disease onset and ameliorates disease severity. discussion: we conclude that netrins enhance bbb stability, and protects the bbb during neuroinflammatory disease. microglial cells were shown to play an important role in many diseases of the central nervous system, not least due to their capability to become activated upon signs of brain injury. they migrate to lesion sites, proliferate, release cytokines and chemokines, and phagocytose cells or cellular debris. therefore, the microglial cytoskeleton has to be highly rearrangeable. one major element of the contractile system in nonmuscle cells is nonmuscle myosin ii (nm ii). as the role of nm ii in glial cells is poorly characterized, it is an aim of this project to study nm ii-dependent functions in microglia. we found that inhibition of nm ii by blebbistatin, which is a highly selective inhibitor of nm ii adenosine phosphatase, prevents any morphological shaping in freshly plated microglia and leads to functional deficits during migration and phagocytosis of fluorescence-labeled beads. using confocal microscopy, we could find diffuse expression of nm ii in resting microglia in vitro, whereas activation with bacterial lipopolysaccharide (lps) led to perinuclear accumulation of nm ii protein. in an activated state, microglial cells release pro-inflammatory cytokines and reactive oxygen species; interestingly, in the presence of blebbistatin the release of nitric oxide (no) as well as rearrangement of nm ii was prevented. taken together, these results illustrate a key role for nm ii in microglial shaping and functioning. ongoing studies will improve our knowledge on spatial and functional aspects of the actomyosin complex during demyelinating processes and open targets for development of therapeutic strategies towards remyelination in the cns. prolonged activation of the nucleus basalis of meynert (nbm), the primary source of cholinergic projection to the cerebral cortex has been reported to cause significant increases in cerebral cortical blood flow (cbf) in rodents. while the nbm also gives rise to gabaergic and glutamatergic projections to the cerebral cortex, the nbm-driven increase of cbf has been described to be dependent in part on muscarinic acetylcholine receptors. lately, several groups reported that astrocytes modulate local cbf via intracellular ca 21 signaling. considering that cortical astrocytes express machrs and in vivo activation of the nbm leads to machr-dependent ca 21 surges in astrocytes, cholinergic modulation of cbf via astrocytic ca 21 surges is conceivable. we report here that a single train stimulation of the nbm (stnbm; 100 hz, 0.5 s, 200 ma) induces a biphasic (a rapid increase, followed by an overshooting slower decrease that goes back to baseline within a minute) laser doppler flowmetry (ldf) response in the somatosensory cortex. moreover, the stnbm-induced ldf response was sensitive to the machr antagonist atropine. stnbm with a weaker stimulation intensity (50 ma) induces a transient decrease. surprisingly, we find that ip3r2 knockout mice, which lack cytosolic ca 21 surges in astrocytes, show similar ldf responses to stnbm. a.-c. boulay, c. giaume, m. cohen-salmon cirb, paris, france astrocytic endfeet are specialized processes, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. one of the most striking properties of astrocyte endfeet is their enrichment in the gap junction proteins connexin (cx) 43 and cx30 allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. however, the role of these proteins at the gliovascular interface is not fully understood. we have recently demonstrated that astroglial perivascular cxs expression starts after birth during bloodbrain barrier maturation and controls its integrity (ezan et al. cx30 d/d purified vessels showed essentially a strong upregulation (fold change > 12) of sgcg. this gene encodes gamma-sarcoglycan, a membrane glycoprotein associated with the dystrophin-dystroglycan complex which is essential for muscle integrity and is impaired in duchenne muscular dystrophy (petrof et al., 1993) . at the gliovascular interface, the dystrophin-dystroglycan complex anchors astrocyte endfeet to the basal lamina (nico et al., 2010) , and is crucial to the bbb integrity. however, expression of sgcg and its role in the brain has until now only been poorly documented. moreover, we found an upregulation of s100a8 and s100a9 (fold change 6,3 and 5,5, respectively) encoding the calgranulin a and b, two proteins present in neutrophils and monocytes, and involved in their migration to inflammatory sites, attachment to endothelial cells and extravasation (ryckman et al., 2003) . altogether, our results suggest that cx30 might modify the dystrophin-dystroglycan complex thereby influencing blood-brain barrier properties, and control the attachement as well as the transmigration of leukocytes through the endothelium, thus contributing to the regulation of inflammatory processes in the brain. the blood-brain barrier is supported by the perivascular glia. a crucial event during the gliogenesis is the replacement of radial glia by astrocytes. immunohistochemical staining of nestin visualized the first gliovascular connections after e16, and by e20 the radial glial endfeet cover the vessels completely. by e20 numerous nestin immunoreactive astrocyte-like cells participated in it. in parallel, nestin immunoreactivity also appeared in the wall of the vessels. by about p10 both radial glia and nestin immunopositive astrocytes disappeared and gave place to astrocytes immunoreactive to s100 protein and glutamine synthetase, markers of mature astrocytes. in the radial glia these markers were not found, and they colocalized with nestin only scarcely. the s100 and glutamine synthetase immunoreactive cells appeared at first in the middle zone of the pallium about p2, and their population gradually extended to both the pial surface and the deep cortical zones, colonizing the whole thickness by p8, in parallel with the disappearance of nestin immunoreactive glial elements, either radial or astrocytic ones. at p6 gfap immunoreactive cells were still confined to the most superficial and deepest cortical zones, so showed no colocalization with s100 and glutamine synthetase, unlike that found after p8. whether this glial 'takeover' results in a transitory increase of leakage of the blood-brain barrier, it remains to be answered. it seems however, to underly that cerebrovascular fibronectin and laminin immunoreactivities disappear at first from the middle zone of the developing cortex and only later from the superficial and deep ones, between p2 and p10. according to krum and rosenstein (1991) disappearance of these immunoreactivities refers to the fusion of the glial and vascular basal laminae, the maturation of the definite gliovascular connections. surrounded by astroglial scars that inhibit such growth. little is known about how astrocytes assemble to form this scar, and a deeper understanding of the cellular and molecular mechanisms that control this process is needed if this goal is to be achieved. in vitro data suggest that certain bioactive molecules can alter astrocyte morphology into a more growth supporting state. other molecules have been shown to promote axon growth. clinical application of many of these molecules would require neurosurgical delivery directly into the spinal cord. injectable biomaterials have the potential to serve as depots and scaffolds for in vivo delivery of bioactive molecules. we are developing di-block co-polypeptide hydrogels (dch) as fully synthetic biomaterials that could safely and easily be injected into specific sites after sci to deliver molecules in order (i) to understand better, and (ii) to manipulate, the mechanisms that control glial scar formation. we have previously shown that dch are biocompatible after injection into brain or spinal cord and selfassemble into structures with finely controllable properties. our current work shows that dch depots can deliver diffusible bioactive growth factors that influence local neurons or astrocytes in predictable ways and form gradients that are effective up to several mm away from depots. we are now testing the ability of dch depots to deliver specific growth factors that will manipulate scar-forming cells when dch are injected at clinically realistic sub-acute times after sci. to investigate the molecular phenotype of esdm, we performed a whole transcriptome analysis of esdm in comparison to primary cultured microglia, flow cytometry-sorted microglia, different myeloid cells, t cells, astrocyte-and neuron-enriched cultures. while being distinct from t cells, neurons and astrocytes, esdm were closest related to primary cultured microglia followed by flow cytometry-sorted microglia and other myeloid cells. esdm and primary cultured microglia showed a strong overlap in their transcriptome by only having 143 gene transcripts differentially expressed. most of these differentially expressed genes were immune-related genes that were up-regulated in primary cultured microglia. this indicates that esdm were in a less activated state rather reflecting the in vivo biology of these cells. flow cytometry analysis of cell surface markers selected from the microarray confirmed the close relationship between esdm and primary cultured microglia. esdm resemble primary microglia not only in respect to surface marker expression and functional properties, but also display a molecular phenotype similar to primary microglia. our data suggest that esdm are a reliable tool for the replacement of primary microglia. spinal cord injury (sci) is a devastating condition which usually leads to paralysis. this outcome of injury in most cases is permanent due to the cascade of immunological factors that create an anti-repair environment around the site of injury and due to the limited capacity of central nervous system (cns) axons to regenerate, creates a daunting challenge to establish an effective therapy. from many years of research it is apparent that a combination of therapies would be the best course of action for treatment of sci; this however greatly increase the animals cohorts and time to establish the most effective course of treatment. for this reason we have developed an in vitro model of spinal cord injury as a moderate throughput screen, which is in accordance with the 3r's. this culture consist of dissociated mixed rat embryonic spinal cord cells plated onto a monolayer of astrocytes this is essential for the spinal cord cells, obtained from rats of embryonic day 15, which then develop into a carpet of neurites that over time become myelinated forming internodes of myelin interspaced with nodes of ranvier. this mixed cns culture was utilized using a scalpel blade to axotomise the axons and a persistently neurite-free area is created which is accompanied by many features of sci, including demyelination and reduced neurite density adjacent to the lesion and the presence of and reactive astrocytes. these finding are congruent with changes seen in vivo after spinal injury. data will be presented regarding the effects from proven and novel pharmacological agents on neurite outgrowth and myelination looking to understand the mechanism in which they act. as well as modifications of the culture system to incorporate methods to align axons and isolate cell bodies from their processes for a more detailed analysis of regeneration. the role of increased activity of astrocyte networks in structural synaptic plasticity following remote axonal injury is unexplored. we set out to investigate the efficiency of synaptic rearrangements in models where astrocyte reactivity is selectively diminished by the inhibition of the main cytokine pathway in transgenic mice (tg) in comparison to that observed in wild type mice (wt). first, synapse densities were compared in the facial nuclei two weeks after unilateral facial nerve transection by synaptotagmin-1/psd-95 immunolabelling and ultrastructural analysis. we found that the recovery of synapse density was decreased by 40% in tg mice compared to wt mice, and that the reduction in synaptic density correlates with a relative loss of neuronal coverage by reactive astrocyte end-feet in the same conditions. specifically, we observed a 30% reduction in excitatory synapse density in the tg mice. second, we developed an organotypic entorinho-hippocampal slice culture system in which astrocyte activation and synaptic plasticity could be more closely monitored in real time. this was assisted by two-photon microscopy, using glial ca 21 -imaging and dendritic spine motility analysis after perforant pathway transection. in summary, we demonstrated that full astrocyte activation is necessary for partial structural recovery of synapses after axonal injury. our work, using simplified models, may help experimental approaches aimed at optimizing adaptive plasticity in cns injuries. pathological loss of myelin in diseases like multiple sclerosis (ms) is usually followed by a phenomenon of remyelination, in which oligodendrocytes synthesize new myelin sheaths to envelope exposed around axons in the adult central nervous system (cns). the importance of remyelination arises from the fact that this process not only restores saltatory conduction, but also protects axons from different insults and thus limits clinical disability in demyelinating diseases. in this scenario, the mammalian subventricular zone (svz) has garnered attention as a potential source of replacement cells after injury. this zone harbours stem cells and supports long-distance migration, and is activated in ms patients to promote gliogenesis. although ng2 1 precursor cells are the first to react to demyelination and show the highest proliferation rate in comparison with other cells, the relative contribution of the svz with respect to the inflammatory demyelination induced by the theiler's virus infection and oligodendrogenesis has never been addressed. in the present study, we have investigated the behavior of the svz in theiler's murine encephalomyelitis virus -induced demyelinating disease (tmev-idd). we report that in this viral model for ms, there is a preclinical phase of the disease with demyelination in the corpus callosum that is followed later by an attempt of remyelination. this phase is accompanied by an activation of the svz with no apparent activation of ng2 1 precursors, and a strong and clear staingalectins (gals) are a family of soluble lectins that, when binding to b-galactosides, form multivalent complexes with glycoconjugates of the cellular surface and induce a modulation of intracellular signaling pathways for differentiation and survival. recently, galectin-1 has been described as a microglial deactivator which prevents neurodegeneration and promotes neuroprotection in an eae model. however, its action at neuronal level is poorly understood. spinal cord injury (sci) also remains a major challenge to neurological research, as several factors such as extracellular matrix molecules inhibit axonal regeneration. among them, semaphorine 3a (sema3a) contributes to the inhibition of axonal regeneration by acting on microtubules and actin cytoskeleton. neuropilin-1 (nrp-1) is a neuronal receptor whose binding of sema3a generates repulsion of axonal growth. in addition, nrp-1 has been described as a target of gal-1 in non-neuronal systems. the aim of this work was to study the role of gal-1 intraumatic sci, considering that sema3a expression is "turned on" in sci and prevents axonal regeneration via nrp-1. gal-1 knockout mice (lgals1 -/-) were submitted to a full traumatic sci at thoracic level (t9-t10) and treated with different concentrations of recombinant gal-1. we demonstrated that lgals1 -/mice treated with gal-1 have a dose-dependent functional recovery of motility as early as 6 days post-treatment, which is structurally associated to neuronal repopulation and high axonal regeneration. these effects were only observed in animals treated with gal-1 with dimerizing capacity, but not in mice treated with a mutant gal-1, only present in a monomeric form. in addition, we observed that microglial response was "turned off" in gal-1-treated mice. moreover, exogenous dimeric gal-1 bound to the surface of injured motoneurons and promoted the spread of nrp-1. 3d confocal microscopy reconstruction showed gal-1 and nrp-1 spatial proximity. to confirm this result, we carried out immunoprecipitation assays, which allowed us to determine that only the dimeric form of gal-1 interacted with nrp-1 and induced a decrease in the levels of sema-3a bound to nrp-1. our results show that gal-1-based treatments combine its neuronal effect on nrp-1 with its deactivating effect on microglia, which leads to a better restorative process after medullar lesion. the findings presented here support the potential of dimeric gal-1 as a therapeutic agent for human sci patients. theophylline and caffeine. it depresses activation of microglial cells and astrocytes which is associated with neuronal damage during inflammation and hipoxia. it is largely known that ethidium bromide (eb) injection into the cns induces local oligodendroglial and astrocytic death, resulting in primary demyelination, blood-brain barrier disruption and schwann cell invasion, and thus serving as an experimental demyelinating model in animals. the aim of this study was to evaluate if prop had the capacity of affecting glial cell behaviour during the process of demyelination and remyelination following gliotoxic injury with eb. wistar rats were divided into 2 groups: i-rats injected with 10 microlitres of 0.1% eb into the cisterna pontina and treated with prop; ii-rats injected with eb and not treated with the xanthine. prop (agener) treatment was done using 12.5 mg/kg/ day by intraperitonial route during all experimental period. the rats were euthanized from 7 to 31 days after eb injection and brainstem sections were collected and processed for light and transmission electron microscopy studies. results from both groups were compared by using a semi-quantitative method developed for documenting in semithin sections the extent and nature of remyelination in gliotoxic lesions. in general terms, by 7-11 days following gliotoxic lesion, the center of the lesion was expanded and filled with huge amounts of myelin debris among foamy macrophages and demyelinated axons. no astrocytic processes were found in this site and some lymphocytes were seen in the neuropil and around blood vessels. the most prominent feature of the 15 th day was the initial association at peripheral locations between naked axons and remyelinating cells. schwann cells were associated with one or multiple demyelinated axons or already forming thin myelin lamellae around single axons in astrocytic-free areas. oligodendrocytes began to form thin myelin sheaths in areas completely or partially filled with astrocytic prolongations. by 21-31 days, it became evident that rats treated with prop presented a small improve in the repair process when compared to untreated animals, the latter presenting greater amounts of myelin-derived membranes in the central area and a lesser extension of oligodendrocyte remyelination, but without any apparent difference regarding to schwann cells. by 31 days results showed that prop administration following eb injection seemed to stimulate oligodendroglial remyelination (mean remyelination scores of 3. peripheral axotomy results in a disconnection of the neuronal cell body from the target organ. a complex rearrangement of the nerve microenvironment, the so called wallerian degeneration, takes place distally to the lesion and involves schwann cell proliferation as well as macrophage infiltration. the process results in the formation of the bands of b€ ungner which guide the axonal sprouts throughout the nerve distal stump. although this process has been extensively studied, a thorough understanding of the three-dimensional nerve tissue reorganization is not completely known. such knowledge may in turn lead to more effective strategies aiming at the repair of peripheral nerves following transection or crushing. in this sense, the tetrahydrofuran (thf)-based tissue-clearing technique has been adapted for sciatic nerves of mice and used for subsequent observation to 2p-lsm. transgenic mice that express fluorescent proteins in neurons, astrocytes, schwann cells and microglia/macrophages as well as double and triple hybrids were used. additionally, by immunohistochemical analysis, it was possible to observe the structure of the nerve after injury by the expression of neurofilament and s100b, in non fluorescent mice. also, increased non neural cell responsiveness after injury was studied with anti-p75 ntr immunostaining. transmission electron microscopy allowed the evaluation of the ultrastructure of the nerve microenvironment, as well as the regenerative process by 3, 7 and 14 days after injury. microscopic examination of cleared tissues of transgenic mice through 2p-lsm consisted in comparing the different cell type interaction before and after injury, with particular interest on the schwann cells, macrophages and growing axons. question: mass spectrometry (ms) has become nowadays an essential tool for the detection and identification of biomolecules in various contexts of biological problematic. ms can be performed from all types of biological materials ranging from biological fluids to tissues. end of 90's it was shown that with certain instrumentation such as those equipped with a maldi ion source it was possible to record ms spectra directly in situ from tissue sections. more, point to point automated acquisition of maldi ms spectra from a whole tissue section was shown to allow for reconstruction of the 2d images of biomolecules contained in the section distribution through ion density maps (caprioli rm et al., anal. chem. 1997). methods: maldi ms imaging (maldi msi) is a molecular imaging modality that presents several advantages including possible imaging of hundreds of biomolecules in one step acquisition and the untargeted character of the method allowing to study molecules without any prerequisite knowledge or use of probes. results: over the past decade maldi msi has demonstrated its potentiality in terms of applications for biology and clinics (franck j et al., mol. cell. proteomics. 2009). maldi msi can be use to study both endogenous molecules namely metabolites, lipids, peptides and proteins or exogenous ones such as drugs or xeniobiotics and was applied to various problematic. understanding of physiological or physiopathological processes requires knowledge on the identity and localization of molecules within a tissue. maldi msi reseals a clear potential to answer this objective and give a unique contribution. indeed, more recently maldi msi has gained new strategies for identification of molecules in situ on tissues. in particular, many efforts were given to develop confident identification of proteins from tissue sections. conclusion: here we will review this novel technology and discuss some of its current and potential contributions for neurosciences application (wisztorski m et al., dev. neurobiol. 2008). this will be illustrated by presentation of the investigation of invertebrate neuroimmune response as well as vertebrate neuropathological disorders using either fresh frozen or formalin fixed paraffin embedded tissues. the sphingosine 1-phosphate (s1p) signaling pathway is known to influence astroglial reactivity in experimental autoimmune encephalomyelitis and the synthetic s1p analog fty720 has been shown to provide neuroprotection in experimental models of acute stroke. however, the effects of a manipulation of s1p-s1p1 signalling at later time points after experimental stroke have not yet been investigated. we examined whether a relatively late initiation of a fty720 treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors. we induced photothrombotic stroke (pt) in adult c57bl/6j mice and allowed them to recover for three days. starting on post-stroke day 3, mice were treated with fty720 (1 mg/kg b.i.d.) for 5 days. behavioral outcome was observed until day 31 after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, taqman v r analysis and immunofluorescence with especial emphasis on markers of astroglial reactivity. fty720 treatment results in a significantly better functional outcome persisting up to day 31 after pt. this is accompanied by a significant decrease in gfap-immunoreactivity (-ir) and an increase in psdsize. however no change in gfap-mrna, gs-ir or cs56-ir was observed. while fty720-treatment leads to a decrease in s1p concentration, it increases vegf-mrna in the periinfarct cortex. the initiation of fty720 treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors. due to the restrictions in regenerative capacity of the brain the tissue most severely damaged after traumatic brain injury (tbi) will end up as a fluid filled cavity. presently, it is an impossibility to effectively restore lost brain tissue, but ongoing research on the stimulation of endogenous neural stem cells has increased the hope to viably and functionally repopulate the injured parenchyma. however, it is crucial that possible therapies can show a long-term effect on both regeneration and functional recovery to be of clinical interest. in this study the enhanced induction of neurogenesis in rats after experimental tbi was evaluated three or six weeks after injury. severe focal tbi was performed using the controlled cortical impact model after which a combination therapy of intracerebroventricular administration of epidermal growth factor (egf) for seven days followed by deposition of extracellular matrix scaffold, containing vascular endothelial growth factor, into the cortical cavity. the treatment was devised to accomplish an optimal effect on the stem cell regeneration. the animals with six weeks survival time were functionally evaluated using the morris water maze (mwm). before sacrifice the animals were injected with bromodeoxyuridine (brdu) to identify newly generate cells. sections were made and immunohistochemically double stained for brdu and cell type markers for neurons, astrocytes and blood vessels. the sections were micrographed and analyzed for hemispheric tissue loss as well as number of new astrocytes, neurons and blood vessels. three weeks after injury there was a significant treatment effect as shown by an increase in neuronal and astrocytic regeneration. however, after six weeks there was no difference in the number of newly generated neurons and astrocytes. evaluation of tissue loss and spatial learning in the mwm corroborated that the treatment had no effect at the later time point. the results clearly show the importance of longterm studies in rodents to ensure that a promising effect on tissue regeneration and functional outcome is not merely temporary. astrocyte activation to extracellular matrix (ecm) -producing cells is inhibitory to regeneration in cns injury or disease. here we show that the cleaved neurotrophin receptor p75 ntr controls transforming growth factor (tgf)-beta-mediated astrocyte activation via regulation of nucleocytoplasmic shuttling of smad2. loss of p75 ntr completely rescues tgf-beta-induced hydrocephaly, astrocyte activation, and ecm production in gfap-tgf-beta transgenic mice. nuclear fractionation of tgfbeta-treated p75 ntr knock out astrocytes revealed reduced nuclear phosphorylated smad2 than their wt counterparts. in accordance to p75 ntr depletion, inhibition of tgf-beta-induced gamma-secretasemediated p75 ntr cleavage also reduces nuclear accumulation of smad2 and the secretion of proteoglycans that inhibit neurite outgrowth. taken together, our results identify regulated intramembrane cleavage of p75 ntr as a mechanism for astrocyte activation resulting in the regulation of tgf-beta signaling and scar formation in the diseased brain. the reaction of glial cells toward brain injury comprises a proliferative response with some reactive astrocytes even reacquiring stem cell potential as indicated by forming long-term self-renewing and mutlipotent neurospheres (for review: robel et al., 2011). here we addressed first the question to which extent the type of injury selectively affects the proliferative response of ng2 glia and astrocytes and to which extent the proliferative reaction of astrocytes may be linked to the stem cell response. interestingly, while both astrocytes and ng2 glia mounted a profound proliferative response after acute invasive injury, such as stab wound and mcao, non-invasive injury models, such as amyloid plaque deposition or widespread neuronal death could not activate the proliferation of these macroglial cells, while microglia proliferated actively in all these lesion models. intriguingly, the proliferative response of astrocytes correlated to the activation of their potential to form self-renewing, multipotent neurospheres with a steep decline with age. we then set-out to determine the signals regulation reactive astrocyte proliferation and neurosphere formation after invasive injury and demonstrate that invasive lesions result in entrance of sonic hedgehog (shh) into the brain from extraneural sources, such as the cerebro-spinal fluid, and activate astrocyte proliferation and neurosphere formation. this response can be blocked by systemic injection of the shh-signaling antagonist cyclopamine as well as inducible astrocyte-specific deletion of the receptor smoothened by glast creert2 . interestingly, shh-agonists can boost the proliferative response of astrocytes in vivo and their subsequent neurosphere-forming capacity, thereby providing a new approach how to activate this response in conditions where it is limited, as e.g. in age or noninvasive injury conditions. taken together, our work highlighted for the first time differences in the proliferative and stem cell response of reactive astrocytes in different injury paradigms and identified the shh signaling pathway as a key signal in this response. tumor necrosis factor (tnf) is a pleotrophic cytokine involved in normal brain function and inflammation, apoptosis, and other responses that occur following injury and disease. there is a considerable amount of confusion as to whether or not tnf activation is beneficial or detrimental following injury to the cns. genetic studies in mice have suggested that inflammation in disease models involves soluble tnf (soltnf) and that maintenance of innate immune function involves transmembrane tnf (tmtnf). these findings suggest that the outcome of selective pharmacologic inhibition of tnf may depend on whether the pharmacologic intervention is targeted towards soltnf or tmtnf. therefore, we took advantage of a dominant-negative inhibitor of soltnf, xpro195, that selectively inhibits soltnf and compared the effect of this inhibitor to a more widely used igg1 fc-tnf receptor (tnfr) 2 fusion protein etanercept, an inhibitor of both soltnf and tmtnf, that has been a successful treatment strategy for several autoimmune diseases. however, etanercept has been shown to display severe side effects by increasing the risk of infections. female c57bl/6 mice were subjected to a spinal cord injury at t9 level. mice were divided into three groups, receiving etanercept, xpro1595, or saline. the drug was delivered directly onto the lesion site using alzet mini osmotic pumps for 3 days starting immediately after introduction of the lesion. mice were allowed to survive 35 days. functional evaluation was performed using the basso mouse scale, rung walk, open field test and hargreaves test. in contrast to etanercept, xpro1595 significantly ameliorated recovery of hind limb function (evaluated by motor recovery score) compared to saline when administered continuously to the lesioned cord, whereas s.c. injections every 3 days for 8 weeks following sci had no effect. our study suggests that selective inhibition of soltnf is beneficial following sci when administered directly to the contused spinal cord and raises the possibility that selective inhibition of soltnf may represent a new therapeutic strategy for the treatment of sci without compromising the innate immune response. here, we show that expression of oncostatin m (osm) is upregulated after spinal cord injury (sci). to reveal the relevance of increased osm signaling in the pathophysiology of sci, osm was applied locally after hemisection. acute osm-treatment significantly improved locomotor recovery after mild and severe sci. delayed osm-treatment in the chronic phase was ineffective. improved recovery in osm-treated mice was associated with a reduced lesion size, less astrogliosis and diminished neuroinflammation. the underlying mechanism involves neuroprotective effects of osm. furthermore, osm promoted nerve fiber regeneration both in vitro and in vivo. thus, local production of osm after sci is an endogenous response that limits cns lesion propagation and promotes recovery. the university of western australia, crawley, australia following neurotrauma, cells beyond the initial trauma site undergo secondary degeneration, with excess ca 21 a likely trigger for loss of neurons, compact myelin and function. treatment of secondary degeneration by limiting excess ca 21 entry into cells using inhibitors of specific ca 21 channels showed promise in preclinical studies, but clinical trials were disappointing and combinatorial approaches are acknowledged as necessary. we assessed efficacy of every possible combination of three ca 21 channel inhibitors at reducing secondary degeneration 3 months after partial optic nerve (on) transection in rat. we used lomerizine to inhibit voltage gated ca 21 channels (for 3 months); oxidised adenosine-triphosphate (oxatp) to inhibit purinergic p2x 7 receptors and/ or 2-[7-(1h-imidazol-1-yl)26-nitro-2,3-dioxo-1,2,3,4-tetrahydro quinoxalin-1-yl]acetic acid (inq) to inhibit ca 21 permeable a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (ampa) receptors (both for the first 2 weeks after injury). only the three ca 21 channel inhibitors delivered in combination completely preserved visual function to levels not different from normal animals. in order to determine the mechanism/s by which the three inhibitors delivered in combination preserved function, we assessed neuroprotection, nerve swelling, myelin compaction and node/paranode complexes. preservation of retinal ganglion cell (rgc) somata and axons is unlikely to have accounted for the full recovery of function as only partial neuroprotection of rgcs vulnerable to secondary degeneration was achieved. a range of the ca 21 channel inhibitor combinations prevented swelling of optic nerve vulnerable to secondary degeneration, with no one agent selectively effective. each of the treatments involving lomerizine significantly increased the proportion of axons with normal compact myelin and decreased the proportion of axons with partially decompacted myelin, indicating the importance of sustained control of ca 21 flux via voltage gated ca 21 channels for prevention of chronic myelin decompaction. nevertheless, limiting decompaction of myelin or formation of atypical node/ paranode complexes was not sufficient for preservation of function in our model. it is likely that the additional prevention of the lengthening of the paranodal gap that was only achieved by treatment with the three ca 21 channel inhibitors in combination was an important effect that contributed to the associated preservation of visual function by this combinatorial treatment strategy. to generate myelinating oligodendrocytes opcs undergo a distinct series of morphological and molecular changes. these are regulated by a combination of extrinsic and intrinsic factors, which have only been partially been revealed so far. in the present study we identify a novel signaling mechanism as potent negative regulator of opc maturation. ephrin b3, a member of the b-class family of ephrins, inhibits opc process formation and blocks the formation of mature oligodendrocytes in vitro. the presence of ephrinb3 results in activation rhoa and pkca signalling and deactivation of fak kinase in opcs in vitro. moreover, epha4 rtk, a receptor responsive to ephrinb3 in opcs, is also expressed by opcs in demyelinating lesions. infusion of recombinant ephrin b3 into demyelinating lesions results in a failure of remyelination caused by an inhibition of opc maturation. in contrast, antibody-mediated masking of ephrin b3 epitopes in vitro as well as in demyelinating lesions of aged rats promotes opc differentiation and accelerates myelin regeneration. our results demonstrate an important role of ephrin b3 for the process of remyelination. after peripheral nerve injury, axons rapidly degenerate and start regrowing only after a few days. full functional recovery, however, is rare. a progressive loss of intact axons also determines the clinical phenotype of chronic demyelinating peripheral neuropathies, such as charcot marie tooth disease type 1a (cmt1a). cmt1a is the commonest inherited neuropathy, caused by a duplication of the peripheral myelin protein 22kda (pmp22) gene. we demonstrate that the transgenic neuronal overexpression of the growth factor neuregulin-1 type i enhances axonal regeneration and functional recovery after experimental acute peripheral nerve injury. similarly, neuregulin-1 type i overexpression preserves axonal loss and improves nerve function in a mouse model for cmt1a. importantly, neuregulin-1 type i induced axonal preservation in cmt1a mice was independent of myelination, as myelin sheath thickness was not altered. we conclude that axonal recovery and preservation relies on common supportive factors and that paracrine neuregulin-1 type i constitutes a beneficial signal in peripheral nerve disorders. g. szab o university of tuebingen, tuebingen, germany question: the enormous capacity for repair of the cns after lesion is one of the most amazing property of lower vertebrates. in the mammalian central nervous system (cns) the astrocytes reveal the so-called orthogonal arrays of particles (oaps); the clusterization of the aqp4 molecules at the astrocytic endfeet. in lower vertebrates these structures are lacking. in the case of the amphibia -anolis carolinensis-the caudal spinal cord has the capability for regeneration after loss of the tail, an ability which might be in relation with the presence of tight junctions (tj) between the glial cells like the olfactory ensheating cells act in mammalian olfactory system through the lifespan. the growing axons of the fish optic nerve are embraced by astrocytes which are also interconnected by tjs. tj connected glial cells could contribute to a growth-supportive microenvironment which might reinforce our hypothesis namely the mutual exclusion of aqp4 occurrence in astrocytes and regenerative growth of nerve fiber. methods: we performed immunocyto-and immunohystochemical staining, western blot, freeze-fracture electron microscopy and pcr on cultured aqp4 knockout and wildtype astrocytes and also on formalin fixed paraffin embedded tissue sections regarding junctional proteins, such as occludin, claudin 1,3,5. result: in the light of our experiments, there is no mutual exclusiveness between the presence of aqp4 and tight junctions in astrocytes, at least in mammals. further investigations need to be completed. glutamate and electrical activity influence opc differentiation and myelination in normal development. opcs receive synaptic input from unmyelinated axons, possibly to initiate myelination. both opcs and mature oligodendrocytes respond to glutamate via ampa and nmda receptors. activation of glutamate receptors in vitro increase opc migration but decreases proliferation. here we examine the role of glutamate signalling in remyelination following experimental demyelination in the adult rodent cns. we voltage-clamped opcs in brain-slices of adult rat cerebellar peduncle containing focal areas of primary demyelination and post-identified these by ng2-immunolabelling. recruited opcs mainly expressed ampa receptors at the peak of the opcs proliferation (5-10 days postlesion), as over 90% of the glutamate evoked current was blocked with nbqx (ampa/kainate receptor antagonist) and unaffected by ap5 (nmda receptor antagonist). the demyelinated axons continued to propagate action potentials with latencies similar to those in unmyelinated axons during development. critically, the demyelinated axons established synapses with opcs expressing voltage-gated sodium currents. these synaptic inputs had identical decay times to those recorded during developmental myelination. pharmacological inhibition of neuronal activity or glutamate signalling decreased remyelination efficiency by impairing differentiation. these results indicate that 1) glutamate currents are mainly mediated via ampa and kainate receptors during the opc recruitment 2) opcs establish synaptic contact with demyelinated axons and 3) the neuronal activity is essential for remyelination. together, these data suggest that demyelinated axons remain active and communicate with opcs through glutamate release during the regenerative process to guide their remyelination. there has been much recent interest in the use of intraocular stem cell transplantation to slow or reverse visual loss in glaucoma. however, migration and integration of stem cells into the retina is limited by reactive gliosis, a major barrier to retinal engraftment. our aim was to identify glial modulators of low toxicity and measure their potential to facilitate stem cell entry into the host retina. an explant organotypic culture system was used as a stem cell transplantation model and screening tool for candidate drugs. gfp1 mouse mesenchymal stem cells (mmscs) were co-cultured on the surface of the explants for 4 days.the microglial contribution to mscinduced reactive gliosis was investigated by assessing microglial activation and proliferation using immunohistochemistry for f4/80 and edu. distribution of chondroitin sulphate proteoglycans (cspgs) and their role in the barrier was also explored by immunostaining for chondroitin sulphate (cs-56) and by chondroitinase abc(chabc) treatment. stat3 inhibitor vi and jak1 inhibitor were used to suppress glial fibrillary acidic protein (gfap) expression. the efficacy of each drug to overcome the barrier was evaluated by immunohistochemistry for the glial markers gfap. mmsc integration was assessed by simultaneous immunostaining for gfp. cell viability after drug treatment was investigated by measuring the preservation of retinal thickness and by immunostaining for the neuronal marker neun and for caspase3. co-culture of retinal explants with mmscs caused a 1.5 6 0.4 fold increase in gfap expression. microglial activation and proliferation was not markedly affected by the presence of grafted mmscs on retinal surface. cspgs, highly expressed in the outer retina but only moderately in the inner retina, do not contribute substantially to the barrierto inner retinal engraftment. in contrast, interfering with the jak/stat pathway successfully reduced gfap expression by 60% 6 10% compared to control and facilitated mmscs integration into the host tissue. no toxic effect on cell viability was detected. on the contrary, over the 4 days ex-vivo culture, stat3 inhibitor vi treatment resulted in a neuroprotective effect on the outer nuclear layer, whose thickness (50.84 6 4.4 mm) is better preserved compared to control (38.4 6 9.0 mm) the results show that microglia and cspgs do not play a dominant role in the barrier to retinal engraftment, but the jak/stat pathway represents a promising pharmacological target.targeting modulators of glia reactivity to promote stem cell integration in the host tissue may facilitate stem cell therapy in glaucoma and other neurodegenerative diseases. oecs possess many characteristics favorable for the repair of sci including the secretion of growth and survival factors, the expression of cell adhesion molecules and the ability to integrate with astrocytes. oecs have also been reported to promote exceptional outgrowth of severed axons across the inhibitory environment of the lesion scar. the unique combination of regenerative properties suggests oecs would be beneficial for the repair of ventral spinal root avulsion injuries. after ventral root avulsion anterior horn neurons often fail to regenerate axons back into the surgically re-implanted nerve root resulting in poor functional recovery. to evaluate the efficacy of oec transplants for the repair of ventral root avulsions, the ability of oecs to promote neurite outgrowth from spinal cord neurons was investigated in a spinal cord organotypic co-culture. the integration of oecs with the spinal cord tissue was also examined by confocal fluorescence microscopy. oecs were harvested from adult sprauge dawley (sd) rats, cultured for 14 days and transfected with gfp. slices of cervical spinal cord were prepared from p8 sd pups and cultured on collagen coated membrane inserts. each slice was surrounded by a collagen gel with or without the addition of gfp oecs and glial derived neurotrophic factor (gdnf). our results indicate that oecs alone and in combination with gdnf have a positive effect on neurite outgrowth and morphology. the aim of this study was to obtain an hydrogel for the controlled delivery of vascular endothelial growth factor (vegf-a165). hydrogels are a class of liquid-gel biomaterials with high water content, typically composed of cross-linked, three-dimensional hydrophilic water-soluble polymer networks. the advantages of injectable hydrogel systems are: biocompatibility, biodegradability, adjustable shape, low cost and similarity to the extracellular matrix; moreover, they can be used as growth factor and cell delivery systems for tissue engineering applications. agarose/gelatin (a/gl) hydrogel was cross-linked with genepin. rheological measurements show that a/gl hydrogel can be injected through a syringe into the inner cavity of a nerve guide. in vitro assays performed to evaluate adhesion, proliferation, viability and migrationusing a fibroblast cell line (nih3t3), neonatal olfactory bulb ensheathing cells (nobec) and a schwann cell line (rt4-d6p2t)show that hydrogel allows cell adhesion, proliferation, viability and migration. vegf-a165 is a potent angiogenic factor and angiogenesis has long been recognized as an important and necessary step during tissue repair. vegf is known to have a positive effect on schwann cell proliferation and migration, neuron survival and outgrowth of regenerating nerve fibers. the hydrogel preparation protocol was optimized to be functionally efficient at physiological conditions, allowing the loading of vegf without the risk of its denaturation. different amounts of vegf (50-100-200 ng/ml) were incorporated in the a/gl hydrogel and the releasing rate in vitro up to 65 days was quantified by elisa . released vegf bioactivity was validated in human umbilical vein endothelial cells (huvec) and in nobec by evaluating phosphorylation of vegf-receptor 2 (vegfr2), akt and erk1-2. moreover, dorsal root ganglia explants were cultured on hydrogel containing different amounts of vegf, and an increased neurite outgrowth was quantitatively assessed. these results demonstrate that vegf can be successfully incorporated and bioactive released from a/gl hydrogel inducing vegfr2 activation and neurite outgrowth. in vivo test are ongoing on adult rats: one centimetre lesions on medial nerve were performed, followed by implantation of porous polye-caprolactone tubes filled with a/gl hydrogel containing vegf-a165. functional tests and histological analysis will be carried out to evaluate peripheral nerve regeneration. .spinal cord injury (sci) often leads to death of oligodendrocytes and considerable demyelination. demyelination of axons compromises signal conduction, and contributes to the functional deficits following sci. enhancing remyelination may serve to restore conduction and likely prevents axonal degeneration. here we focussed on a potential therapeutic intervention to promote remyelination, the transplantation of human oligodendrocytes precursor cells (opc)s capable of differentiating into mature oligodendrocytes and remyelinating denuded axons. human platelet derived growth factor (pdgf)-responsive neural precursor (prp) cells were isolated from the fetal forebrain and had been previously shown to differentiate into mature oligodendrocytes with a higher efficiency in vitro than traditional egf/fgf responsive neural stem cells (chojnacki and weiss 2004; deleyrolle et al. 2006 ). therefore, we tested the potential of human prps to differentiate into new oligodendrocytes and remyelinate axons in a rodent thoracic contusion spinal cord injury model. one week following injury, human prps were transplanted rostral and caudal to the lesion site. subsequent histological examination at two, seventeen and forty-two days post-transplantation demonstrated that human prps survived and integrated well into host tissue, particularly when nk cells were depleted. transplanted human prp's labelled with the mature oligodendrocyte marker apc, and 45% of the cells colabelled with the oligodendroglial marker olig2. astrocyte differentiation was also observed, suggesting human prps function as a multipotent glial progenitor following sci. human-specific immunoreactive processes were seen in close association with axons expressing the myelin protein mbp, providing evidence of remyelination by the transplanted cells. overall, these findings indicate that human prps are a source of new oligodendrocytes capable of producing myelin in response to spinal cord injury. remyelination is a regenerative process during which demyelinated axons are enwrapped by newly formed myelin sheaths. in the central nervous system (cns), this process takes place in two stages: first, during the recruitment stage, oligodendrocyte precursor cells (opcs) divide, migrate and engage the demyelinated axons. then, during the differentiation stage, opcs differentiate to myelin-forming oligodendrocytes. under pathological conditions such as multiple sclerosis (ms), remyelination often fails and as a consequence chronic demyelinated areas accumulate in ms. post mortem evidence suggests that opc differentiation is often impaired in ms patients. however, the reason why opc differentiation fails in ms is not completely understood. it has been proposed that ms lesions contain factors that act as specific inhibitors of opc differentiation. amongst them, myelin associated inhibitors play an important role. we have demonstrated that the inhibitory effect of myelin protein extracts (mpe) is mediated by activation of signalling cascades including pkca-marcks in vitro. furthermore, modulation of pkca has been shown to rescue opc differentiation in the presence of inhibitory mpe. here we demonstrate that tamoxifen, a drug that is used in various clinical settings because of its pkc-inhibitory activity, is able to promote opc differentiation in vitro. in a series of in vivo experiments, we tested the hypothesis that inhibition of pkca by tamoxifen promotes cns remyelination. demyelination was induced in the caudal cerebellar peduncle (ccp) of young-adult sprague-dawley rats by the administration of ethidium bromide. animals received daily doses of tamoxifen. although tamoxifen treatment did not induce changes in the number of opcs expressing immature markers (ng2 and pdgfr-a), a significant increase in the number of cells expressing mature marker of oligodendrocytes (cc1/olig2 and plp) was detected. furthermore, analysis of resin-embedded tissue demonstrated that tamoxifen significantly promotes remyelination in the cns when compared to controls (mann-whitney's test, p < 0.05). although further investigations are required to elucidate the mechanisms by which tamoxifen exerts cns remyelination, our results demonstrate that administration of tamoxifen represents a potent and clinically accessible strategy to promote endogenous myelin regeneration. induced pluripotent stem (ips) cell technology now allows development of autologous oligodendrocyte precursor cells for putative remyelination cell therapy for multiple sclerosis (ms). we have successfully developed mouse and human ips cells and converted these into oligodendrocyte precursor cells (opcs) that can differentiate into fully mature oligodendrocytes. furthermore, we have shown that (intracerebral) implantation of ips-derived opcs in mouse ms models enhances remyelination.before implantation-induced remyelination therapy with autologous ips-derived opc can proceed to a clinical trial for remyelination cell therapy in ms patients, we have to provide solid evidence for long term efficacy and to establish the safety thus ultimately excluding contamination of teratogenic (pluripotent) stem cells. these issues are best examined in a nonhuman primate model. the biomedical primate research center (bprc), rijswijk has developed a welldocumented experimental autoimmune encephalomyelitis (eae) model in marmoset monkeys that most closely approximates the pathology of ms in humans. in this animal model we will verify that intrathecally administered ips-derived opcs migrate to and remyelinate eae lesions and determine their long-term fate.we have developed an efficient protocol to induce marmoset ips cells from skin fibroblasts. for that we have used a lentiviral polycistronic construct, containing the four (human) reprogramming genes oct4, klf4, sox2 and cmyc, that is excisable using cre-recombinase. the pluripotent nature of the marmoset ips cells was established based on the endogenous expression of pluripotency transcription factors and the ability to differentiate in-vitro (via embryoid bodies) and in-vivo (subcutaneous injection for teratoma formation) into all the different tissues of the three germ layers. presently we are developing protocols for the differentiation of these marmoset ips cells into an oligodendrocyte cell lineage. published protocols have been modified for production of opcs for research purposes, and produced cells are used for characterization of opcs. ultimately the aim is to define an opc population with optimal myelination capacity. results and discussion: currently, human oligodendrocytes and opcs can be produced from pluripotent stem cells. these cells are routinely characterized in protein expression level, and methods for more comprehensive characterization are under development. produced opcs can be purified with fluorescence-activated cell sorting based on ng2 protein expression in order to study if this cell population is heterogeneous in terms of myelination capacity. conclusions: oligodendrocytes and opcs can be produced from human pluripotent stem cells. studies are ongoing to determine the molecular characteristics and myelination capacity of the differentiated and purified opc-population. remyelination, the regenerative process in which myelin is restored to demyelinated axons, is carried by oligodendrocyte precursor cells (opcs). in a previous study we have shown that oligodendrocyte precursor cells are multipotent and they can differentiate into oligodendrocytes as well as to schwann cells and occasionally astrocytes in the remyelinating lesions. we observed that schwann cells derived from opcs in the central nervous system occupied almost exclusively tissue around blood vessels in astrocyte deficient areas. therefore, we postulated the occurrence of specific niches or microenvironments that modulate opc fate. the aim of present study was to identify the factors and their downstream effectors that significantly discriminate vascular and nonvascular niche and determine the fate of oligodendrocyte precursor cells. we microdissected tissue from vascular and non-vascular regions of lesion areas at 6, 10 and 14 days after bilateral stereotaxic injection of ethidium bromide into the brain white matter of adult rats and performed microarrays to profile the whole transcriptome of both niches separately. this approach allowed us to distinguish between mrnas that are enriched within two separate environmental niches within the same lesion area. the study identified 138 differentially expressed transcripts. comparative bioinformatic analysis of global gene expression combined with signalling transduction pathway structure and genes function analysis revealed specific candidate signaling pathways differentially expressed in local peri-vascular niche compared to nonvascular one. results of rt-pcr-based validation of microarray data have proven that expression gradient of bmp4, bmp7, their antagonist sostdc1, as well as wnt2b, wnt6, dhh and scube1 act as the major regulators of opcs fate in the vascular niche. moreover, we observed that blood vessels undergo substantial regeneration in the course of remyelination and postulated the important role of blood vessels reconstruction in creating the specific microenvironment of neurovascular niche during tissue regeneration. our analysis shown that unique properties of tissue around blood vessels differ from the rest of the lesion which can be one of the factors favoring opcs alternative differentiation in this area of lesion. . however, key aspects of dynamic behavior, such as cell migration or process orientation and motility, can only be monitored by live imaging. to elucidate these key aspects of opc behavior after injury, we used repetitive in vivo two-photon laser scanning microscopy (2plsm) to follow ng2 1 -cells after smaller and larger stab wound injuries in the mouse somatosensory cortex. we therefore used the bac transgenic mouse line sox10 icreert2 (simon et al., 2012) crossed to the inducible reporter line cag-gfp (nakamura et al., 2006) selectively labeling cells of the oligodendrocyte lineage. gfp 1 opcs were imaged through a cranial window at the day of injury and at different time points thereafter. identification of the same cells was possible based on blood vessels labeled by rhodamine dextran as stable landmarks. live imaging revealed that the majority of opcs reacts within 2 days after injury with hypertrophy (enlarged soma and processes), polarization (elongation and concentration of processes towards the injury site), directed migration towards the injury site (defined as movement of the cell body for at least 10 mm) and proliferation (defined by a single cell being replaced by 2 or even more daughter cells). only a small proportion of cells within $500 mm of the injury did not react in any detectable manner. we noted that polarization and hypertrophy occur rather fast after inflicting the injury, while proliferation peaks 4 days after injury. taken together, the live observation of opcs reacting to stab wound injury supports the concept of opc heterogeneity and reveals new insights into the functional role of these cells after injury: the fast process orientation towards the injury site implies a contribution to wound closure and their substantial proliferation sometimes for several rounds amplifies the number of ng2 1 -cells surrounding the injury site with implications for scar formation. the aim of this study was to create a non-invasive tool for cell visualization and cell population tracking that can be used in long term studies. methods: this study was performed using human pluripotent stem cell lines derived in institute of biomedical technology (former regea), university of tampere, finland. cells were differentiated to neural cells using neural differentiation protocol described earlier by lappalainen and colleagues. fluorescent probes used in this study were 1) 5chloromethylfluorescein diacetate (celltracker green cmfda, ct) and 2) 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindodicarbocyanine perchlorate (did). cell viability and proliferation of the labeled populations was briefly studied. also, the labeled cells were characterized using immunocytochemistry and neuronal functionality was studied using micro electrode array (mea). results: during labeling optimization a wide range of different labeling parameters was tested for both probes to obtain long term labeling. most suitable parameters for human neural cells were 10 mm ct with 72 hours incubation time and 10 mm did with 2 hours incubation time. with these concentrations the labeling was visible up to 4 weeks and had no statistical effect on cell viability. ct labeling was not affecting to cell proliferation but with did slight decrease in proliferation was seen in long term culture. labeled populations contained both map-2 positive neurons and gfap-positive astrocytes. also, the ct or did labeled population and their mixed-cultures expressed normal spontaneous network activity when cultured on mea-platform. conclusions: this study indicated that fluorescent probes ct and did are suitable for long-term labeling of human derived neural cultures in vitro. the labeling had no negative effects on cell behavior with the exception of did labeled cells having slight decrease on cell proliferation. importantly, the labeled neural networks were electrically functional. axonal regeneration after spinal cord injury (sci) is limited mainly due to the presence of an inhibitory microenvironment, especially reactive astrogliosis and glial scar formation. overcoming this inhibitory barrier of reactive astrocytes and inhibitory substances making the microenvironment of the damaged neurons permissive for axonal regrowth might be crucial for cns repair. single-dose irradiation has been proved to be neuroprotective in cns trauma through elimination of reactive astrocytes, however, the irradiation conditions with singledose protocols are not in clinical use. in the present study, we aimed to investigate whether low-dose-fractionated irradiation, which is currently being used widely for clinical treatment of several tumor types, could regulate cell cycle elements of reactive astrocytes just like its use in cancers, inhibit glial scar formation, attenuate astrogliosis-mediated inhibition of axonal regeneration and facilitate recovery of motor function after spinal cord hemi-section in beagle dogs. as expected, our results demonstrated that low-dose-fractionated irradiation reduced reactive astrogliosis, attenuated astrogliosis-associated neural injuries, ameliorated microenvironment of axonal regeneration, and benefited recovery of the animals subjected to spinal cord trauma. accordingly, irradiation might be a promising therapeutic strategy targeting at excessive astrogliosis for sci in the near future. mesenchymal stem cell transplantation has been proven to have beneficial effects in various degenerative diseases, including demyelinating models. both in our lab as well as others have shown that they express a number of trophic factors that are capable of inducing remyelination, mainly by activating nearby oligodendrocyte progenitors towards mature oligodendrocytes that in turn remyelinate the damaged area. however, this effect was only observed locally, in the area surrounding the graft, thus in order to achieve general remyelination in various brain structures simultaneously, we decided to perform bone marrow-derived mesenchymal stem cell injections into the lateral ventricles. in this manner, the cells may attach to various areas such as the hippocampus, corpus callosum and fornix, among others, all of which are demyelinated. previous to the graft, the cells were incubated with iron nanoparticles. this way, it is possible to track in vivo the grafted cells by magnetic resonance imaging (mri). as a result, the cells were observed at different time points (0-15-30-60-90 days) by mri. also, the demyelinated areas can also be visualized and myelin quantified using image analysis software. this allows the quantification myelin density in the same individual mice at different moments before and after transplantation. j. muenzel 1,2 , c. becker 2 , t. becker 2 , a. williams 1 1 university of edinburgh, centre for regenerative medicine, edinburgh, united kingdom 2 university of edinburgh, centre for neuroregeneration, edinburgh, united kingdom central nervous system (cns) remyelination is important for the restoration and protection of proper nervous system function in demyelinating diseases such as multiple sclerosis but is poorly understood, and inefficient in humans. in contrast, zebrafish are able to regenerate many tissues very efficiently. as zebrafish are being increasingly used to study myelination, we aimed to develop and characterise an adult zebrafish model of cns de-and remyelination, to answer the question if remyelination is also very efficient, to describe the biology and to identify similarities and differences with mammalian remyelination. this then may allow us to better understand why zebrafish have a high regenerative capacity compared to rodents and humans. previous studies in teleost fishes have used optic nerve crush as a paradigm of myelin injury to investigate myelin repair. this, however, involves the de novo myelination of regenerated axons, rather than deand remyelination of the same axons. lysophosphatidylcholine (lpc) is a detergent-like toxin, which is widely used in rodent models to demyelinate axons. we administer lpc to the optic nerve of adult zebrafish and observe less myelin by both immunoreactivity and electron microscopy at the lesion site at 8 days post lesion (dpl) and microglia activation along the optic pathway. immunoreactivity and electron microscopy suggest complete regeneration of myelin at 28 dpl. we cannot identify any axonal injury in this model. in young zebrafish (aged 4-6 months), the myelin thickness of remyelinated fibres shows no difference to the pre-lesion state, which is different to mammals, where the myelin thickness is reduced. however, in old fish (aged 18 1 months), although the axon diameter is the same as pre-lesion and in young animals, , remyelinated fibres have thinner myelin, suggesting that the regenerative capacity of zebrafish declines with age. we believe that this new zebrafish model of cns remyelination can be added to the suite of current models to better understand the remyelination process, why it fails, and to test for compounds to improve it, with the added benefit that zebrafish are rapid breeders, transgenesis is easy and there is a high potential for live imaging. although often described as a hard-wired component of the vertebrate body, the nervous system is a plastic and considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity of its core components. unlike the cns, the pns is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that underlie this phenomenon. peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. they are ensheathed by myelinating or non-myelintaing schwann cell glia, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. this structure forms from centrally-derived glial cells and serves as a protective barrier that is essential for nerve function. following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris, regenerate and re-innervate targets. schwann cells have been shown to play an important role in this process by trans-differentiating, proliferating, clearing debris, and guiding re-growing axons, but less is known about the potential role of perineurial glia. to investigate the role of perineurial cells in pns regeneration, we have developed an injury response assay that uses a micropoint laser to create injuries along the motor nerves in live transgenic zebrafish. time-lapse imaging of injured nerves reveals that perineurial glia rapidly respond to nerve injury and extend processes toward the injury zone. this is in contrast to schwann cells, which we observe orienting towards the distal stump where they engulf and clear axonal debris. these data demonstrate that perineurial glia respond immediately to motor nerve injuries in a manner distinct from schwann cells, and future work is aimed at defining the molecular mechanisms that mediate the cellular responses of perineurial glia and schwann cells, as well as determining if developmental paradigms are recapitulated in these glial populations during the regenerative process. we have previously shown that in schwann cells the transcription factor c-jun acts as a master regulator of wallerian degeneration and is required for successful repair following nerve injury (arthur-farraj et al., methods. we here investigate on the release of the gliotransmitter glutamate from superfused isolated purified glial processes (gliosomes) obtained from adult rat cerebellum. intracellular ca 21 levels were measured by a microfluorimetric technique. immunocytochemical confocal and western blot analysis were also carried on cerebellar gliosomes. results. confocal and western blot analysis confirmed that cerebellar gfap-positive gliosomes are a purified preparation of glia processes. more then 80% of gliosomes were positive for brain-specific lipid binding protein (blbp), indicating that they derived from bergmann cells. by measuring the release of glutamate we obtained evidence for the presence of glutamate release-facilitatory ampa receptors on the bergmann glial processes. in fact, ampa evoked [ 3 h]d-aspartate or endogenous glutamate release which was abolished in ca 21 -free medium; effectiveness of the selective inhibitor naphthylacetyl spermine indicated the involvement of ca 21 -permeable, glua2-lacking ampa receptors. ca 21 imaging confirmed the presence of ca 21 -permeable, glua2lacking ampa receptors on bergmann glia processes. activation of the glua2-lacking ampa receptors triggered vesicular exocytotic glutamate release: inhibition of vesicular loading by the vesicular glutamate transporter (vglut) inhibitors rose bengal or trypan blue, or by the h 1 -atpase inhibitor bafilomycin a1 prevented the ampa-evoked glutamate release (see figure) . confocal analysis confirmed that blbpand gfap-positive processes expressed vglut1 and vglut2. conclusions. we here report evidence that: 1. functional processes of bergmann cells are recovered in purified preparation of adult rat cerebellar gliosomes; 2. ampa receptors located on bergmann glia processes show features of ca 21 permeable, glua2-lacking ampa receptors; 3. activation of the ampa receptors evokes ca 21 entry in isolated bergmann glia processes and ca 21 -dependent vesicular glutamate release from the processes. activation of ca 21 -permeable, glua2lacking ampa receptors coupled to vesicular glutamate release might represent a mechanism by which bergmann glia processes modulate synaptic transmission at parallel fibers/purkinje cell synapses. the formation of brain edema, which accompanies ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to extracellular k 1 elevation and neurotransmitter accumulation in the extracellular space. an increased uptake of these osmotically active substances, predominantly provided by astrocytes, is accompanied by intracellular water accumulation via aquaporin-4 (aqp4). previously, it has been shown that the removal of perivascular aqp4 via the deletion of alpha-syntrophin, a protein responsible for anchoring aqp4 on the astrocytic membrane, delays edema formation and k 1 clearance (amiry-moghaddam et al.,2003, pnas 11;100(23):13615-20). therefore, we aimed to elucidate the impact of alpha-syntrophin deletion on astrocyte volume changes in the cortex during pathological states, such as hypoosmotic stress or oxygen-glucose deprivation (ogd), using three-dimensional (3d) confocal morphometry in situ. in addition, single cell rt-qpcr profiling was carried out to reveal possible differences in the expression profiles of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. in order to visualize individual astrocytes that lack alpha-syntrophin, double transgenic mice were generated by crossbreeding gfap/egfp mice ( . 3d-confocal morphometry revealed that alpha-syntrophin deletion results in significantly smaller/slower astrocyte swelling when induced by 20 min hypoosmotic stress (210 mosm), 30 min ogd or by high extracellular k 1 (50 mm), while alphasyntrophin deletion had no effect on the astrocytic shrinkage evoked by hyperosmotic stress (350 mosm). the volume recovery of cortical astrocytes from gfap/egfp/alpha-syntrophin knockout mice was significantly slower following their exposure to hypo-or hyperosmotic stress, whereas no differences were found in astrocyte volume recovery following ogd or after their exposure to high extracellular k 1 . compared to the cortical astrocytes of gfap/egfp mice, single cell rt-qpcr analyses revealed that astrocytes from gfap/egfp/alpha-syntrophin knockout mice express higher mrna levels for two-pore domain k 1 channels (twik-1, task-2, task-3), inwardly or outwardly rectifying k 1 channels (kir3.1, kir5.1, kv1.3, kv1.6) and chloride channels (clc1,clc4), while mrna expression for the glutamate transporter glt-1 is lower. in summary, the deletion of alpha-syntrophin slowed down astrocyte swelling during hypoosmotic stress, ogd or high k 1 ; however, it also resulted in alterations in astrocytic gene expression profiles. supported by grants ga cr 13-02154s and p304/12/g069 from the gacr. microglia undergo a process of activation in any type of pathology which is controlled by many factors such as cytokines, chemokines or growth factors, but also by neurotransmitters. we found that a subpopulation (16%) of freshly isolated microglial cells from the adult brain respond to the muscarinic acetylcholine receptor agonist carbachol with a ca 21 response, indicating the expression of functional receptors. the carbachol-sensitive population increased in microglia/ brain macrophages isolated from the tissue of mouse models for stroke (57%) and alzheimer's disease (26%), but not for glioma and multiple sclerosis. microglia cultured from adult and neonatal brain contained a similar carbachol-sensitive sub-population (17% and 10%) which was increased by treatment with interferon-c to 60% within 12 hours, and was sensitive to blockers of protein synthesis. carbachol was a chemoattractant for microglia and decreased phagocytosis activity, indicating that microglia are a functionally heterogeneous population. t13-05a astrocytes and s1p receptors the family of sphingosine 1-phosphate receptors (s1prs) are g protein-coupled comprising five subtypes (s1p1r-s1p5r). these receptors are expressed in cells of the immune, cardiovascular, and central nervous systems (cns), in addition to others. s1prs play important roles in celular proliferation, differentiation, survival and migration. recently, the immunomodulatory drug, gilenya v r has been approved as the first oral therapy for multiple sclerosis (ms), after proving efficacious in clinical trials. the active ingredient of gilenya v r is the phosphorylated compound fty720 (pfty720), which is a potent agonist on all s1prs, except s1p2rs. pfty720 has been suggested to work as a 'functional antagonist' causing s1p1r internalisation in lymphocytes, thus limiting t cell auto-immunity. in addition to regulating the immune system, the lipophilic nature of the pro-drug fty720 allows it to readily cross the blood-brain-barrier (bbb) where it may also activate s1prs expressed on both neurons and glia. here, the role of s1prs in the cns was investigated, by specifically investigating their role in astrocyte function. a range of methods were used to examine the effects of s1pr activation and their roles in astrocytes, including: (i) s1p1r subtype trafficking, (ii) transient and continued intracelular signalling, (iii) astrocyte cell migration, (iv) release of pro-inflammatory cytokines, (v) oligodendrocyte cell differentiation and survival and (vi) myelination state in brain slice cultures. the data showed that activation of the s1p1r subtype leads to (i) its internalisation and (ii) continued signalling in astrocytes, and that s1p1rs were found to (iii) promote astrocyte migration, (iv) limit release of pro-inflammatory cytokines, (v) promote oligodendrocyte survival and (vi) limit demyelination. these studies demonstrate a role for s1p1rs in regulating astrocyte function and suggest their use a drug targets for neuroinflammtory and neurodegenerative disorders. in particular, both astrocytes and oligodendrocytes express kir4.1, which are essential for setting their strongly negative rmp, as well as the maintenance of [k 1 ] o following neuronal activity. notably, strong expression of the kir7.1 subtype was determined during a whole genome microarray analysis of the mouse optic nerve, a typical cns white matter that contains mainly astrocytes and oligodendrocytes. the kir7.1 subunit is known to be involved in potassium transport in epithelial cells and has been identified in cerebellar purkinje neurons, but has not been reported in glia. here, we examined functional expression of kir7.1 in mouse brain and optic nerve. rt-pcr and western blot confirmed expression of kir7.1 mrna and protein expression in the brain and optic nerve. positive kir7.1 immunostaining was observed in astrocytes and oligodendrocytes in brain sections and in optic nerve explant cultures and the results indicated a developmental increase in kir7.1 expression. in astrocytes, kir7.1 was localised to perivascular end-feet, supporting a potential role in k 1 regulation. in oligodendrocytes, kir7.1 were localised to cell somata, suggesting a developmental role in setting their rmp. in addition, oxygen and glucose deprivation (ogd) experiments were performed on isolated intact optic nerves from mice aged p10 and examined for the effects of the kir7.1 channel blocker vu590. after 60 min ogd, blockade of kir7.1 resulted in increased cell death of optic nerve glia, measured using propidium iodide (pi) labelling, from 80 6 30 in controls to 189 6 49 in vu590 (n 5 4 nerves per group, 6 fov per nerve; p < 0.001, anova). our results demonstrate expression of kir7.1 in glial cells, and indicate they are important in protecting glial cells from ischemic damage. is an eight transmembrane domain protein highly expressed in brain. human mutations in mlc1 lead to the rare genetic disorder "megalencephalic leukoencephalopathy with subcortical cyst" (mlc). characteristic for the disease are the onset of macrocephaly within the first year of life, and a slowly progressive loss of motor skills with epilepsy and cognitive decline. at the cellular level, countless fluid-filled vacuoles occur within myelin sheaths surrounding axons and in astrocytic endfeet. in cell culture models, mlc1 mutations are associated with defects in chloride currents and cell volume regulation. thus far, the expression and function of mlc1 in intact murine and human brain are still controversial. to understand the role and developmental expression of mcl1 in the brain, we have developed a mlc1 mutant null mouse model carrying an egfp reporter gene under the mlc1 promotor. we now show the exclusive expression of mlc1 in astrocytes throughout the brain with specifically higher expression around blood vessels, at the sub-ventricular zone and at the glia limitans. the functional loss of mlc1 in mutant mice recapitulates in part the human mlc disease. ko mice show macrocephaly with high brain wet weight. water filled vacuoles develop in the white matter of the cerebrum and in large fiber tracts of the brainstem. by contrast, the heterozygous loss of mlc1 has no consequences on myelin structural integrity. both heterozygous and homozygous mlc1 ko mice have dysmorphic peri-vascular and peri-ventricular astrocytes. in contrast to humans, ko mice have no motor deficits, but are hyperactive and show an anxiety-like behavior. in the mlc1 ko mouse, a dysfunction of an astrocytic protein causes loss of myelin structural integrity leading to vacuolating myelinopathy. therefore, the mlc mutant mouse could be a key model to study the astrocytic involvement in brain water and ion homeostasis. gaba activated slowly desensitizing responses in ng2 cells which were mimicked by muscimol and inhibited by bicuculline. to elucidate the subunit composition of the receptors we tested its pharmacological properties. co-application of pentobarbital, benzodiazipines and zolpidem all significantly increased the gaba responses. the presence of small tonic currents indicated the presence of extrasynaptic gaba a receptors. to further analyze the subunit expression, single cell transcript analysis was performed subsequent to functional characterization of ng2 cells. the subunits a1, a2, b3, c1 and c2 were most abundantly expressed, matching properties resulting from pharmacological characterization. importantly, lack of the c2 subunit conferred a high zn 21 sensitivity to the gaba a receptors of ng2 cells. to determine the effect of gaba a receptor activation on membrane potential, perforated patch recordings were performed. in the current-clamp mode, gaba depolarized the cells to about 230 mv, indicating a higher intracellular clconcentration (about 50 mm) than previously reported. gaba-induced depolarization in ng2 cells might trigger ca 21 influx through voltage-activated ca 21 channels. schwann cells (sc) play important roles in the development and regeneration of the peripheral nervous system (pns) following injury. several molecules such as neurosteroids and neurotransmitters have been suggested as potential pharmacological targets in regulating sc physiology and regenerative potential. nevertheless, the slow growth rate and difficulties in harvesting limit sc applications in regenerative medicine. adipose-derived stem cells (asc) can be differentiated into a sc-like phenotype (dasc) sharing morphological and functional properties in the present study, we analysed changes in glutamate transporter expression and function following a mechanical lesion in organotypic slice cultures of the mouse hippocampus using immunohistochemistry, western blots and dynamic imaging. the lesion was positioned perpendicular to the stratum pyramidale in the ca1 area and comprised the entire hippocampus proper. after three-six days, a glial scar had formed along the lesion site. activated astrocytes in close proximity (100-150 mm) to the lesion ("scar cells") directed long, palisading gfap-positive processes towards the lesion, had significantly swollen somata and lost their ability to take up sr101. furthermore, some exhibited distinct clustering of glast and glt-1 immunoreactivity. scar cells showed greatly diminished increases in intracellular sodium in response to application of d-aspartate, an agonist for glutamate transporters. astrocytes in the periphery to the lesion, in contrast, maintained their ability to take up sr101 and showed only slight upregulation of gfap, as well as less swollen cell bodies. cells in the periphery displayed only marginal changes in glutamate transporter immunoreactivity and unaltered amplitudes of sodium changes in response to d-aspartate. taken together, our data show that mild astrogliosis in the periphery of a mechanical lesion is not accompanied by a significant change in glial glutamate uptake capacity. at the scar itself, a strong clustering of glutamate transporters is observed that apparently goes along with a severe functional reduction in glutamate uptake. transport of base equivalent (hco 3 -) across the membrane is a crucial process to maintain the intracellular and extracellular proton concentrations within a narrow physiological range. the electrogenic sodium-bicarbonate cotransporter (nbce1, slc4 a4) is a major base transporter in eukaryotes and expressed in many tissues, especially in epithelial cells. nbce1 may transport significant amounts of bicarbonate into and out of the cell. in the brain, nbce1 is predominantly expressed in glial cells and operates with a transport stoichiometry of 1na:2hco 3 -. we have studied the bicarbonate sensitivity of nbce1, in primary cultured mouse cortical astrocytes (c57bl6/n) using live cell fluorescence imaging with confocal microscope and bcecf-am as a proton sensitive fluorescence probe. we have also used the primary cortical astrocyte culture from nbce1-ko mouse to compare the nbce1-mediated processes in wt astrocyte culture. bicarbonate dose response protocols suggest that nbce1 has a very high affinity for bicarbonate (k m <1 mm). due to this high affinity for bicarbonate, nbce1 can sensitize even residual bicarbonate concentrations of 150-300 lm present in nominally bicarbonate-free extracellular solutions (buffered with hepes). the removal of residual bicarbonate by saturating extracellular buffer (hepes) with 100% o 2 reduced these nbce1-mediated intracellular proton changes significantly. in astrocytes of nbce1-ko mice, cytosolic h 1 changes could not be detected at hco 3 concentrations lower than 3 mm. it has been reported that astrocytic nbce1 activity mediates the activation of astrocytic glycolysis by a mechanism dependent of intracellular bicarbonate and ph (ruminot et al 2011 j neurosci 40: 64-71). using a genetically encoded fret-based glucose nanosensor, we show that glucose metabolism can be activated at low millimolar bicarbonate concentration, which was largely absent in astrocytes from nbce1-ko. our results demonstrate the highest bicarbonate sensitivity yet described in animal cells, mediated by nbce1 in cortical astrocytes. nbce1 may function as a bicarbonate sensor in these cells, e.g. for modulating energy metabolism. supported by the deutsche forschungsgemeinschaft de 231/24-1. inward currents which were significantly blocked by the l-type calcium channel antagonist nimodipine. to better investigate the role of l-type calcium channels in ng2 glia at different developmental stages of the cns, we take advantage of the inducible cre-lox system by breeding homozygous cav1.2 floxed mice and cav1.3 flexed mice with ng2-creert2 knock-in mice. we are currently investigating how the removal of cav1.2/1.3 alters proliferation, migration and survival of ng2 glia. a particular focus lies on the analysis of behavioral abnormalities generated by cav1.2/1.3-deficient ng2 glia. chronic neuropathic pain is a frequent consequence of spinal cord injury (sci). yet despite recent advances, upstream releasing mechanisms and effective therapeutic options remain elusive. previous studies have demonstrated that sci results in excessive atp release to the peritraumatic regions and that purinergic signaling, among glial cells, likely plays an essential role in facilitating inflammatory responses and nociceptive sensitization. we sought to assess the role of connexin 43 (cx43) as a mediator of cns inflammation and chronic pain. to determine the extent of cx43 involvement in chronic pain, a weight-drop sci was performed on transgenic mice with cx43/cx30 deletions. sci induced robust and persistent neuropathic pain including heat hyperalgesia and mechanical allodynia in wild-type control mice, which developed after 4 weeks and was maintained after 8 weeks. notably, sciinduced heat hyperalgesia and mechanical allodynia were prevented in transgenic mice with cx43/cx30 deletions, but fully developed in transgenic mice with only cx30 deletion. sci-induced gliosis, detected as upregulation of glial fibrillary acidic protein in the spinal cord astrocytes at different stages of the injury, was also reduced in the knockout mice with cx43/cx30 deletions, when compared with littermate controls. in comparison, a standard regimen of post-sci treatment of minocycline attenuated neuropathic pain to a significantly lesser degree than cx43 deletion. these findings suggest cx43 is critically linked to the development of central neuropathic pain following acute sci. since cx43/cx30 is expressed by astrocytes, these findings also support an important role of astrocytes in the development of chronic pain. mast cells (mcs) are immune cells that reside in normal brain tissue. they store in granules an array of pro-inflammatory mediators, which are rapidly released to the extracellular milieu upon activation through an extracellular ca 21 -dependent mechanism. neurotoxic amyloid peptides are known to induce mcs degranulation but the membrane transduction mechanism remains unknown. here, the possible role of pannexin 1 hemichannel (panx1 hc) known to be permeable to ca 21 was studied. objective: since ca 21 influx is essential for mcs degranulation, we evaluated if the neurotoxic amyloid fragment ab 25-35 peptide promotes mcs degranulation via activation of panx1 hcs. methods: primary na€ ıve mcs were differentiated from bone marrow precursors of wild type (wt) and panx1 knock out (panx1 -/-) c57bl/6 mice using il-3 present in wehi3 conditioned medium. the presence of panxs 1, 2 and 3 mrnas presence was evaluated by rt-pcr analyses. the hc activity was assessed using the 4',6-diamidino-2-phenylindole (dapi) uptake assays and measurements of membrane current using whole cell patch clamp while intracellular ca 21 signal was evaluated using fura-2am. degranulation was assessed by quantification of extracellular histamine as well as release of toluidine blue (tb) from tb preloaded mcs from wt and panx1 -/mice. results: only panx1 mrna was detected in wt mcs. stimulation with ab 25-35 but not ab 35-25 peptide induced histamine and tb release. it also enhanced dapi uptake and total membrane current in wt mcs. these responses were drastically reduced in wt mcs pretreated with 10 mm carbenoxolone and 200 mm 10 panx1, blockers of panx hcs and were absent in panx1 -/-mcs. in wt mcs treated with ab 25-35 increase the intracellular ca 21 signal and was drastically prevented by 10 panx1, absence of extracellular divalent cations and in panx1 -/-mcs. conclusion: these findings indicate that mcs express functional panx1 hcs, which are essential for ca 21 influx required for the degranulation response induced by ab 25-35 . thus, inhibition of panx1 hcs might avoid spreading of mc-dependent inflammatory mediators released during alzheimer's disease progression. gliomas are the most frequent primitive cns tumors and are thought to derive from astrocytes or from neural progenitors/stem cells. however, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. tgf alpha is frequently over-expressed in the early stages of glioma progression. sharif & al (oncogene. 2007 (19):2695-706) previously demonstrated that prolonged exposure of normal astrocytes to tgf alpha is sufficient to trigger their reversion to a neural progenitor-like state. when astrocytes de-differentiated with tgf alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. our study aims to identify and characterize the protein signature of those in vitro transformed cells in an attempt to understand their neoplastic behavior and the effect of transformation on metabolic processes. this involves a global proteomic analysis using the 2d-dige methodology and a study of the metabolism of these cells (carbon source, lactate, glucose and glutamine use, ros metabolism…) by 1hand 13c-nmr nmr quantification of metabolites. such approaches are expected to provide information allowing understanding the metabolic reprogrammation that occurred during transformation. the comparative 2d-dige proteomic analysis of normal and transformed astrocytes shows that during transformation, the cells increase their expression of glycolytic enzymes, thus acquiring the ability to use aerobic glycolysis (warburg effect). moreover, the transformed cells reduce their capacity for tricarboxylic acid oxidation and for neurotransmitters (glutamate and gaba) metabolism. ingenuity pathway analysis indicates major effects on carbohydrates, amino acids and nucleotides metabolic components. using enzymatic activity measurements and the detection of protein isoforms by 2d-western blot and zymography, we document a change in expression and activity of various isoenzymes that may be responsible for those metabolic reprogrammations. r.e. k€ alin, a. jarczewski, f. apel, r. monk, s. kraft, j. radke, f.l. heppner charit e -universit€ atsmedizin berlin, neuropathology, berlin, germany question: glioma are the most frequent malignant primary tumors of the brain. glioma survival and growth is determined by the interaction with brain parenchyma, which includes intense tumor angiogenesis. this process is supported by glioma-invading myeloid (gim) cells, namely brain resident microglia or brain-invading macrophages. depletion of gim cells leads to a significant decrease in glioma volume. in a previous study, we established the novel neuroendocrine hormone apelin as an angiogenic factor and described its upregulation in human glioblastoma multiforme. our aim is thus to study the role of apelin signaling in tumor angiogenesis but also its possible effect on brain monocytes for the regulation of glioma growth. methods: to investigate apelin signaling during gliomagenesis we took advantage of an orthotopic mouse model for tumor formation. intracerebral xenotransplantation of the human glioma cell line u87mg, expressing lentiviral control or apelin shrna, allows us to study the specific contribution of glioma-derived apelin to gliomagenesis. to further dissect a putative immune function of apelin, we used the isogenic mouse glioma cell line gl261 for intracerebral implantation into immunocompetent mice lacking (apelin-ko) or overexpressing apelin (apelin-tg). results: loss-of-apelin expression in u87mg cells resulted in a reduced glioma volume and attenuated the formation of the xenograft vasculature. gl261 implants in apelin-ko mice produced a similar phenotype. in addition, invasion of glm cells was significantly reduced. interestingly, expression analysis showed an upregulation of the apelin receptor apj in brain myeloid cells making them competent to respond to glioma-derived apelin. conclusions: our findings demonstrate that apelin plays an important role in tumor angiogenesis and glioma growth. we show here that both, glioma cell-derived apelin and apelin expressed by the tumor neovasculature are contributing to tumor angiogenesis, gim invasion and glioma growth. we anticipate that our study will provide insights whether apelin signaling may serve as a future novel target for antiangiogenic tumor therapy. tumor infiltrating microglia/macrophages (tims) constitute the largest population of infiltrating cells in glioblastoma (gbm), the most aggressive brain tumor. data from the clinic and from experimental work performed in murine and human models indicate that tims play a significant role in gbm biology as they support proliferation, migration and invasion of tumor cells. evidence is amounting that tumor cells actually polarize tims towards this m2-like, tumor-supportive phenotype. we showed that toll-like receptor 3 ligand reverses this m2-into a m1-like phenotype. pre-activated, m1-like polarized tims incubated with spheroids of gbm cells reduced migration, killed and phagocytosed tumor cells over a 15 day-period, indicating a sustained m1 activation of tims in absence of exogenously added stimuli. in order to analyse in more detail tims-gbm cells interactions, we have undertaken two approaches. we use spheroids of cells (tumor with/without tims) embedded in collagen matrix, in which tims can be implanted, as an experimental model. this three-dimensional in-vitro system is well suited for a qualitative and quantitative monitoring of cell proliferation, death, migration and invasion. data experimentally generated are then implemented in a mathematical model that is, to our knowledge, the first one proposed to take into account tumor cells and tims in order to simulate gbm progressive behaviour. in a first step, the capacity of spheroids made of murine glioma cells to invade collagen matrices was evaluated in absence and presence of microglia. invasion was monitored by photography of the spheroids and images were processed with photoshop cs5 software. in order to achieve a precise determination of invasion, we chose to measure the diameter of the core plus the invasive rim as a read out of expansion rate. untreated microglia promoted growth and invasion of tumor cells. data generated through the proposed in-vitro system were comparable to and reproduced the insilico simulations obtained with the mathematical model, hence validating our mathematical and experimental approaches. we currently evaluate the rate of proliferation and death of tumor cells in various settings that modulate tims polarization, using flow cytometry and confocal (live) imaging. data contributed by these two approaches should facilitate the delineation of a predictive model for tumor progression in a tims-enriched microenvironment with possible therapeutic fallouts. sialic-acid-binding immunoglobulin-like lectins (siglecs) are type 1 membrane proteins displaying an amino-terminal immunoglobulin-like variable (v-set ig-like) domain that binds sialic acid and variable numbers of immunoglobulin-like constant region type 2 (c2-set ig-like) domains. siglec-h is a recently identified mouse-specific cd33-related siglec that signals via the itam linked adaptor protein dap12. so far the function of siglec-h and its ligand are unknown. in this project, we demonstrate gene transcription and protein expression of siglec-h by mouse microglia after interferon-c (ifn-c) treatment or polarization into a m1-subtype. targeting of beads to siglec-h by specific antibodies triggered the phagocytic uptake of the beads by the microglia. a siglec-h fc fusion protein generated by our laboratory selectively bound to cells with an altered glycocalyx, particularly glioma cells, but not to normal control cells. m1-polarized microglia phagocytosed glioma cells and also limited their proliferation in a co-culture system. interestingly, no signs of apoptosis were observed before phagocytosis of the glioma cells by siglec-h expressing microglia. phagocytosis of glioma cells was reduced after shrna mediated siglec-h knock down in microglia. furthermore, microglial clearance of glioma in vitro was dependent on the interaction of siglec-h with its corresponding adaptor protein dap12. our data show that m1-polarized microglial cells can engulf glioma cells via a dap12-mediated and siglec-h dependent uptake. glioblastomas (gbm) are the most common brain tumors in humans. although advances in the chemotherapeutic management of glioblastoma have been made, almost 80% of the patients die within the first 2 years after diagnosis. on the basis of these observations, the identification of new molecules that can counteract the gbm growth and invasiveness appears relevant. previously, we have demostrated that m2 muscarinic receptor agonist arecaidine inhibited cell proliferation in a time and dose-dependent manner and induced a severe apoptosis both in two glioma stable cell lines (u251 and u87) and in primary cultures derived from human biopsies. in order to clarify the mechanisms causing the decreased cell proliferation, we have evaluated the ability of m2 receptor activation to counteract the notch-1 and egfr pathways. the analysis by real time pcr and western blot have demonstrated that, in both cell lines, m2 receptor caused a decreased expression of egfr and notch-1 and its ligands (e.g. delta-1, jagged-1 and 22), suggesting that the decreased cell proliferation may dependent on altered expression and activity of these pathways. although facs analysis have showed that arecaidine induced an arrest of cell cycle progression, we observed, in both cell lines, a significant increase of dividing cells already after 24 hours of treatment. in particular, the number of metaphases increased significantly, while the percentage of anaphases diminished. furthermore we observed in both cell lines, a dis-regulation of mitotic spindle assembly, misalignment of chromosomes and the presence of multipolar spindles. moreover, due to prolonged activation of the promethaphase/methaphase mitotic checkpoint, chromosomes appeared more condensed in cells treated with arecaidine. facs analysis and immunocitochemistry using anti-histone c-h2ax, a marker of double strand breaks, have also demonstrated that arecaidine treatment induced dna damage. on the other hand, m2 agonist induced also oxidative stress, followed by an increased expression of the enzyme superoxide dismutase and sirtuins (sirt1 and sirt2). in conclusion, the data obtained suggest that the activation of m2 receptor induces cytostatic and cytotoxic effects in glioblastoma cell lines, opening new therapeutic perspectives for this receptor in glioblastoma therapy. univdersidade federal do rio de janeiro, rio de janeiro, brazil glioblastoma (gbm) is one of the most aggressive human cancers. despite current advances in multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome for patients with high grade glioma remains fatal. there is now a growing awareness that the main limitations in understanding and successfully treating gbm might be bypassed by the identification of a distinct cell type that has defining properties of somatic stem cells, as well as cancer-initiating capacitybrain tumor stem cells, which could represent a therapeutic target. in addition, experimental studies have demonstrated that the combination of antiangiogenic therapy, based on the disruption of tumor blood vessels, with conventional chemotherapy generates encouraging results. emerging reports have also shown that microglial cells can be used as therapeutic vectors to transport genes and/or substances to the tumor site, which opens up new perspectives for the development of gbm therapies targeting microglial cells. finally, recent studies have shown that natural toxins can be conjugated to drugs that bind to overexpressed receptors in cancer cells, generating targeted-toxins to selectively kill cancer cells. further, extensive effort is being dedicated to characterize the molecular basis of gbm resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. we show that a non-cytotoxic concentration of equinatoxin ii (eqtx-ii), a pore-forming toxin from the sea anemone actinia equina, potentiates the cytotoxicity induced by temozolomide (tmz), a first-line gbm treatment, and to etoposide (vp-16), a second-or third-line gbm treatment. we also suggest that this effect is selective to gbm cells and occurs via pi3k/akt pathway inhibition. finally, magnetic resonance imaging (mri) revealed that a non-cytotoxic concentration of eqtx-ii potentiates the vp-16-induced inhibition of gbm growth in vivo. these combination therapies constitute a new and potentially valuable tool for gbm treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir-302-367, which induces the exit of gsc from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, 2012). we postulated that such a drastic change in the cell phenotype should be accompanied with metabolic alterations that could be instrumental in the loss of functional properties of gscs induced by the micro-rna cluster. metabolome profiling by mass-spectrometry of gscs and gsc-mir-302-367 pointed to changes in the gaba synthesis pathway (see abstract el-habr et al). remarkably, exposure of na€ ıve-gsc to metabolites of the gaba synthesis pathway found to be overproduced in gsc-mir-302-367 reproduced at least in part the effects of mir-302-367, including inhibition of clonality, down-regulated expression of selfrenewal markers, and loss of self-renewal properties. the metabolites studied acted by interfering with progression of the cell cycle and the nuclear localization of transcription factors crucial for maintenance of gsc stem-like properties. studies assaying the effects of the metabolites on gsc tumor-initiating properties are under way. these results demonstrate that metabolic regulations can participate in the control of gsc properties, and opens novel paths in therapeutic targeting of glioblastoma. *lgd and eae contributed equally. glioblastomas are the most frequent and aggressive form of adult primary brain tumors. glioblastoma stem cells (gscs) are thought to play key roles in the development and resistance of the tumor to existing radio-and chemotherapies. our previous results showed that the cluster of micrornas, mirna-302-367, induces loss of gsc stem-like and tumor-inducing properties (fareh et al. 2012). to further understand the molecular pathways controlling the peculiar properties of gscs, we sought for metabolic changes likely to accompany the drastic change in cell phenotype induced by mir-302-367 expression. we measured the intracellular and secreted levels of 271 metabolites by mass spectrometry. reconstitution of the metabolomes revealed significant and coordinated changes in components of the krebs cycle, and the glutamine/glutamate neuropeptide metabolic pathway that suggested altered turnover of the gaba synthesis pathway. exon array hybridization and western blot analysis, revealed changes in expression of several enzymes of the gaba synthesis pathway in mir-302-367-gscs. of note, none of the analyzed enzyme transcripts appeared as a direct target of mir-302-367. our results could be integrated in a complex multistep schema compatible with enhanced turnover of the gaba synthesis pathways, resulting in decreased cell gaba levels and increased levels of gaba by-products, as observed in mir-302-367 gscs. further studies showed that these metabolic changes participate in the induced loss of gsc properties (see abstract by dubois et al). * eae and lgd contributed equally colonization of the brain by carcinoma cells is barely understood, in particular when considering interactions with the host tissue. we recently demonstrated that microglia assist carcinoma cell invasion. current findings indicate that this is part of a danger response of the entire brain tissue. in the brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia as well as astrocytes comparable to that seen at the interface of human cerebral carcinoma metastases. this response tries to protect the brain from intrusion of benign epithelial cells by inducing apoptosis. however, this is ineffective against malignant cells which did not undergo apoptosis and actually exploited this reaction to invade instead. gene expression and functional analyses revealed that c-x-c chemokine receptor type 4 (cxcr4) and wnt signaling are involved in this process. furthermore, cxcr4 regulates the recruitment of microglia towards brain injury in a zebrafish model and was expressed in human stroke patients, suggesting a conserved role of cxcr4 in various danger reactions. we propose that glia-assisted malignant invasion takes advantage of the physiological two-stage glial defense reaction. carcinoma cells escape the second step of cell killing and misuse the damage response to colonize the brain. university of tuebingen, tuebingen, germany aquaporin-4 (aqp4), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. in contrast, most cultivated glioma cell lines do not express aqp4, and primary cell cultures of human glioblastoma lose it during the first passages. accordingly, in two glioma cell lines of the rat (c6 cells and rg2 cells) and in sma mouse glioma cell lines we found no aqp4 expression. this let us consider the possibility that aqp4 expression depends on brain microenvironment. aqp4 negative rat glioma cells were implanted into rat brain. within two weeks, a tumor developed. aqp4 staining of the tumor cells was positive. however, if the identical cells were implanted into the rat's flank, they did not express aqp4. in contrast to the normal brain, where aqp4 staining is polarized in the astrocytic endfoot membranes, the aqp4 staining in c6 and rg2 tumors was distributed over the whole glioma cell as in human glioblastoma. we conclude that the micro-environment is crucial for aqp4expression in brain and brain-tumor. glioblastomas (gbm) are infiltrative and highly vascularised brain tumors containing a subpopulation of multipotent stem-like cells. here we questioned whether notch pathway activation in these cells influenced the extent of tumoral vascularisation and dissemination. we used two cd133 1 sox2 1 gbm stem-like cultures generating highly infiltrative but poorly vascularised tumors upon intracranial grafts. the use of a hes5 promoter reporter construct indicated that the notch pathway was only active in a small fraction of cells. sustained notch activation in these cultures, using an activated form of the notch-1 receptor (nicd), induced a profound alteration of their morphology, phenotype and properties. a severe decline of their migration and proliferation rates was observed in vitro and in vivo. intracranial and subcutaneous transplantation revealed that nicd triggered an extensive vascularisation of tumoral cells indicated by the presence of wellformed vessels with large lumens. close interactions between gbm cells and host endothelial cells were readily observed but no evidence for endothelial transdifferentiation was found either in vitro or in vivo. microarray and proteomic analyses showed that nicd induced the expression of vascular adhesion proteins (icam-1, vcam-1, itga9), proangiogenic cytokines (plgf, il-8, hb-egf), metalloproteinase (mmp-9), a-smooth muscle actin and dll4, a notch ligand essential for vascularisation. this was associated with a remarkable transcription factor switch whereby cells became klf9 1 snai2 1 while ascl1, olig2, and sox2 were reduced or lost. thus in addition to its well-described role in vascular development and remodelling, the notch pathway is also central in controlling proliferation, migration and vascularisation of gbm-stem like cells. t04-04b extracellular space diffusion parameters in the mouse thalamus in bral2 knock-out mice retinal wholemounts were immunostained with antibodies against gfap, iba-1 and mhc-ii. results: in the na€ ıve retinas, weak constitutive mhc-ii expression was scarcely found in some iba-11 microglial cells and rarely in gfap1 astrocytes. only a small dendritiform subpopulation of iba-11 cells, located in the juxtapapillary area and in the marginal region of the retina, had a strong mhc-ii immunoreaction. in comparison with na€ ıve both, in contralateral and in oht-eyes: i) gfap was upregulated in m€ uller cells and microglia was activated; ii) mhc-ii was upregulated on macroglia and microglia. in microglia, it was similarly expressed in contralateral and ohteyes. by contrast, in macroglia, mhc-ii upregulation was observed mainly in astrocytes in contralateral eyes and in m€ uller cells in oht-eyes conclusions: both, contralateral and oht-eyes had macro-and microglial retinal changes in mhc-ii expression after two weeks of laser-induced oht approximately 70% of the nmo patients harbor antibodies against the water-channel aquaporin-4, aqp4-igg (seropositive nmo), expressed mainly by perivascular astrocytes. however, some patients diagnosed with nmo don't have aqp4-igg (seronegative nmo). the aim of this work is to compare in vitro the neuroinflammatory profile of igg from seropositive (igg-nmo1) and seronegative (igg-nmo-) nmo patients. pool of purified igg from igg-nmo1 and igg-nmo-patients fulfilling diagnostic criteria of 2006, and from multiple sclerosis (igg-ms) patients and healthy controls (igg-hc) were include in the study. mixed glial cell cultures of astrocytes (40%), oligodendrocytes (40%) and microglia (15%) were obtained from spinal cord of postnatal c57bl6/j mice. after 21 days in vitro, cells were treated with the igg pools for 12 hours for rt-pcr, and 24 hours for western blot and immunocytofluorescence (if) igg-nmo1 caused a stronger upregulation of c/ebpb mrna and nos2 protein than igg-ms and igg-nmo-. all these data show that igg-nmo1 and igg-nmo-induce a different proinflammatory glial activation. the proinflammatory pattern of igg-nmo-is quite similar to that induced by igg-ms patients. these findings raise the question if igg-nmo-could be a different disease. such as gdnf, artemin, bdnf and sonic hedgehog, adhesion molecules including p75ntr, n-cadherin and n-cam and the transcription factor olig1. c-jun also controls the structure of the regeneration tracks. in schwann cell c-jun null mice, the initial rate of axonal regeneration after injury is impeded and long term recovery of function measured by footprint analysis, toe pinching , von frey hairs, and the hargreaves test is not seen 10 weeks after injury. there is widespread death of both large and small drg neurons and although spinal cord motor neurons survive there is widespread failure of both sensory and motor neurons to reinnervate target muscles united states mutations in the neurofibromatosis 2 (nf2) gene cause neurofibromatosis type 2 (nf2) characterized by formation of multiple schwannomas and meningiomas. nf2 encodes a tumor suppressor protein called merlin. loss of function of merlin is associated with an increased level of active rac and p21-activated kinases (pak). the lim domain kinases (limk1 and 2) are rac-pak substrates and modulate actin dynamics and cytoskeletal organization by phosphorylating cofilin, an actin severing and depolymerizing agent acknowledgements: this work was supported by grant ncn 2011/03/b/ nz4/00042 and statutory funds. phenotypes, their functions are fundamentally different in a model of brain injury. these data provide new insights into the protective role of microglia after brain injury. . although the pathogenesis is still not clear, activation of the immune system at some point of the disease process plays a crucial role. it is known that myelin-specific autoreactive t-cells and monocytes cross the blood-brain barrier (bbb) and migrate into the cns. within the cns, these cells secrete proinflammatory cytokines which stimulate local glial cells to produce inflammatory factors, including reactive oxygen species that contribute to demyelination and ultimately axonal loss. the destruction of the bbb and the influx of monocytes and t-cells from the circulation are key events in the progression of ms pathology. tissue transglutaminase (tg2) is a multifunctional enzyme that has been shown to play a role in monocyte/macrophage adhesion and migration onto extra cellular matrix proteins in vitro. this binding occurs via interaction with bintegrins. previously, we observed the appearance of tg2 in monocytes in active human post-mortem ms lesions in the cns.in the present study we question whether tg2 expression is regulated in primary human monocytes by inflammatory mediators and whether it is modulated in ms patients. methods: to this end, tg2 in monocytes was detected by semiquantitative rt-pcr.results and conclusions: our preliminary results show that activation of primary human monocytes with lps or lps1ifnc results in a time-dependent increase in tg2 mrna whereas the expression of factor xiiia (another member of the transglutaminase family) remains stable over time. moreover, the tg2 mrna level is higher in unstimulated monocytes derived from ms patients compared to control subjects while factor xiiia mrna level remains unaffected.these initial data are promising with respect to a possible role for monocyte-derived tg2 being involved in adhesion to and migration across the bbb under inflammatory conditions (e.g. multiple sclerosis). this hypothesis has not been proven yet. in addition, by increasing the number of patients, a consistent difference in tg2 expression in monocytes between ms patients and control subjects may point to tg2 as a novel biomarker for disease. an important aspect of chronic neurodegenerative diseases, such as alzheimer's, parkinson's, huntington's and prion disease, is the generation of an innate inflammatory response within the central nervous system (cns). microglial and astroglial cells play a key role in the development and maintenance of this inflammatory response, showing enhanced proliferation and morphological activation. using a laboratory model of chronic neurodegeneration (me7 murine model of prion disease), we studied the time-course and regulation of microglial proliferation. our results show that resident microglial cells have an increased proliferation rate during the development of the disease, leading to a significant increase in the population, without a contribution from circulating cells. microglial proliferation is differentially regulated in diverse regions of the cns, pointing to a heterogeneous development of the pathology. we have identified novel molecular regulators of the proliferative response, and addressed the significance of the contribution of microglial cells to the pathological course of the disease by modifying their proliferation. we also found a correlation of our results with the scenario present in chronic human neurodegenerative conditions variant creutzfeldt-jakob disease (vcjd) and alzheimer's disease. our results demonstrate that microglial proliferation is an important feature of the evolution of chronic neurodegenerative disease, with direct implications for understanding the contribution of the cns innate immune response to disease progression. here, we examined the underlying mechanism of fibronectin aggregation and address whether inflammatory mediators, such as toll like receptor (tlr) agonists or pro-inflammatory cytokines induce fibronectin aggregation by astrocytes. our findings reveal that only tlr3 and 24 agonists, including the endogenous tlr3 agonist stathmin, increased stable fibronectin aggregation. interestingly, only white matter-derived astrocytes displayed fibronectin aggregation whereas in cortical astrocyte cultures fibronectin remained unaffected, suggesting regional differences in the functional response of astrocytes. furthermore, in rat cerebellar slice cultures, the tlr3 agonists only induce aggregation of fibronectin after lysolecitin-induced demyelination. in this manner, efficient remyelination is impeded and, consequentially, progressive neuronal decline occurs.taken together, tlr3 and 24 agonists promote fibronectin aggregation by primary rat astrocytes. since tlr3 on astrocytes and stathmin are both upregulated in ms lesions, stathmin, as an endogenous tlr3 agonist, might play a role in inducing fibronectin aggregation after c-jun reprograms schwann cells of injured nerves to generate a repair cell essential for regeneration. neuron. 75(4): 633-47). in this study we identify a novel role for c-jun in the activation of notch signalling in the denervated schwann cell. we find that c-jun is required to activate notch signalling, leading to upregulation of the bhlh protein hes1. hes1 then plays two functions in the denervated cell, promoting myelin breakdown and acting as part of a negative feedback loop to reduce c-jun levels. as a result of this, ablating notch signalling specifically in schwann cells acts to increase c-jun levels. we show that this upregulation of schwann cell c-jun accelerates axon outgrowth, target re-innervation and remyelination by generating a cell, which promotes faster than normal functional recovery. these results identify novel functional links between the c-jun and notch signalling pathways. they also show that not only is schwann cell c-jun necessary for successful nerve regeneration, but that nerve repair can be improved by enhancing normal c-jun signalling. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). after injury of the peripheral nervous system (pns), schwann cells and macrophages phagocyte myelin debris in wallerian degeneration in order to regenerate axons to distal targets. the immunoreceptor cd300f is normally expressed in the myeloid line and in nervous system in microglia, oligodendrocytes and neurons under certain conditions. however, little is known about the cd300f ligands. by using a cd300f-fc fusion protein we have analyzed the expression of the cd300f ligands in the pns. moreover, we have also analyzed the possible role of the cd300f immunoreceptor in peripheral nerve regeneration by blocking the interaction between cd300f and its ligands with the same fusion protein. thy1-yfp-h mice sciatic nerves were injected with cd300f-fc, control migg2a or pbs immediately before a crush injury. the results show that cd300f ligand is expressed in schwann cells. moreover, after a sciatic nerve injury, animals injected with the cd300f-fc protein show a lower number of yfp-positive fibers growing into the tibial nerve after 10 days post-lesion (dpl) than control groups. moreover, the cd300f-fc group shows a higher number of macrophages and cd206-positive cells at 10 dpl when compared to control groups. we do not see these differences in axon regeneration and macrophage infiltration after 28 dpl. we have also evaluated the mrna expression of the pro-inflammatory cytokine il-1b at 24 hours after crush and injection of the fusion protein. q-pcr shows an up-regulation of the mrna in control groups and a lower mrna expression level in the cd300f-fc protein group. together these results show that the pair cd300f receptor and ligand is implicated in some aspects of wallerian degeneration and nerve regeneration such as the modulation of both the influx and phenotype of macrophages. in response to the central nervous system (cns) injury, hematopoetic cells migrate to the lesion where they can potentially contribute to tissue regeneration. following cns injury, these cells can secrete a plethora of immunomodulating cytokines and/or pro-regenerative growth factors as well as phagocyte proinflammatory tissue debris. nevertheless, the role of primitive hematopoetic stem/progenitor cells (hspc) -derived from bone marrow -to support cns regeneration has not been studied in detail. therefore, the aim of the present study was to examine characteristics of hspc in vitro as well as to test proregenerative potential of these cells to support cns repair in vivo.highly pure population of primitive hematopoetic stem/progenitor cells was isolated from the bone marrow and the cns with fluorescence activated cell sorting (facs). the number of hspc in the cns lesion was significantly increased compared to intact cns tissue. sorted-bone marrow hspc were co-cultured with primary astrocytes to examine their phenotype according to the presence of specific antigens and gene expression as well as to test their phagocytosis potential. in the presence of astrocytes, bone marrow-derived hspc differentiated in vitro into microglia-like cells expressing specific myeloid/microglia markers and showing high ability to ingest fluorescent microbeads. the in vivo potential of hpsc for supporting regeneration was examined by their transplantation into the cns lesion.results of our study demonstrated that primitive hematopoetic stem/progenitor cells are the source of microglia-like cells which can support regeneration in the central nervous system. when the brain or spinal cord is injured, glial cells in the damaged area undergo complex changes resulting in the formation of the glial scar. whether the scar is beneficial and detrimental to recovery remains controversial. the signals that initiate the formation of the glial scar are unknown. because both canonical and non-canonical wnts are increased after spinal cord injury (sci), we examined the role of canonical wnt signaling in the glial reactions to cns injury. to disrupt b-catenin-dependent wnt signaling specifically in opcs, we created transgenic mice carrying an opc-specific conditionally deleted bcatenin gene. after moderate contusion injuries to the thoracic spinal cord of control mice, opcs proliferate and accumulate in the penumbra region surrounding the injury epicenter. in the b-catenin-depleted mice, there is reduced proliferation and accumulation of opcs after sci, reduced accumulation of activated microglia/macrophages and reduced astrocyte hypertrophy. using a crushed optic nerve model, we show that these reduced glial reactions create an environment that is permissive for axonal regeneration. these results suggest that canonical wnt signaling in glia after cns injury is necessary for the formation of the glial scar and they identify wnt signaling as a new therapeutic target for promoting axon regeneration. the crucial role of central nervous system (cns) glial cells in the integrity and physiology of neuronal networks, is well known. however, little is known about glial cells in the peripheral nervous system (pns). one of the main glial cell types in the pns is the perineuronal satellite glial cells (sgcs), that surround drg neurons in an envelope-like fashion. despite their abundance in peripheral nerves, having stem cells properties and playing a role in neuropathic pain, there is limited information on their physiology. although sgcs have similarities to astrocytes in terms of purinergic-and gap junction-mediated signaling, their membrane properties and ionotrophic glutamate receptor expression are more or less unknown. our aim was therefore to characterize the membrane properties and glutamatergic ion channel expression of sgcs. we performed patchclamp experiments on sgcs wrapped around sensory neurons in the rat dorsal root ganglion (drg) in vitro. whole-cell recordings revealed that sgcs are slightly hyperpolarized in comparison to the neurons they ensheath, with a resting membrane potential of approximately $80mv and a high input resistance (&gt;1gx). moreover, upon membrane depolarization no detectable voltage-gated na 1 , ca 21 or k 1 currents were detected. extracellular application of glutamate agonist, kainic acid, n-methyl-d-aspartic acid (nmda) and 2-amino-3-(3hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (ampa) during the recording, did not evoke any response from the cells, indicating their lack of ionotropic glutamatergic receptor (iglur) expression. double immunocytochemistry on lucifer yellow (ly) filled scgs revealed that they express the scip/oct6 transcription factor, which is also expressed by promyelinating schwann cells.drg ganglion sgcs differ from cns perineuronal cells as they lack glutamatergic receptors, but are similar to astrocytes as they have no voltage-gated ion channels, are gap-junctionally coupled and express glutamine synthetase. thus, we are currently addressing if sgcs respond to neuronal activity in a similar manner to astrocytes in the cns with the use of calcium imaging. these studies will provide further insights into the physiological role of sgcs in the pns. multiple sclerosis (ms), a chronic neuroinflammatory disease with presumed autoimmune etiology, is the most common demyelinating disorder of the central nervous system among young adults. progressive neurological impairment of the patients are associated with accumulation of characteristic brain lesions characterized by inflammation, demyelination, gliosis and axonal damage. demyelination and loss of oligodendrocytes contributes to axonal loss and permanent neurological deficits. remyelination occurs but is limited; one contributing factor is the incapability of oligodendrocyte precursor cells (opc) to differentiate and form new myelin sheaths. k 1 channels are not only known to predominantly set and maintain the membrane potential but also to be important regulators of various cell functions like proliferation and maturation. specific regulation of these cellular functions is based on high channel diversity and their ability to form heteromers as well as alternate mrna splicing and posttranslational modifications. so far the expression and function of potassium channels in cells of the oligodendroglial lineage has not been clearly identified. previous studies indicate that an unspecific blockade of k 1 channels in opc suppresses maturation as well as proliferation. moreover, for one distinct channel (k ir 4.1) a functional relevance has been shown, as deficiency of kir4.1 leads to altered opc maturation and hypomyelination in mice. here, we elucidate the expression and functional relevance of k 1 channels in oligodendrocytes. our first electrophysiological recordings from primary murine opcs revealed the presence of different k 1 channels with both inward and outward rectification. in future experiments, we aim at identifying k 1 channel subtypes that specifically regulate opc cell functions and might influence cell maturation under neuroinflammatory conditions. thereby, we want to offer new insights in the functional role of k 1 channels in opcs/oligodendrocytes and contribute to novel therapeutic strategies for the treatment of ms. aqp4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. aqp4 has been described as an important entry and exit site for water during formation of brain edema and regulation of aqp4 is therefore of broad interest. phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. protein kinase (pk)-dependent phosphorylation of ser 111 has been reported to increase the water permeability of aqp4 expressed in an astrocytic cell line. this possibility was, however, questioned based on the crystal structure of the human aqp4. our study aimed to resolve if ser 111 was indeed a site involved in phosphorylation-mediated gating of aqp4. the water permeability of aqp4-expressing xenopus oocytes was not altered by a range of activators and inhibitors of pkg and pka. mutation of ser 111 to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of aqp4. pkg activation had no effect on the water permeability of aqp4 in primary cultures of rat astrocytes. molecular dynamics simulations of a phosphorylation of aqp4.ser 111 recorded no phosphorylation-induced change in water permeability. a phospho-specific antibody, exclusively recognizing aqp4 when phosphorylated on ser 111 , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. thus, our data indicate a lack of phosphorylation of ser 111 and of phosphorylation-dependent gating of aqp4. undocked connexons may form open hemichannels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules with masses lower than 1 kda. a range of physiologically relevant molecules, smaller than the assumed molecular cut-off of 1 kda, have therefore been proposed to permeate connexin 43 (cx43) in its hemichannel configuration. however, the permeability profile of cx43 hemichannels remains unresolved as does the molecular substrate for hemichannel activity in astrocytes. exposure of cx43-expressing xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. in spite thereof, a range of smaller biological molecules, such as water, glutamate, lactate, glucose, and atp, did not gain detectable access through the pore. in contrast, permeability of glutamate, glucose and atp was observed in oocytes expressing the other major astrocytic connexin, cx30. exposure to low divalent cation solutions also induced a robust membrane conductance in cx30-expressing oocytes but none in cx43-expressing oocytes. expression of cx43 in c6 glioma cells failed to induce hemichannel activity. our results thus call for caution when assigning molecular identity to astrocytic channel activity and when interpreting hemichannel-mediated dye-uptake as equal to permeability of physiologically relevant molecules. atp-gated p2x4 receptor channels expressed in spinal microglia actively participate in central sensitization, making their functional regulation a key process in chronic pain pathologies. p2y6 metabotropic g q -coupled receptors, also expressed in microglia, are involved in the initial response to nerve injury, triggering phagocytosis upon activation by udp. it has been reported recently that expression of both p2x4 and p2y6 is upregulated in activated microglia following nerve injury. we show here, in resting as well as lps-activated primary microglia, that p2y6 decreases p2x4-mediated calcium entry and inhibits the dilation of p2x4 channels into a large-conductance pore measured with a yo-pro-1 uptake assay. furthermore, p2y6 activation modulates the atp-dependent migration of microglia, a process likely involved in their shift from migratory to phagocytic phenotype.reconstituting the p2x4-p2y6 interaction in recombinant systems shows that p2y6 activation decreases p2x4 current amplitude, activation and desensitization rates, and reduces p2x4 channel permeability to the large cation nmdg 1 . phospholipase c-mediated hydrolysis of the phosphoinositide pi(4,5)p 2 , a necessary cofactor for p2x4 channel function, underlies this inhibitory crosstalk. as extracellular levels of both atp and udp are increased in the spinal cord following nerve injury, the control of p2x4 activity by p2y6 might play a critical role in regulating neuropathic pain-inducing microglial responses. with sc, thus representing a valid sc alternative. we have previously shown that dasc express c-aminobutyric-acid (gaba) receptors (i.e. gaba-a and gaba-b receptors), which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitter systems in asc.in this study we investigated the expression of purinergic receptors in dasc. using rt-pcr, western blot analyses and immunohistochemistry we have demonstrated that asc express p2x 3 , p2x 4 and p2x 7 purinoceptors. interestingly, differentiation of asc towards glial phenotype was accompanied by up-regulation of p2x 4 and p2x 7 receptors. using ca 21imaging techniques, we have shown that stimulation of purinoceptors with adenosine-5 0 -triphosphate (atp) results in intracellular ca 21 signals, indication functional activity of these receptors.. moreover, we have shown that the increase of intracellular ca 21 leads to sc death, an effect that can be prevented using specific p2x 4 or p2x 7 antagonists.altogether, these results show, for the first time, the presence of functional purinergic receptors in sc-like derived from asc and their link with critical physiological processes such as cellular death and survival. the presence of these novel pharmacological targets in dasc might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dasc for peripheral nerve repair. university of freiburg, freiburg, germany intracellular ph homeostasis is a vital function shared by all cells and is mainly regulated by the coordinated action of several acid-base transporters. in the brain, ph homeostasis is of particular importance since changes of extracellular ph (pho) are events associated with both physiological conditions and pathological states in brain function. transient pho changes usually accompany physiological processes, including neuronal activity, and astrocytes respond to a rise in extracellular k 1 with plasma membrane depolarization and intracellular alkalinization. on the other hand, loss of brain ph homeostasis can lead to severe pathological conditions and significant changes of pho have been observed during seizures and spreading depression.in the present study, we sought to investigate regulation mechanisms of the electrogenic sodium/bicarbonate cotranspoter 1, nbce1, and of the vacuolar h 1 -atpase (v-atpase) in primary hippocampal astrocytes following extracellular acid-base changes, following neuronal activity, and using the 4-aminopyridine (4-ap) model of epilepsy in vitro.we show that extracellular acidosis, but not extracellular alkalosis, increased the number of activated astrocytes. our data also show differential regulation of acid-base transporters in the hippocampal glial cells during extracellular acid-base changes. intracellular ph measurements revealed a crucial role for v-atpase in regulation of intracellular ph, a process accompanied by increased membrane expression of the transporter, as assessed by immunofluorescence and surface biotinylation. nbce1-b is the nh2-terminal variant of nbce1 predominantly expressed in glial cells and is transcriptionally regulated followed neuronal activity or treatment of the cells with 4-ap.these data propose transcriptional and post-translational modification of acid-base transporters as putative regulatory mechanisms in astrocytes to cope with changes of extracellular ph serving the maintenance of intracellular ph homeostasis. these astroglial networks fulfil a variety of functions in the brain, e.g. potassium buffering and metabolite transport. in this study we compared glial networks in different brain regions to investigate site specific effects on network formation. we combined electrophysiology and immunohistochemstry with semi-quantitative rt-pcr and western blot analysis to investigate connexin expression, gap junction coupling and antigen profiles. experiments were performed in wild type and transgenic mice with glia specific fluorescence labelling as well as in cx30ko mice. astrocytes were investigated between postnatal days 12-60. gap junction networks in the ca1 region of the hippocampus and the ventrobasal thalamus show abundant coupling in hgfap-egfp mice. intriguingly, we found significant coupling between oligodendrocytes and astrocytes in the thalamus, while in the hippocampus panglial coupling was less abundant. other glial cells did not participate in the networks. we also found that a fluorescent glucose analog, 2-nbdg, propagates through the thalamic panglial network. the function of these panglial networks remains largely unclear.in heterozygous cx43-ecfpki mice, deletion of one allele of the major hippocampal cx43 significantly reduced the number of coupled astrocytes only in the hippocampus, while the thalamic networks remained unchanged. sr101 labelling of astrocytes and subsequent 2p microscopy identified a significant subset of thalamic sr1011 cells lacking cx43-ecfp expression. sr101 did not label oligodendrocytes as analysed in plp-gfp mice. semi-quantitative rt-pcr and western blot analysis revealed stronger expression of cx30 in thalamic nuclei while cx43 levels were higher in the hippocampus. this indicates a minor role for cx43 in gap junction coupling of astrocytes in the thalamus. consistent with these findings, the thalamus of cx30ko mice displayed a strong decrease in astrocytic cell coupling compared to wild type littermates.together, these results indicate that thalamic astrocytes differ in various aspects from their counterpart in other brain regions and support the emerging concept of astrocyte heterogeneity. the mechanism of secondary damage spread after brain trauma remains unresolved. in this work, we redirected the attention to astrocytic communication pathways. using an in vitro trauma model that consists of a scratch injury applied to a rat astrocyte monolayer, we found a significant induction of connexin43 hemichannel activity, demonstrated by ethidium uptake, in regions distal from the injury ($17 mm away and maximal $1 h after injury). this response was abolished by two connexin hemichannel blockers, la 31 and the peptide gap26. in addition, the trauma-induced increase in hemichannel activity was prevented by inhibition of purinergic p2 receptors. the scratch-induced increase in hemichannel activity was absent in astrocytes from cx43 knockout mice. this activity took place with a singular spatial distribution, since cells located at $17 mm away from the scratch gained connexin hemichannel activity. however, the functional state of gap junction channels (dye coupling) was not significantly affected in the same locations. the connexin43 hemichannel activity was also enhanced by the acute extracellular application of 60 mm k 1 . the increase in hemichannel activity was correlated with an increment in apoptotic cells, measured by immunofluorescence to annexin v at 24 h post-trauma, which was totally prevented by gap26 peptide. these findings could open a new approach to prevent or reduce the secondary cell damage due to brain trauma. l. schlosser, a. scheller, f. kirchhoff institute of physiology, saarland university, homburg, germany current research suggests that astrocytes represent heterogeneous neuroglial populations in different regions of the brain. this is particularly evident when the expression pattern of various transmitter receptors is analyzed. hippocampal astrocytes, for example, do not express ampa receptors, while cerebellar bergmann glia is loaded. functional analysis of glial receptors requires dedicated efforts for each distinct brain region and developmental age. unfortunately, the molecular identification using antibody approaches is often hampered by either poor antibody quality or abundant receptor expression on adjacent neuronal membranes. therefore, we are using cre/lox-mediated gene ablation for proper functional analysis.here, we are focusing on the characterization of astrocyte-specific gene deletion of the gaba b1 receptor. metabotropic gaba b receptors consist of two subunits gaba b1 and gaba b2 forming a heteromeric receptor complex of which gaba b1 is essential. in contrast to neurons, activation of astroglial gaba b receptors leads to transient rises of intracellular ca 21 . the functional impact of these glial gaba b -receptors is largely unknown.to address the gaba b receptor function in astrocytes we took advantage of the cre/lox system and crossbred glast-creert2 knockin mice with floxed gaba b1 receptor mice. first immunohistochemical analysis reveals a selective deletion of gaba b1 on a majority of astroglial processes. functional studies addressing the role of these receptors in astroglial ca 21 signaling are in progress. purinergic signaling is the most diverse system in astrocytes to communicate with other glial cells and neurons. astrocytes express a variety of p2x (ionotropic) and p2y (metabotropic) receptors. especially in case of brain injury, atp levels and receptor expression on astrocytes are increased. atp is immediately degraded to adp, amp and adenosine. these nucleotides/nucleosides act back on the preferred purinoreceptor expressed on glial and neuronal cells. in the last decade researchers tried to evaluate the functional impact of astrocytic purinoreceptors on the complex information flux at the tripartite synapse. a prominent role has been suggested for the p2y1 receptor subtype. we generated conditional mouse mutants to investigate the function of p2y1 receptors in astrocytes in vivo and crossbred mice carrying the floxed p2y1 gene with mice that express the inducible dna recombinase (creert2) under the control of the glast (l-glutamate/ l-aspartate transporter) locus. ablation of the p2y1 receptor is induced in development and adulthood by intraperitoneal tamoxifen injections. successful recombination of the targeted p2y1 receptor is evaluated by qrt-pcr on genomic dna and mrna usually 21 days after tamoxifen application. at the genomic level we find $25% and $50% recombination in astrocytes of the cerebellum and hippocampus, respectively. similar recombination frequencies are observed in young (p11) or adult mice (6-10 weeks). when looking at the transcript level, the mrna is reduced to 57% in the cerebellum of adult mice and to 76% in the hippocampus. in young mice we determined a reduction to 40% and 42% mrna expression in the cerebellum and hippocampus, respectively. given the high level of p2y1 expression on other cells of the brain these findings indicate a successful astrocyte-specific knockout. further fluorescence-activated cell sorting (facs) of recombined cells expressing the red fluorescent protein td-tomato will be used to verify the knockout. the potential function of these astroglial receptors in neuronal networks will be as well investigated using histological (em and light microscopy), twophoton ca 21 imaging in situ and in vivo and behavioral approaches. we recently showed that they invade the cortex at 11.5 days of embryonic age (e11.5). they first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. the absence of the expression of mac-2 and mhc ii suggest these cells have a na€ ıve/quiscent phenotype during embryonic development of the cortex. besides immunohistochemical markers is the presence of different k 1 channels on microglia also an indication of their activation stage. however most studies have been conducted on postnatal and adult microglial cells. therefore we aimed at determining the electrophysiological activation phenotype of these embryonic microglia.at the age of e13.5 and e15.5, microglial cells display a small inward rectifying k 1 current and this independent of their location in the embryonic cerebral cortex and their cell morphology. these cells also express functional p2x receptors, which based on the profile of the response are most probably p2x7 receptors. time-lapse analysis showed that embryonic microglia are highly dynamic cells. suggesting that these cells are already very active during fetal development. rapid extracellular removal of glutamate, the major excitatory neurotransmitter in the cns, is essential for normal brain function. this task is primarily accomplished by the action of the sodium-dependent, high-affinity transporters glast and glt-1 (rodent analogous of eaat1 and eaat2), which are mainly expressed by astrocytes. impairment or failure of glast and glt-1 plays an important role in many pathological conditions. question: pelizaeus-merzbacher-like disease (pmld) is a hypomyelinating leukodystrophy caused by mutations in the gjc2 gene encoding the gap junction (gj) protein connexin47 (cx47). cx47 is expressed in oligodendrocytes and forms most gj channels to astrocytes and to other oligodendrocytes. since pmld-associated gjc2 mutations cause loss of cx47 function, gene replacement strategies may be promising for developing future treatments. a mouse model for plmd, the cx32/ cx47 double knockout (ko), is characterized by early onset of severe leukodystrophy at 4 weeks of age leading to death by 6 weeks, offering the possibility to test therapies. the aim of the present study was to generate a lentiviral vector to allow gene delivery specifically to oligodendrocytes, and to examine the transduction efficacy, distribution, duration and levels of egfp reporter gene and cx47 expression throughout the cns, in order to establish a method for oligodendrocyte-targeted gene therapy.methods: the expression cassette with the cx47 gene along with the ires-egfp as a reporter gene under the control of oligodendrocyte-specific cnp promoter was cloned into the lentiviral vector pcclsin.ppt.hpgk.gfp.pre. mock vectors lacking the cx47 gene were also generated as controls. viral particles were produced to high titers and purified, before injection. lentiviral vectors were delivered into the brain of wild type (wt) and cx47ko mice at postnatal day 1 (p1), intraventricularly and in the stratum radiatum of the dorsal hippocampus. egfp and cx47 expression was assessed using immunochemistry and immunoblot analysis at different time points post injection.results: we found widespread expression of virally delivered egfp in different brain regions colocalizing with oligodendrocyte markers (cc1 and olig2). the expression of egfp was detected from p15 until 3 months post-injection. on average 20.3 6 2.56% of oligodendrocytes were egfp positive, with highest rates in the subventricular zone (29.3 6 6.31%, n 5 6) and olfactory bulb (19.9 6 4.36%, n 5 8) and lower rates in the cortex (16.1 6 2.60%, n 5 6) and corpus callosum (12.3 6 3.40%, n 5 3). in cx47ko brain expression of virally delivered cx47 was also found after p21 with formation of gj plaques in a subpopulation of oligodendrocytes.conclusions: our results show that neonatal lentiviral gene delivery may result in stable and widespread cns expression targeted to oligodendrocytes by using cell specific promoters. thus, gene therapy approaches using lentiviral vectors may be feasible for future treatment of leukodystrophy and should be further studied. recent reports identified mrna coding for different cav subtypes like 1.2 and 1.3 in hippocampal ng2 glia. using acute brain slices prepared from developing and mature ng2-eyfp mice, we also found that all ng2 glia upon depolarization in different brain regions elicited a. grimaldi, g. d'alessandro, c. lauro, m. catalano, c. limatola sapienza university of rome, rome, italy glioblastoma (gbm) is one of the most aggressive tumor of the central nervous system, being characterized by a great invasiveness and a very low survival rate of patients. this work wants to better define the role of tumor microenvironment in the modulation of tumor invasiveness. it is known that tumor migration and invasiveness can be modulated by the chemokine cxcl12 and its receptor cxcr4. the expression of both these proteins has been demonstrated in various human gbm cell lines and it has been shown that, blocking this pathway, tumor cells greatly reduce their invasive ability. we have recently demonstrated that an important molecule directly implicated in tumor migration is the intermediate conductance calcium-activated potassium channel (kca3.1). given the expression of these channels also in other cell types infiltrating the tumor mass, like microglia, we investigated the effect of kca3.1 inhibition on microglia-glioblastoma interaction. preliminary data indicate that kca3.1 activity on microglia is involved in the movement of these cells toward the tumor mass. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in human glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir-302-367, which induces the exit of gscs from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, 2012) . to elucidate the gene networks that govern gsc exit from their stem state, we used chip-seq (chromatin immunoprecipitation followed by next generation dna sequencing), to identify gene loci showing enrichment of the active (h3k4me3) and repressive (h3k27me3) epigenetic marks in gsc-mir-302-367 as compared to na€ ıve gscs. functional significance of the chip-seq results was evaluated with confrontation of the chip data with the transcriptomes of the corresponding cells derived from exon array hybridization. western blot analysis showed that global h3k4me3 and h3k27me3 expression levels were similar in gsc and gsc-mir-302-367. chip-seq analysis revealed similar gross number of gene loci associated with h3k4me3 or h3k27me3 (13000 and 5000 genes, respectively) in either cell type. interestingly, 16% (2381 genes) of the analyzed genes exhibited a change in either h3k4me3 or h3k27me3, or both in mir-302-367-gscs as compared to na€ ıve gscs. these changes resulted into a transcript variation in 14% of the cases (332/2381) considering as significant an increase or a decrease of at least 2-fold and the 77% of these genes translated into congruent alterations in the corresponding transcript levels.analysis of the functional significance of the changes observed using functional annotation databases identified novel gene networks, likely to participate in the regulation of gsc properties. studies under way focus on members of these networks specifically activated in mir-302-367 gscs that might alter gsc dialog with their microenvironment. malignant gliomas are the most frequent primary tumors of the brain with poor clinical prognosis. infiltrating peripheral macrophages and resident microglia contribute significantly to the tumor mass. we have previously shown that microglia as the intrinsic immune competent brain cells promote glioma expansion by up-regulating metalloprotease mt1-mmp through toll-like receptor (tlr) and its adaptor protein myd88. in this study we identified tlr2, as the main tlr controlling mt1-mmp expression and pro-tumorigenic signaling in microglia. glioma-derived soluble factors and synthetic tlr specific ligands induced mt1-mmp expression in microglia from wt mice but not tlr2-/-mice. by using the organotypic brain slice model, we found that tumor expansion depended on both parenchymal tlr2 expression and the presence of microglia. tlr2 is also highly expressed in human gliomas and inversely correlates with patient survival. in search for an endogenous tlr2 ligand released from glioma cells, we screened glioma conditioned medium (gcm) by mass spectrometry for endogenous tlr2 agonists produced by glioma cells and found versican, an extracellular matrix proteoglycan and a reported ligand of tlr2. to examine if versican is the factor mediating the glioma-microglia interaction, we silenced versican expression in gl261 cells. primary microglia were then stimulated with gcm from versican sirna and non-target sirna transfected gl261 cells and microglial mt1-mmp expression was analyzed by real-time pcr. an almost 3-fold up-regulation in microglial mt1-mmp expression was observed using gcm from non-target transfectants while the expression was reduced with gcm from versican knock-down gl261 transfectants. our results show that tlr2 activation is an essential part of the signaling cascade employed by glioma cells to convert microglia into a pro-tumorigenic phenotype and tlr2 thus may be a novel target for glioma therapies. two ipsdms were used to determine their responses in the presence of glioblastoma cells. using lc-ms/ms method, proteomic profiles were generated and compared between ipsdms exposed to human glioblastoma cells and ipsdms exposed to normal human astrocytes as control.results: immunolabeling of the two ipsdm cell lines showed specific expression of microglial markers such as iba-1, cd11b, and cd68. the comparative proteomic analyses indicated that microglia exposed to glioblastoma cells showed differential protein expressions relevant for cytoskeletal activity, energy production, and cell survival compared to control.conclusion: this is the first study showing the proteomes of human induced pluripotent stem cell-derived microglia exposed to glioblastoma cells, and the findings could provide further insight of the interaction between microglia and glioblastoma. key: cord-004534-jqm1hxps authors: nan title: abstract date: 2009-06-09 journal: eur biophys j doi: 10.1007/s00249-009-0478-1 sha: doc_id: 4534 cord_uid: jqm1hxps nan standard proteomics techniques are unable to describe the stoichiometry, subunit interactions and organisation of assemblies since many are heterogeneous, present at low cellular abundance and frequently difficult to isolate. we have combined two existing methodologies to tackle these challenges: tandem affinity purification (tap) and nanoflow esi-ms. we use methods designed to maintain non-covalent complexes within the mass spectrometer to provide definitive evidence of interacting subunits based on the masses of complexes and subcomplexes generated by perturbation both in solution and gas phases. structural models will be presented for three oligomeric protein complexes of unknown structure: the yeast exosome and the human u1snrnp and eif3 complexes. these models will then be examined within the context of their function. recent developments in mass spectrometry have added a further dimension to our studies of protein complexes: that of their collision cross-section. using ion mobility mass spectrometry we have been able to add spatial restraints to our models validating our models with measurements of collision cross-sections. very recently we have had a considerable breakthrough which has enabled us to preserve intact membrane complexes in the gas phase . this enables us to establish lipid and nucleotide binding and to define the stoichiometry and post translational modifications within the intact transmembrane regions of a number of complexes. in vivo molecular sensing: fluorescence beyond labeling f. beltram scuola normale superiore, i-56126 pisa, italy fluorescent molecules are powerful reporter tools that have much extended the impact of optical microscopy, particularly thanks to the flexibility of genetically encoded tags. detection can now target single molecules even in the complex environment of intact live cells offering unprecedented insight on biological processes in real time in live cells and tissues. fluorescent labels, however, can do more that this. our increased ability to tailor molecule and, more in general, nanosystem properties allows us to design, produce and exploit intelligent tags that can actually analyze the cellular environment. today multifunctional nanosystems can be produced that provide a signal dependent on the value of a specific biochemical parameter. importantly these nanosystems can target specific subcellular domains and have the ability to be used also in the case of live organisms. recent results will be discussed that highlight the impact of nanobiotechnology in this context with a particular emphasis given to methods suitable for in vivo studies that can be transferred to the biomedical world. far-field optical nanoscopy s. w. hell max planck institute, göttingen, germany the resolution of a far-field optical microscope is usually limited to d = λ/ (2 n sin α) > 200 nm, with n sin α denoting the numerical aperture of the lens and λ the wavelength of light. we will discuss lens-based fluorescence microscopy concepts that feature a resolving power on the nanoscale. all these concepts share a common basis: exploiting selected (pairs of) states and transitions of the fluorescent marker to neutralize the limiting role of diffraction. specifically, the fluorophore is switched on and off, that is, between a bright and a dark state, to detect the emission of adjacent features sequentially in time. the first viable concept of this kind was stimulated emission depletion (sted) microscopy in which the fluorescence ability of the dye is switched off by stimulated emission. in the sted microscope, the extent of the region in which the molecule is able to fluoresce follows d ≈ λ/ 2 n sin α 1 + i/i s , meaning that fluorophores that are further away than d can be separated. i is the intensity that drives a fluorophore from the bright fluorescent state to the dark ground state by stimulated emission. i s depends (inversely) on the lifetime of the states. for i/i s → ∞, it follows that d → 0, meaning that the resolution can be molecular). altogether, far-field optical 'nanoscopy' is a fascinating development in optics with high relevance to the many areas of sciences, in particular the life sciences. since it has already been a key to answering important questions in biology, and owing to its simplicity and commercial availability, we expect far-field fluorescence 'nanoscopes' to enter most cell biology and many nanoscience laboratories in the near future. grabbing the cat by the tail: discrete steps by a dna packaging motor and the inter-subunit coordination in a ring-atpase c. bustamante, j. moffitt university of california, berkeley, california, u.s.a. as part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid. bacteriophage ϕ29 packages its 6.6 mm long double-stranded dna into a 42 nm dia. x 54 nm high capsid using a multimeric ring motor that belongs to the asce (additional strand, conserved e) superfamily of atpases. a number of fundamental questions remain as to the coordination of the various subunits in these multimeric rings. the portal motor in bacteriophage phi29 is ideal to investigate these questions and is a remarkable machine that must overcome entropic, electrostatic, and dna bending energies to package its genome to near-crystalline density inside the capsid. using optical tweezers, we find that this motor can work against loads of up to ∼55 piconewtons on average, making it one of the strongest molecular motors ever reported. we establish the force-velocity relationship of the motor. interestingly, the packaging rate decreases as the prohead fills, indicating that an internal pressure builds up due to dna compression attaining the value of ∼6 megapascals at the end of the packaging. we show that the chemical energy of atp is converted into mechanical work during phosphate release. using ultra-high resolution optical tweezers, we determined the step size of the motor and established the coordination of the polymerases around the ring. we propose a comprehensive model of the operation of this motor. watching proteins function in real time via timeresolved x-ray diffraction and solution scattering p. a. anfinrud laboratory of chemical physics/niddk, nih, bethesda, maryland, u.s.a. to generate a deeper understanding into the relations between protein structure, dynamics, and function, we have developed x-ray methods capable of probing changes in protein structure on time scales as short as 150 ps. this infrastructure was first developed on the id09b time-resolved x-ray beamline at the european synchrotron and radiation facility, and more recently at the id14b biocars beamline at the advanced photon source. in studies of ligand-binding heme proteins, a picosecond laser pulse first photolyzes co from the heme, then a suitably delayed picosecond x-ray pulse passes through the protein and the scattered x-rays are imaged on a 2d detector. when the sample is a protein crystal, this "pump-probe" approach recovers time-resolved diffraction "snapshots" whose corresponding electron density maps can be stitched together into movies that unveil the correlated protein motions that accompany and/or mediate ligand migration within the hydrophobic interior of the protein. when the sample is a protein solution, we recover timeresolved small-and wide-angle x-ray scattering patterns that are sensitive to changes in the size, shape, and structure of the protein. scattering studies of proteins in solution unveil structural dynamics without the constraints imposed by crystal contacts; thus, these scattering "fingerprints" complement results obtained from diffraction studies. this research was supported in part by the intramural research program of the nih, niddk dc-sign is a trans-membrane protein expressed on antigen presenting cells and recognizes pathogens like hiv-1, hepatitis c virus and ebola. by electron microscopy and near-field optical nanoscopy, we demonstrated that at the plasma membrane dc-sign is organized in well-defined nanodomains of 100-180 nm in diameter. intensity-size correlation analysis revealed remarkable heterogeneity in the nanodomains molecular packing density. we constructed and characterized several dc-sign mutated forms lacking specific molecular domains. by immunogold labeling and spatial point pattern analysis, we show that the extracellular neck domain is essential for dc-sign nanoclustering. finally, we present a model that describes the probability of a cell to have a certain number of receptors joining the contact site in the initial encounter with an external object. monte carlo simulations subsequently define the parameters that are determinant in the object-cell encounter. our results show that receptor nanoclustering is of particular importance for binding objects of sizes comparable to the nanocluster size, indicating that the nanoscale spatial organization of dc-sign is optimized for binding to virus-sized objects. imaging of mobile stable lipid rafts in the live cell plasma membrane m. brameshuber 1 , j. weghuber 1 , v. ruprecht 1 , h. stockinger 2 , g. j. schuetz 1 1 johannes kepler university linz, austria, 2 medical university of vienna, austria the organization of the cellular plasma membrane at a nanoscopic length scale is believed to affect the association of distinct sets of membrane proteins for the regulation of multiple signaling pathways. based on in vitro results, conflicting models have been proposed which postulate the existence of stable or highly dynamic platforms of membrane lipids and proteins. here we directly imaged and further characterized lipid rafts in the plasma membrane of living cho cells by single molecule tirf microscopy. using a novel recording scheme for "thinning out clusters while conserving stoichiometry of labeling" 1 , molecular homo-association of gpi-anchored mgfp was detected at 37 • c and ascribed to specific enrichment in lipid platforms. the mobile mgfp-gpi homo-associates were found to be stable on a seconds timescale and dissolved after cholesterol depletion. having confirmed the association of mgfp-gpi to stable membrane rafts, we attempted to use an externally applied marker to test this hypothesis. we used bodipy-gm1, a probe that was recently reported to be enriched in the liquid-ordered phase of plasma membrane vesicles. when applied to cho cells at different surface staining, we found that also bodipy-gm1 homo-associated in a cholesterol-dependent manner, thus providing further evidence for the existence of membrane rafts. [1] appl phys lett 87, 263903 (2005) . synapsin knock-out mice as an in vitro model of human epilepsy studied with multi-electrode arrays d. f. boido, p. farisello, p. baldelli, f. benfenati department of neuroscience and brain technologies, italian institute of technology, genova, italy mutant mice lacking synapsins (syn), a family of synaptic vesicles (sv) proteins implicated in the regulation of neurotransmitter release and synapse formation, are epileptic. the attacks appear after the third month of age and severity increases with age. several mutations of syn genes have been found in families of patients with epilepsy. we used micro-electrode arrays (meas) to study spontaneous and chemically evoked epileptiform activities in cortico-hippocampal brain slices obtained from wild-type (wt) and synko mice. 6-months old synko mice show sporadic ictal (ic) events in the entorhinal cortex. a potassium channel blocker, 4aminopyridine (4ap), elicits ic and inter-ictal (i-ic) events in both wt and synko slices. in the hippocampus of young synko (15-days old) mice, 4ap induces i-ic events at higher frequencies than in wt mice. also the frequency of ic events, mainly observed in the cortex, is higher in synko. the analysis of adult (1-year old) mice, revealed a clear age-related aggravation, which paralleled the increase in the severity of the epileptic phenotype observed in vivo. many slices from adult synko mice showed an ic event, while wt slices were refractory at this age to experience ic activity. synko mice are useful to study how neuronal network hyperexcitabilty due to mutations in sv proteins leads to the development of epileptiform activity. meas proved themselves to be useful tools to characterize the epileptic signals foci and patterns of propagation. high electron mobility transistor (hemt) structures were used to bridge the gap between the analysis of biological reactions and biophysical characterization. the combination of nanotechnological measurement approaches with biological reactions provides new possibilities for living cell examinations after exposure to ionizing radiation and basically during the irradiation experiments itself. in this transdisciplinary approach experimental data and handling of biological material enables the identification and specification of systems properties of biological responses to ionizing radiation at different hierarchical levels. gan/algan-heterostructures form a hemt with a gate very sensitive to ph-value changes and potential changes in general. to record cell membrane potentials and ion fluxes during and after irradiation experiments living cells are cultivated on the functionalised biocompatible chip surface. here, we present results of x-ray stimulated cell responses grown on gan-chip surfaces. we recorded transistor signal changes of 0.13 µa within 60 s caused by an irradiated cell monolayer. to measure cell potentials, not only after irradiation experiments but also during the irradiation itself expands the examination restrictions in an enormous way. measuring diffusion by spatial-cross-correlation e. gratton, m. a. digman laboratory for fluorescence dynamics, university of california, irvine, u.s.a. fluorescence correlation spectroscopy (fcs) has emerged as a very powerful method to study the motions of proteins both in the interior and exterior of the cell. it provides information at the single molecular level by averaging the behavior of many molecules thus achieving very good statistics. single particle tracking (spt) is also a highly sensitive technique to measure particle movement. however, the fcs method suffers in spatial resolution while the spt technique only allows for the tracking of isolated molecules. here we propose a change of paradigm in which using spatial pair cross-correlation functions we can overcome this limitation. our method measures the time a particle takes to go from one location to another by correlating the intensity fluctuations at specific points on a grid independently on how many particles are in the imaging field. therefore we can trace the average path of the particles. for example, our method could be used to detect when a protein passes the nuclear barrier and the location of the passage. this information cannot be obtained with the frap (fluorescence recovery after photobleaching) technique or the image correlation spectroscopy method. the interaction of the bax c-terminal domain with membranes j. c. gomez-fernandez, s. sanchez-bautista, a. perez-lara, s. corbalan-garcía departamento de bioquimica y biologia molecular. universidad de murcia, murcia, spain the c-terminal domain of the pro-apoptotic protein bax (bax-c) acts as a membrane anchor during the translocation to the membrane of this protein leading to programmed cell death. we have used static and mas-nmr techniques to show that the interaction of bax-c with membranes is modulated by the presence of a negatively charged phosphatidylglycerol. the width of the resonance peaks were considerably more increased by bax-c, in the presence of phosphatidylglycerol. bax-c substantially decreased the t 1 relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol but they were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. 13 c-mas-nmr showed that t 1 values were decreased when bax-c was incorporated and, when phosphatidylglycerol was also present, the decrease in t 1 affected considerably more to some carbons in the polar region. these results indicate that bax-c interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of bax-c with the membrane determines the membrane disposition of this domain. fluorescence spectroscopy showed that the trp residues of bax-c were located in a microenvironment more hydrophobic when phosphatidylglycerol was present. h. g. franquelim 1 , l. m. s. loura 2 , n. santos 1 , m. castanho 1 1 instituto de medicina molecular, univ. lisboa, portugal, 2 faculdade de farmácia, univ. coimbra, portugal since the efficacy of hiv fusion inhibitors was previously reported to be related to an ability to interact with membranes, we studied the interaction of the hiv fusion inhibitor sifuvirtide, a 36 aa negatively charged peptide, with lipid vesicles. since this peptide has aromatic residues, fluorescence spectroscopy techniques were used with no need for attached probes. results showed no significant interaction with both zwitterionic fluid phase and cholesterol-enriched membranes; however extensive partition to fluid phase cationic membranes were observed. in the dppc gel phase, however, an adsorption at the surface of these membranes was detected by using a differential quenching approach with lipophilic probes, as well as by fret. moreover, the interaction with gel phase membranes seems to be specific towards pc vesicles, since no significant interaction was retrieved for membranes composed by shingomyelin and ceramide. besides fluorescence, atomic force microscopy and zeta-potential were used to further investigate this issue. our results show a selectivity and specificity of the peptide towards rigid domains, where most of the receptors are found, and help explain the importance of the interaction with membranes in the improved efficacy of sifuvirtide compared to other fusion inhibitors, by providing a local increased concentration of the peptide near the fusion site on both cellular and viral membranes. the study of molecular dynamics at the single-molecule level with fluorescence far-field optics offers new detailed insights into scientific problems, especially in living cells. unfortunately, the resolution of common far-field techniques is limited to about 200nm in the lateral direction by diffraction. in recent years, several concepts such as stimulated emission depletion microscopy (sted) have been successfully applied to overcome the diffraction barrier by exploiting the photophysical properties of fluorescent labels. we present the combination of high resolution sted microscopy with different fluorescence fluctuation techniques providing the unique ability to study molecular dynamics with high spatial (<40nm) and temporal resolution (<1ms) in living cells. using fluorescence correlation spectroscopy (fcs) and general single-molecule analysis, we were able to explore single-molecule dynamics in up to 70-fold reduced focal volumes on two-dimensional samples such as lipid membranes with excellent signal-to-noise ratios. special attention is drawn to inhomogeneous lipid diffusion on the plasma membrane of living cells 1 . by extending the available spatial scale of standard single-molecule fluorescence far-field spectroscopy techniques, our experiments outline a new way of approaching scientific problems. modulation of the properties of membrane microdomains as a control mechanism in cellular physiology p. o'shea cell biophysics group, institute of biophysics, imaging & optical science, school of biology, university of nottingham, nottingham ng7 2rd, u.k. this presentation will outline the molecular-physical rationale of how membrane microdomains may modulate the behaviour of membrane receptor systems as a controlling mechanism in cell signaling. a number of external factors that modulate these properties will be indicated that have a bearing on controlling cellular behaviour. some of key questions will be considered such as the factors that control the assembly and disassembly of microdomains, the size and numberdensity of the microdomains and the lifetime that they exist within the membrane. the technical challenges that these questions identify will also be outlined with some possible solutions. throughout this presentation, correlations will be made between theory and experiment as well as between model membrane systems and real cellular systems. structural and dynamic properties of caveolin-1 and -2 fragments at the membrane interface c. le lan 1 , j. gallay 2 , m. vincent 2 , j.-m. neumann 1 , b. de foresta 1 , n. jamin 1 1 cea, ibitecs, sb2sm, & ura cnrs 2096, gif-sur-yvette, france, 2 ibbmc, université paris-sud, umr8619-cnrs, ifr115, orsay, france caveolins are major protein components of caveolae, microdomains of the plasma membrane involved in a large number of biological functions, including signal transduction, cholesterol homeostasis and transport. the consensus topological model of caveolin-1 includes a small central intramembrane region (102-134) flanked by two cytosolic amphiphilic domains (82-101 and 135-150) which probably constitute in-plane membrane anchors. we investigated the interaction of the cav-1(94-102) juxta-membrane segment with various membrane mimics, using fluorescence, cd and nmr. this segment partitioned better in dpc and in dm/anionic lipids micelles than in dm micelles and this partitioning was coupled with the formation of an amphipathic α-helix. this amphipathic helix was located in an average shallow position, in the polar head group region of the dpc micelle, as shown by fluorescence data and intermolecular noes, with the aromatic doublet w98-f99 probably pointing towards the inside of the micelle on average. the peptide encompassing the homologous sequence of cav-2 was also localized to the dpc micelle polar head group region, in which it adopted a more stable helical conformation than cav1(94-102) . these data brings experimental support for the role of this segment as an interfacial membrane anchor. resveratrol (trans-3,4',5-trihydroxystilbene) , a phytoalexin present in grapes and its analogue piceatannol (trans-3,4,3',5'-tetrahydroxystilbene) are biologically active compounds and possess potential chemopreventive and anticancer properties. the activity of resveratrol and piceatannol can be mediated by membrane effects since structure of lipid membrane domains may play an important role in cell signalling pathways. drugs interactions with dmpc bilayers was investigated using a combination of esr spectroscopy and differential scanning calorimetry. spin probes used in epr experiment were located in different part of lipid bilayer. study was performed at temperatures below and above phase transition temperature (t m ). epr spectra were simulated and displayed with ghost condensation method. the values of ϑ and ϕ (the main and asymmetry cone angles of wobbling spin probe, respectively) were taken for free rotational space parameter (ω) calculation. the decrease of ω values was observed in the presence of both compounds and the effect was more pronounced in lipid gel phase. order parameter and correlation time were also determined and presented in form of ghost patterns. using this approach the differential influence of studied compounds on membrane heterogenity was revealed. separating hydrostatic pressure from cellular strain: development of an in vitro model system r. sulley 1 , j. whatmore 1 , c. p. winlove 2 , a. shore 1 , j. tooke 1 , r. ellis 2 , k. gooding 1 1 peninsula medical school, 2 school of physics, university of exeter, uk endothelial cells (ecs) line blood vessels & are constantly subjected to haemodynamic and mechanical stresses and strains. these stimuli are known to influence ecs, modifying their morphology, intracellular signalling & gene expression. most reported systems exposing ec to mechanical forces in vitro alter pressure & strain simultaneously, making it impossible to distinguish the two potentially independent stimuli. this distinction is particularly relevant when examining the interaction of haemodynamic forces on microvascular ecs, which are exposed to low hydrostatic pressure but significant strains. this research aims to create an in vitro system that can independently examine the effects of pressure and strain, over a range experienced by ecs in the microvasculature. human ecs are seeded (12x10ˆ4cells/cmˆ2) on to the inner surface of compliant 4mm diameter tubing. which is mounted on a perfusion rig inside a sealed, fluid-filled chamber. a continuous sinusoidal cyclical strain of 5-10% is created by a pump attached to the external chamber. lumenal pressure is generated using two hydrostatic pressure heads. validation experiments show that pressure & substrate strain can be independently varied & controlled over a physiological range. the system is now being used to investigate the effects of pathophysiological haemodynamic abnormalities on ec function. membrane potential dynamics of living cells in response to femtosecond laser irradiation n. i. smith 1 , j. ando 2 , k. fujita 2 , s. kawata 2 1 photonics advanced research center, osaka university, suita, osaka 565-0871, japan, 2 dept of applied physics, osaka university, suita, osaka 565-0871, japan the ultrashort pulsed near-infrared femtosecond laser has had a large impact in biomedical research fields and in microscopy, where it has enabled new imaging methodologies. at high intensities, the focused beam of a femtosecond laser has been used to irradiate specific locations inside a cell, often beneath the cell membrane, exploiting the inherent penetration and localized absorption that comes from the multiphoton absorption physics. this has been applied in photobleaching, photouncaging, laser surgery and other experiments where the light is used not merely for observation but is instead an integral tool to interact with the dynamics of cells, to probe and perturb the cell condition. in this talk i will discuss biological and mechanical effects that can be generated by short exposures to femtosecond laser irradiation, such as calcium waves, membrane hyperpolarization, and cell contraction. this talk will concentrate on the changes in membrane potential that can occur when the cell is subjected to focused femtosecond laser beams. both depolarization and hyperpolarization of the membrane potential could be evoked, depending on the laser parameters and on the position of the laser focus. these results have implications for the use of laser beams in microscopy, optical gene transfection, and laser nanosurgery. in a recent study we showed that the melting behavior of supported lipid bilayers (slbs) on mica can be influenced by the solution ionic strength and by the slb preparation temperature [1] . by changing these parameters we could control the coupling between the two bilayer leaflets obtaining a coupled or decoupled melting behavior. thus, we could provide evidence that the slb model system is also suited for the study of lipid/protein interactions which had been questioned in the past. then we investigated the mutual interactions between the membrane lipids (pope:popg 3:1) and the kcsa potassium ion channel by studying kcsa proteins reconstituted in slbs. in particular, we studied the melting behavior of the slb and the ion channel distribution relative to the different membrane phases by temperature controlled atomic force microscopy (afm). by decreasing the temperature we found that the proteins underwent diffusion so to be excluded from the growing solid ordered regions. further, the ion channels tended to accumulate at the domain boundaries or they aggregated in the liquid disordered phase. both effects have been suggested to affect protein function. when we started from a low temperature at which the membrane was mainly in the solid ordered phase the membrane melting processes started in the vicinity of the included ion channels. we report fluorescence recovery after photobleaching (frap) measurements performed at variable spot radius for t7-egfp-hmop receptors on sh-sy5y neuroblastoma cells in the presence of ligands. two different agonists, damgo and morphine, caused markedly different changes to receptor diffusion as compared to the basal state. like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joint semi-permeable domains, but with smaller domain size and diffusion coefficient. this effect was inhibited by pertussis toxin, suggesting that this dynamic behaviour is associated with early steps of signaling. in the presence of damgo, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. hypertonic sucrose buffer suppressed this effect which we attribute to receptor entry into clathrin-coated pits. it is likely that the observation of distinct receptor dynamics in the presence of damgo and morphine involves the agonist-selective phosphorylation of the receptor. the alteration of the physiological transcriptional program is one of the constant features of cancer cells. however, the characterization of the chromatin changes at single-gene level requires going beyond the diffraction limit affecting conventional fluorescence microscopy. the ability of molecular biology techniques to obtain a detailed view of the chromatin status at a sub-promoter resolution has to pay instead the price of averaging over a cell population. we report here the application of an approach based on high-resolution cytometry, chromatin immuno-precipitation and transcriptional profiling (dna microarray) for the characterization of the transcriptional and chromatin changes induced by the oncogenic transcription factor pml/rarα. the presentation will focus on the imaging protocol employed to observe the effects on the chromatin status and the extent of the deregulation induced on transcriptional activity in acute promyelocytic leukemia cells. this multiple-approach examination provides a further step towards the comprehension of the hierarchy of chromatin modifications leading to the establishment of a malignant transcriptional program. dna is rigid negatively charged polymer and in solution exists in extended conformation. i n vivo, volume occupied by dna must be reduced to fit to tiny space of cell nucleus. to condense dna, dna-dna electrostatic repulsion must be cut off that is achieved by interaction with cationic ligands. in binding to dna, oligocations compete with salt cations (k + , na + , mg 2+ ). description of salt dependence of oligocation-induced dna condensation is still lacking. we studied dna condensation by model oligocations, ε-oligo(l-lysines), with variation of charge from +3 to +31. combination of light scattering, uv-monitored precipitation assay and isothermal titration calorimetry allowed covering wide range of dna (c dna ) and salt (c kcl ) concentrations. salt dependence of dna condensation efficiency of the ligand, ec 50 (ligand concentration at the transition midpoint) displays two regimes: salt-independent at low c kcl and salt-dependent at higher c kcl (steep increase of ec 50 with c kcl ). simple formula describing ec 50 as function of ligand charge, c dna and dissociation constant of ligand-dna complex (k d ), was proposed. in the salt-independent regime ec 50 is defined by c dna . salt-dependence of ec 50 is rooted in the variation of k d with c kcl earlier described in ligand-dna binding studies. importance of our findings for description of chromatin is discussed. interaction between proteins from linker region of nucleosome in presence/absence of dna in solution i. b. kipenko 1 , e. v. chikhirzhina 2 , a. m. polyanichko 1 1 faculty of physics, saint-petersburg state university, russia, 2 laboratory of cell biochemistry, institute of cytology, ras, saint-petersburg, russia interactions in the linker region of the nucleosome play a key role in the structural organization of the chromatin. the most fascinating and least understood is the interplay between non-histone chromatin protein hmgb1 and a linker histone h1. it is known that both h1 and hmgb1 bind the linker region of the dna in vivo. however it is still a matter of debate as to whether these proteins assist each other or compete upon binding. the main attention in this work is paid to the investigation of the interactions between the hmgb1 and h1 proteins in physiological environment. using circular dichroism (cd) spectroscopy we have studied the interactions between hmgb1 and h1 at various hmgb1/h1 ratios (r). it has been shown that there is a cddetectable interaction between the proteins h1 and hmgb1 at r < 1. we have demonstrated that the interaction between these proteins results in changes of their secondary structure. cd indicates that the structural impact of the unordered fragments decreases while the net α-helicity of the proteins increases upon the interaction. we have also shown that large higher order structures are formed in solution. in this work we have also discussed the dna-binding properties of the hmgb1 and h1 proteins. the work was supported by a rfbr grant (09-08-01119) and the government of staint-petersburg. t. u. rodionova 1 , a. m. polyanichko 1 , v. i. vorob'ev 2 1 faculty of physics, saint-petersburg state university, russia, 2 institute of cytology of the russian academy of sciences, saint-petersburg, russia hmgb1 is a nonhistone chromosomal protein. data regarding the structure of the hmgb-proteins obtained so far are rather different. thermodynamic experiments reveal predominant α-helical structure of the proteins only at temperatures below +5 • c, i.e. under physiological conditions they are mainly disordered. despite a lot of experimental data biological role of hmgb1 still remains unclear. it is believed that the proteins perform structural functions in chromatin and participate in various regulatory processes in cell. using circular dichroism spectroscopy and dna melting analysis we have shown that hmgb1 changes its structure upon binding to dna. it was shown that at room temperature only about 25% of amino acid residues form α-helices, while in dna-hmgb1complex the degree of the α-helicity of the protein increases to approximately 50%. based on the data obtained we estimate the size of hmgb1 binding site as 80-100 b.p. we have also demonstrated that despite of strong dna-bending properties of hmgb1 its binding to dna results in increase of the double helix termostability. the authors are grateful for the financial support from the russian foundation for basic research (grants 09-08-01119, 07-04-01072) and the government of saint-petersburg. structural organization of supramolecular complexes of dna with chromosomal proteins hmgb1 and h1 a. m. polyanichko 1 , h. wieser 2 1 faculty of physics, saint-petersburg state university, russia, 2 dept. of chemistry, university of calgary, canada a combination of uv and ir absorption and circular dichroism spectroscopy together with atomic force microscopy was applied to investigate the structure and formation of large supramolecular dna-protein complexes. this combination of techniques was used to overcome limitations of uv-cd spectroscopy due to considerable light scattering in such solutions. based on the analysis of ftir and uv circular dichroism spectra and afm data the interaction of dna with highmobility group non-histone chromatin protein hmgb1 and linker histone h1 was studied. it is believed, that hmgb-domain proteins perform both structural and regulatory functions in chromatin. however, the particular mechanisms of it functioning remain unclear/ our data show that histone h1 facilitated binding of hmgb1 to dna by interacting with the sugar-phosphate backbone and binding of asp\glu amino acid residues of hmgb1. acting together, hmgb1 and h1 stimulated the assemblage of supramolecular dna-protein structures. the organization of the ternary complexes is modulated by the interactions between hmgb1 and h1 molecules. the dna-proteins interactions in the presence of metal ions were different, causing prominent dna compaction and formation of large intermolecular complexes. the work was supported by rfbr (grant 09-08-01119). biophysical properties and mechanisms of phage dna ejection t. mdzinarashvili 1 , m. khvedelidze 2 , a. ivanova 1 , t. partskhaladze 1 , n. shengelia 1 1 i. javakhishvili tbilisi state university, tbilisi, georgia, 2 institute of molecular biology and biophysics, tbilisi, georgia to determine the requirements for phage adsorption on bacterial cell and for the realizing resources of following dna ejection thermodynamic and hydrodynamic methods were employed. the temperature, bacterial membrane fragments and receptors had been chosen as such external factors. the phages with short and long tail, both contractile and noncontractile have been studied. our viscometric studies of the phage dna ejection induced by receptor by the example of t5 phage and its receptor fhua have shown that the minimum protein-to-phage ratio necessary for complete dna release is 300 to 1. the viscometric study of ddvi phage dna ejection induced by membrane fragments obtained from its host cells has shown that the environmental conditions play significant role in ejection process. both methods show that the thermally induced phage dna ejection for all investigated by us phages have shown that this process is nonenthalpic. finally from our experimental results we conclude that the start of the dna ejection process from the phage particle occurs without additional energy from either a physical or chemical source. we thank gnsf for the financial support. nitroxides induce apoptosis through caspase-3 activation and collapse of mitochondrial potential k. matczak 1 , a. koceva-chyla 1 , k. gwozdzinski 2 , z. jozwiak 1 1 department of thermobiology, 2 department of molecular biophysics, university of lodz, lodz, poland nitroxides are new class of antioxidants that have been proved to show high reactivity toward free radicals. they act as superoxide dismutase mimics dismutating superoxide anions, but can also exert pro-oxidative properties. in view of their possible dual activity nitroxides could be of great importance in medicine. we have investigated pro-apoptotic activity of pyrroline and pyrrolidine nitroxides pirolid (pd) and pirolin (pl) in human breast cancer cells. in cancer, it is the failure of malignant cells to undergo apoptosis that is crucial. using microplate fluorescence methods, we estimated kinetics of changes in mitochondrial transmembrane potential and caspase-3 activity in breast cancer cells mcf-7 treated with pirolin or pirolid. these features are connected with induction of apoptosis in some type of cancer cells. we observed steady-state increase in caspase-3 activity up to 12 h of postincubation that was followed by a decrease in the enzyme activity at 24 h. caspase-3 activation was considerably greater in cells treated with pirolid. both nitroxides also caused notable decrease in mitochondrial transmembrane potential, which suggest that they can induce apoptosis in breast cancer cells through mitochondrial pathway. o. chernyavskiy 1 , l. vannucci 2 , p. bianchini 3 , f. difato 4 , l. kubínová 1 1 dept. of biomathematics, inst. of physiology, as cr, prague, czech republic, 2 dept. of immunology, inst. of microbiology, as cr, prague, czech republic, 3 lambs, dept. of physics, university of genoa, italy, 4 dept. of neuroscience and brain technologies, the italian institute of technology, genoa, italy the second harmonic generation (shg) imaging along with confocal laser scanning microscopy in reflectance mode can be applied to imaging unstained tissues in vivo, so it can be considered as a fast and non-invasive tool for in vivo studies. murine b16f10 melanoma cells after subcutaneous inoculation in syngeneic mice were let to develop into tumor up to 15-20 mm in diameter. microscopic images were taken before and after microwave hyperthermia treatment (mwht). the microscopic images were acquired by 1-photon imaging in reflectance mode, shg imaging and 2-photon imaging of tissue autofluorescence. the evaluation of changes in the images after mwht of the tumor demonstrated changes in the architecture and organization in both the tumor capsule and tumor mass. the presented study was supported by the academy of sciences of the czech republic (grant iaa500200510, institutional research concepts no.av0z50200510, av0z50110509), and ministry of education, youth and sports of the czech republic (research program lc06063). intracellular delivery and fate of peptide-capped gold nanoparticles: towards cellular biosensors y. cesbron 1 , v. sée 1 , p. free 1 , p. nativo 1 , d. g. spiller 1 , m. r. h. white 1 , m. brust 1 , b. lounis 2 , r. lévy 1 1 liverpool institute for nanoscale science, engineering and technology, liverpool, uk, 2 université bordeaux i / cnrs, bordeaux, france gold nanoparticles (nps) have extraordinary optical properties that make them very attractive single molecule labels. although understanding their dynamic interactions with biomolecules, living cells and organisms is a prerequisite for their use as in situ sensors or actuators. while recent research has provided indications on the effect of size, shape, and surface properties of nps on their internalization by living cells, the biochemical fate of nps after internalization has been essentially unknown. here we show that peptide-capped gold nps enter mammalian cells by endocytosis. we demonstrate that the peptide layer is subsequently degraded within the endosomal compartments through peptide cleavage by the ubiquitous endosomal protease cathepsin l. preservation of the peptide layer integrity and cytosolic delivery of nps can be achieved by a combination of cathepsin inhibition and endosome disruption. this is demonstrated using a combination of distance-dependant fluorescence unquenching and photothermal heterodyne imaging. these results prove the potential of peptide-capped gold nps as cellular biosensors. current efforts focus on in-vivo labeling of nps, nanoparticle-based real-time sensing of enzyme activity in living cells, and the development of photothermal microscopy for single nanoparticle imaging in living cells. towards intravital two photon microscopy study of lymphocytes mobility in lymphonodes m. caccia 1 , l. sironi 1 , m. collini 1 , i. zanoni 2 , t. gorletta 2 , m. di gioia 2 , g. francesca 2 1 dipartimento di fisica, università di milano bicocca, italy, 2 dipartimento di biotecnologie e bioscienze, università di milano bicocca, italy during the last 30 years the edge between optical fluorescence based microscopy and the world of bio-medical research has become thinner and thinner and two photon laser scanning microscopy (tplsm) is one of the most powerful tool for immunological and medical research. one of the most limiting step in intravital microscopy is the preparation of the animal model and the number of animals to be sacrificed to get good statistics. alternative routes must be searched. we employ tplsm to explanted lymphnodes. two photon excitation and a non descanned detection mode allow to increase, respectively, the excitation and detection efficiency while the explanted organs are kept very close to the condition they experience in live animals by means of an home-made temperature controlled box surrounding the microscope and a system for the flux of physiological fluids. experiments performed on explanted lymphonodes, kept under constant flux of co 2 -o 2 saturated buffers and at 36 o c, agree with literature for what concern t-cell homing and motility. this seems to confirm that t-cells behavior in explanted organs mantained in physiological conditions is very similar to that observed in live animals. we then believe that our tplsm microscope would allow to study cell behaviors in in-vivo with high efficiency and a little technical effort. a generalized quantitative frap method with no restriction on the size of the photobleached area heterochromatin protein 1 in dna damage response -recruitment or dissociation from repair sites? j. w. dobrucki division of cell biophysics, faculty of biochem. biophys. and biotechnol., jagiellonian university, kraków, poland we studied recruitment of dna repair proteins to damage sites in live cells, by microscopy approaches, using a new method of inflicting local, sublethal damage in nuclei of live cells. oxidative damage, which was inflicted by exciting dna-intercalated ethidium with focused green light, triggered recruitment of base excision repair enzymes. surprisingly, an epigenetic regulator, heterochromatin protein 1 (hp1) (zarebski et al., 2009 ) was recruited to damage as well. hp1 is a constitutive component of heterochromatin, and plays an important role in transcriptional repression and regulation of euchromatic genes, however it was not known to be required for repair of oxidative damage. the finding of hp1 recruitment is particularly puzzling, since in another study hp1 was shown to dissociate from chromatin as a result of dna damage (ayoub et al. 2008) . technical aspects of live cell imaging that may explain these contradictory results will be discussed. in this presentation, we report a method for determining both the presence and the stoichiometry of protein complexes at pixel resolution and apply it to disassembling focal adhesions. the method is derived from fluorescence fluctuation methods that have single molecule sensitivity and is based on our previously described n&b (number and brightness) method that measures the number and brightness (aggregation state) of fluorescent molecules in every pixel of a confocal microscope image. the new method exploits the correlation of fluorescence amplitude fluctuations for two colors and detects the presence of molecular complexes and their stoichiometry. while the original n&b method was developed for one color, i.e., a single molecular species, the new method, ccn&b, extends the analysis to two colors and introduces the concept of cross-variance. this method is similar in concept to the two-color pch analysis. however, the covariancebased ccn&b method also generates pixel resolution maps of protein complexes and can be used on commercial confocal microscopes. the method is highly sensitive and has relatively high temporal resolution. we have applied this method to adhesion complexes in cells. in addition of their structural role, to link the extracellular substratum to actin filaments, they also serve as signaling centers that regulate many cellular processes including their own assembly and turnover, migration, gene expression, apoptosis, and proliferation. spatio-temporal analysis of membrane lipid remodeling during phagocytosis s. de keijzer 1 , d. kilic 1 , c. g. figdor 1 , s. grinstein 2 , a. cambi 1 1 department of tumor immunology, radboud university of nijmegen, nijmegen, the netherlands, 2 department of biochemistry, university of toronto, toronto, canada the constant threat posed by pathogens and cell debris is tackled by phagocytosis, the process through which cells engulf and destroy dangerous material. the signaling, targeting and trafficking during phagocytosis is dependent on cytoskeleton rearrangements and membrane remodeling. it is becoming increasingly evident that lipids play an important role and can affect the phagocytic response. they assemble microdomains which can act as signaling platforms and confer charge and curvature to the membrane surface promoting electrostatic attraction and retention of proteins. little is known about the mechanism(s) regulating membrane lipid remodeling during phagocytosis. here we used fluorescently labeled biosensors based on k-ras and h-ras proteins to obtain spatio-temporal information on phagosomal membrane lipid remodeling during fcreceptormediated phagocytosis. the results show that cell activation by cytokines modulates the kinetics of anionic lipids thus affecting the membrane charge and the recruitment of cytosolic proteins to the phagosomal membrane. our data emphasize the fundamental role of lipids in the generation and transduction of signals in phagocytosis, and we believe this can be extrapolated to many important processes in a cell. fluorescence correlation spectroscopy (fcs) is a useful technique for characterizing the mobility and concentration of fluorescent molecules both in vitro and in vivo. we utilize two-photon fcs to characterize the concentration and mobility of fluorescent molecules within living cells of bacillus subtilis. autocorrelation functions were measured in bacteria expressing green fluorescent protein (gfp) under the lac promoter in both nutrient rich and nutrient poor culture medium. although considerable heterogeneity was evident from cell to cell, on average, both intracellular concentration and mobility were found to be dependent upon culture medium. we also investigated bacteria expressing gfp under control of native promoters for involved in the regulation of the carbon metabolic cycle in bacillus subtilis. the gfp concentration, which should be related to promoter activity, was investigated for single cells and cell populations under different metabolic conditions. some photobleaching was observed during the course of the measurements as a decrease in the average fluorescence intensity. this is due to the small size of the bacteria (∼1 fl) and low basal expression levels of gfp (∼100 nm) in the absence of iptg. methods to take this into account during data analysis are discussed. a. fassio 1 , s. congia 2 , p. baldelli 2 , f. benfenati 2 1 dimes, university of genoa, genoa, italy, 2 dnbt , iit, genoa, italy several mutations have been discovered in the synapsin i (syn) gene in families with epilepsy, but the mechanism inducing the epileptic phenotype is unknown. syn is a protein associated with synaptic vesicles (svs) that control sv trafficking and neurotransmitter release. the syn mutations subject of this study are a non sense (ns-1) and of two missense (ms-2, ms-3) . syn knockout (ko) hippocampal neurons were transfected with the either wild type (wt) or mutated syns. all syns presented a common punctate pattern of expression at the level of axonal arborizations but, while ns-1 syn targeted to synapses as wt-syn, ms-2 and ms-3 syns reached the presynaptic terminal less efficiently. we next set up a live cell imaging experiment using synaptophysin-phluorin and evaluated the effects of ns-1, ms-2 and ms-3 syns on the size of sv pools. restoring wt-syn in syn i ko terminals led to a increase of all sv pools. restoring ms-2 and ms-3 syns resulted in a phenotype not significatively different from syn i ko background whereas restoring ns-1 syn caused a decrease of all sv pools as compared either with wt-syn or syn i ko background. these data suggest an alteration in the subcellular distribution and function of syn in patients carrying ms-2 e ms-3 mutation and a more severe effect on synaptic activity in patient carrying ns-1 mutation. imaging dynamics of dc-sign at the plasma membrane of hiv-1-stimulated dendritic cells o understanding how viruses interact with host receptors is essential for developing new antiviral strategies. dendritic cells (dcs) can efficiently capture and take up hiv-1 through multiple attachment factors, such as the c-type lectin dc-sign. however, the initial interactions between hiv-1 and this receptor on dcs are not fully understood yet. in this work, we have used single-molecule epi-tirf microscopy in combination with fluorescently labelled hiv-1 virus like particles (vlps) and dc-sign-specific antibodies to image the dynamic interaction between hiv-1 and dc-sign nanoclusters at the plasma membrane of dcs. by tracking individual trajectories of dc-sign on the cell surface of living dcs we have found heterogeneity in the modes of motion of dc-sign: some clusters are immobile whereas others move very quickly, with a few ones showing a directed motion on the cell membrane. to investigate how such motion might be correlated to dc-sign function as virus attachment factor, we have developed glass platforms functionalized with hiv-1 vlps to locally stimulate living dcs. these virus platforms have allowed us to measure dc-sign diffusion rates and motion modes over the cell surface of stimulated dcs and represent a powerful tool for studying the dynamic interactions between hiv-1 and the dc membrane. a. esposito 1 , t. tiffert 2 , j. mauritz 1 , s. schlachter 1 , j. n. skepper 2 , v. l. lew 2 , c. f. kaminski 1 1 dept. of chemical engineering and biotechnology, univ. of cambridge, u.k., 2 dept. of physiology, development and neuroscience, univ. of cambridge, u.k. plasmodium falciparum (pf ) causes the most lethal form of malaria in humans. early research exposed two paradoxes: 1) during its intraerythrocytic cycle pf permeabilizes the host cell so much that a comparably permeabilized healthy red blood cell (rbc) would lyse prematurely, and 2) pf digests far more hemoglobin than needed for its metabolism. a model of the homeostasis of a pf infected rbc suggested a common explanation of both puzzles: excess hemoglobin digestion is required to reduce the colloidosmotic pressure within the host cell thus ensuring its osmotic stability to the end of the pf asexual cycle. we investigated these predictions with direct measurements of [hb] and volumes of parasitized rbcs. reliable volume and morphological data was obtained by confocal microscopy and quantitative surface reconstruction. furthermore, we developed a new fret-based method to measure hemoglobin molecular crowding by exploiting the reduction in fluorescence lifetime of a donor fluorophore loaded in the rbc cytosol. fret imaging techniques are powerful tools for probing the biophysics of living cells. these tools provided a first validation of the colloidosmotic hypothesis and a deeper understanding of the homeostasis of the intraerythrocytic stage of pf. hiv-1 assembly and release occur at the plasma membrane of infected cells and are driven by the gag polyprotein. using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated assembly of fluorescently labeled hiv-1 at the plasma membrane of living cells with high time resolution. gag assembled into discrete clusters corresponding to single virions. after their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1-2 hours. using a photoconvertible fluorescent protein, we determined that assembly was nucleated by membrane bound gag molecules, while both membrane-bound and cytosol derived gag polyproteins contributed to the growing bud. assembly kinetics were rapid and three phases are observed. in phase i, the number of gag molecules at a budding site increases following a saturating exponential with a rate constant of ∼ 5 x 10 −3 s −1 . hence, gag assembly is complete in ∼ 200 s. in phase ii, a plateau in fluorescence intensity is observed with no exchange of gag protein. the fluorescence intensity decays in phase iii. this decay, in some cases, corresponds to the release of a virion. the time scale from the onset of assembly to release of extracellular particles was measured to be ∼1,500+/-700 s. fast 3d chromatin dynamics studied in living yeasts using a novel lab on chip technology h. hajjoul 1 , m. dilhan 1 , i. lassadi 2 , k. bystricky 2 , a. bancaud 1 1 laas-cnrs, université de toulouse, france, 2 lbme, cnrs umr 5099, toulouse, france we present a novel lab-on-chip technology for 3d particle tracking yeast abased on v-shaped mirrors, which are used to observe fluorescent specimens from multiple vantage points, providing stereo-images that can be recombined for 3d reconstruction. our technology is based on v-shaped mirrors, which are fabricated by wet etching of silicon wafers, and used as optical and fluidic components. after rigorous optical optimization of the device in vitro, the lab-on-chip is applied to study chromatin dynamics in vivo using budding yeasts as a model system. yeasts cells are visualized using gfp fused to the associated repressor protein. we confirm earlier observations that telomeric sequences, i.e. located close to chromosome ends, accumulate at the nuclear periphery, whereas genes found midway along chromosome arms are mostly present in the nuclear lumen. the dynamics of these sequences is followed in 3d with an unpreceded temporal resolution of 10 ms, showing that chromosome dynamics is non linear in the short time regime and switches to a linear regime at larger timescales. notably, this behavior is reminiscent of universal responses observed in polymer solutions, and is related to the confinement and the structure of chromosomes. this technique shows a great potential for studying dynamic processes in small living organisms. retrovirus induced remodeling of the host cell actin architecture m. gladnikoff, i. rousso department of structural biology, weizmann institute of science, rehovot, israel retrovirus budding is a key step in the virus replication cycle. yet, despite substantial progress in the structural and biochemical characterization of retroviral budding, the underlying physical mechanism remains poorly understood, primarily due to technical limitations preventing visualization of bud formation in real time. using atomic force-, fluorescence-and transmission electron microscopy we find that both hiv and moloney murine leukemia virus (mlv) remodel the actin cytoskeleton of their host cells and utilize the forces it generates to drive their assembly and budding. highly dynamic actin-filamentous structures which varied in size over the duration of budding appeared to emanate from the assembled virion. these actin structures assemble simultaneously or immediately after the beginning of budding, and disappear as soon as the nascent virus is released from the cell membrane. analysis of sections of cryo-preserved virus infected cells by tem reveals similar actin filaments structures emerging from every nascent virus. substitution of the nucleocapsid domain implicated in actin binding by a leucine-zipper domain resulted in budding of virus-like particles that was not accompanied by remodeling of the cell's cytoskeleton. notably, budding of viruses carrying the modified nucleocapsid domains was an order of magnitude slower than that of the wild type. the results of this study show that retroviruses utilize the cell cytoskeleton to expedite their assembly and budding. t. gensch institute of structural biology and biophysics (isb-1) cellular biophysics, research centre jülich, germany the determination of ion concentrations in cells -in particular in neurons -is very important for understanding cell function and life. ca 2+ is an ubiquitous messenger in almost all cell types, cl − has several roles, e.g. plays an important role in some neuronal signaling pathways including olfaction, nociception and vision. fluorescence lifetime imaging (flim) is of advantage over intensity based fluorescence microscopy, when comparisons between micro-domains of one cell or between different cells of one cell type are performed. several (organic chromophores and fluorescent protein based) ca 2+ -and cl − -sensors have been tested in culture cells with respect to their applicability in flim studies. the ca 2+ -and cl − concentration in rodent olfactory sensory neurons, dorsal root ganglion neurons and neurons of the retina is investigated by time-resolved flim with two-photon excitation. viscosity is one of the main factors which influence diffusion in condensed media. in a cell viscosity can play a role in several diffusion mediated processes, such as drug delivery and signalling. previously, alterations in viscosity in cells and organs have been linked to malfunction; however, mapping viscosity on a single-cell scale remains a challenge. we have imaged viscosity inside individual cells using novel fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent. 1, 2 this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to 100× higher than that of water. 1 we have also shown that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt). 2 we have demonstrated the effect of such viscosity increase on intracellular reactions by directly monitoring the rates of formation and decay of a short lived toxic intermediate, crucial in pdt, called singlet molecular oxygen, in light perturbed cells. characean algae, close relatives of higher plants, represent a convenient model for studying the effect of propagating electrical signals, action potentials (ap) on photosynthesis. illuminated chara corallina cells produce coordinated spatial patterns of chlorophyll fluorescence (chl fl) and extracellular ph. photosynthesis is higher in the cell regions adjacent to acid zones compared to alkaline zones. under physiological conditions, the electrically induced ap differentially and reversibly suppresses photosynthesis in the alkaline and acid regions. in this work we examined the effects of an artificial psi acceptor, methyl viologen (mv), on chl fl with imaging-pam technique. mv is a divalent cation and poorly permeates through biological membranes. the presence in the medium of mv had no effect on fl as well as on p700 + absorbance signals until the application of a single excitatory stimulus. once an ap was generated in the presence of mv, it induced irreversible inhibition of native (nadpdependent) electron flow and a strong non-photochemical fl quenching all over the cell. this indicates that ap redirects electron flow from the main pathway to the artificial acceptor. we concluded that ap generation opens access for permeation of mv from the medium to the chloroplast stroma across two membrane barriers, plasmalemma and chloroplast inner membrane. we suggested that mv might enter chara cell via plasmalemma ca 2+ -channels activated during ap. evaluation of cell damage after photodynamic and sonodynamic treatment h. kolarova, k. tomankova, s. binder, p. kolar, r. bajgar, m. strnad department of medical biophysics, faculty of medicine and dentistry, palacky university, hnevotinska 3, 775 15 olomouc, czech republic photodynamic therapy (pdt) and sonodynamic therapy (sdt) is a new, combined therapy for treating cancer. the basis of the therapy is to administer a small amount of photosensitizer and sonosensitizer, which are selectively taken up by cancer cells, and then expose the body to light and ultrasound to activate these sensitizers. when the sensitizers absorb light of an appropriate wavelength, it may cause their excitation with subsequent energy transfer to oxygen; the oxygen then becomes highly reactive in cancer cells and produces reactive oxygen species (ros). the resulting damage to organelles within malignant cells leads to tumor ablation. sdt uses an agent that is sensitive to ultrasound, allowing deeper penetration and destruction of abnormal cells. changes in human melanoma cells were evaluated using fluorescence microscope and atomic force microscopy.we focused on obtaining pictures of the topography and pictures involving elastic properties of cell surface. the production of ros was investigated with the molecular probe cm-h2dcfda, and morphological changes in cells were evaluated using fluorescence microscope. the quantitative ros production changes in relation to phthalocyanine concentration, irradiation doses and ultrasound intensity were measured by a fluororeader. activity correlation imaging: visualizing function and structure of neuronal populations s. junek 1 , t.-w. chen 1 , m. alevra 1 , d. schild 2 1 department of neurophysiology and cellular biophysics, university of göttingen, germany, 2 dfg research center for molecular physiology of the brain (cmpb), university of göttingen, germany understanding a neuronal network relies on knowledge about both the function and the structure of its neurons. while he simultaneous observation of hundreds of neurons is possible by densely staining brain tissue with functional dyes, the low contrast of these stainings does not allow the identification of neuronal processes from the raw fluorescent images. however, as neurons are known to exhibit complex temporal patterns of activity, fluctuations in signal intensity over time could be exploited to generate contrast in densely stained tissue. we demonstrate that the uniform calcium signals within individual neurons of the olfactory bulb can be exploited to visualize the morphology and projection patterns of these cells in tissue slices. as different neurons exhibit distinct time patterns, it is possible to generate a highcontrast multi-color visualization of the network's active neurons, solely based on the specificity of temporal fluctuations of a single-color calcium dye. it is thus possible to use other spectral channels for additional labelling, for example using cell-type or protein specific markers. the ability to map function and structure of neuronal populations online opens up a number of intriguing applications, such as selecting cells or cell pairs with certain functional or projection profiles for targeted recordings, ablation or stimulation. -live cell imaging investigation of the dynamics of redox elements in live cells by using fluorescence ratio microscopy g. maulucci 1 , g. pani 2 , v. labate 2 , m. mele 2 , e. panieri 2 , m. papi 1 , g. arcovito 1 , t. galeotti 2 , m. de spirito 1 1 istituto di fisica, università cattolica s. cuore, roma, italy, 2 istituto di patologia generale, università cattolica s. cuore, roma, italy oxidative stress or signaling events can affect cellular redox environment, which act simultaneously as regulator and indicator of key cellular functions in both physiological and pathological settings. by using a redox-sensitive protein (rxyfp), employed ratiometrically, it is possible to generate high resolution redox maps of cells. the spatial distributions of oxidized and reduced elements have been discriminated in human embryonic kidney cells by the deconvolution of the histograms of redox maps. by transfecting cell with glutaredoxin v (grx-v), a significant shift towards more reduced state with respect to that recovered from non-transfected cells is observed. despite such large differences, a common behaviour in the spatial distribution of reduced and oxidized couples can still be observed: oxidized population shows a pronounced localization on the cell borders, near the plasma membrane, while reduced population appears as a collection of well separated spots homogeneuosly distributed thoroughly the inner part of the cell with a mean dimension of 2 um. furthermore we observe that the role of grx-v consists in causing a shift towards reduced values of the highly reduced region, while leaving unaltered the redox-balance of the intracellular side of the plasma membrane. fluorescence resonant energy transfer (fret) is a popular approach to studying molecular interactions. fluorescence lifetime imaging (flim) provides a robust read-out of fret but has largely been restricted to fixed cells owing to relatively long acquisition times. we present a highspeed wide-field multidimensional fluorescence microscope for flim fret in live cells on second timescales for high content analysis and studying fast dynamics. the system uses a nipkow disc for optical sectioning and a time-gated image intensifier for wide-field flim. a spectrally selected supercontinuum excitation source facilitates versatile realtime flim of live cells transfected with fluorescent proteins. for cell signalling studies we have developed a multiplexed fret approach. ras activation at the plasma membrane is demonstrated using flim to read out tagrfp-raf rbd interaction with mplum-h-ras. simultaneously, a spectral ratiometric read-out is used for a second fret (cfp/yfp cameleon) probe to monitor the downstream calcium flux. to further develop multiplexed fret as a tool for imaging multiple components of cell signalling networks, we are working to include polarisation-resolved imaging for steady-state and time resolved fluorescence anisotropy measurements. monitoring gene expression via novel nucleic acid and delivery methods k. lymperopoulos 1 , c. spassova 1 , a. seefeld 1 , h. s. parekh 2 , d. p. herten 1 1 bioquant institute,heidelberg university, germany, 2 school of pharmacy, university of queensland, australia application of single-molecule and high-resolution fluorescence methods to monitor gene expression in living cells increase the demand on novel probes and delivery methods.they require fluorophores with high photostability and quantum yield and highly-efficient delivery methods that ensure the minimum interference with cell processes such as metabolism and signal transduction. we used a novel class of dendrimers with varying generations and branching factors and different number of positive charges due to different moieties and functional groups.these different properties were tested for their efficiency to transfect eukaryotic cells with fluorescent oligonucleotides (odns).different parameters (temperature,concentration of dendrimers, ratio of dendrimers and odns) were evaluated and optimised.we utilised these established optimal conditions to deliver a modified concept of smartprobes to mammalian cells targeting mrnas involved in signal pathways.we tested different mrna targets and we optimised the fluorescence signal by varying a range of parameters,namely the fluorescent label and the intrinsic properties of the smartprobe (length of the loop and stem, conformation and number of guanosines).in the near future,we plan to use these probes for monitoring gene expression levels using diffusion imaging microscopy. we present a novel near-field scanning microwave microscope (smm) capable of providing surface impedance measurements of samples with nanometric resolution. the instrument is the integration of a microwave vector network analyzer (vna) and a scanning probe microscope (afm/stm). a key point is that our software, controlling and synchronizing both the instruments, creates simultaneously images of the sample at several frequency points. this can be used to extract several features of the sample depending on the frequency. moreover, close frequencies show the same features, added to random noise. exploiting this redundancy of information, we have achieved remarkable results. we have been working on the optimization of this system for biological applications, to detect functional characteristics of cells generating a variation of their dielectric properties. this instrument offers the possibility of performing local impedance measurements on a single live cell and, if correctly calibrated, it provides also quantitative information (e.g. absolute measurements of membrane permittivity). the system was demonstrated to work on saccharomyces cerevisiae. a better model for a full test of the potentialities of the new technique is given by excitable cells, characterized by a greater variability of dielectric properties. a challenging objective could be directly imaging ion channels. hiv-1 to efficiently complete a replication cycle has to integrate its genome into the host cellular dna.after hiv-1 enters target cells,neosynthesized viral dna forms along with other proteins the pre-integration complex (pic).pics are then transported into the nucleus where integration,catalyzed by the viral integrase,takes place.hiv-1 viral particles engineered to incorporate integrase fused to egfp have proven effective to study pics within nuclei of infected cells.in this study we report the live imaging analysis of nuclear pic dynamics obtained by time-lapse microscopy.intranuclear trajectories of in-egfp-labeled pic were collected in three dimensions and examined by both mean squared displacement (msd) and cage diameter (cd) analysis.in cd the maximum distances measured between two positions occupied by a pic in a time window of 2 minutes were calculated while in our msd analysis 5-minute long trajectory segments were considered.remarkably,msd revealed the presence of an underlying active transport mechanism.to test the possible role of actin filaments,pic nuclear trafficking was analyzed in cells treated with latrunculin b (actin polymerization inhibitor).preliminary results suggest that the disruption of actin function impairs the active nuclear movement of pics. second harmonic generation microscopy reveals sarcomere contractile dynamics of cardiomyocytes n. prent 1 , c. a. greenhalgh 1 , r. o. cisek 1 , j. aus der au 2 , s. elmore 3 , j. h. van beek 3 , j. squier 2 , v. barzda 1 1 department of physics and institute for optical sciences, university of toronto, toronto, canada, 2 department of physics, colorado school of mines, golden, usa, 3 department of molecular cell physiology, vrije universiteit, amsterdam, netherlands cardiomyocytes, like other striated muscles, exhibit strong inherent second-order nonlinear optical properties that make them exemplar for live cell dynamic studies with second harmonic generation (shg) microscopy. laser scanning shg microscopy with an incident wavelength around 1µm, enables fast imaging for extended periods of time with negligible tissue damage. strong shg signal originates from the anisotropic (a-) bands which are comprised of regularly arranged myosin molecules, while actin molecules, the main constituent of the isotropic (i-) bands, produce negligible shg. consequently, the alternating bands along the myofibril are clearly visualized, therefore, enabling the determination of individual sarcomere lengths and the study of sarcomere length dynamics during macro-scale contractions. shg intensity is shown to positively correlate to sarcomere length, which leads to the development of real-time inherent force sensors for in vivo myocytes. rich sarcomere dynamics can be observed during myocyte contraction, which can be used for medical diagnostic purpose of muscular degenerative diseases. imaging cellular communication in insulin secretion ex vivo and in vivo d. w. piston vanderbilt university, nashville, tennessee, u.s.a. the islet of langerhans is the functional unit responsible for glucose-stimulated insulin secretion (gsis), and thus plays a key role in blood glucose homeostasis. the importance of the islet is demonstrated by the proven ability of islet transplants to reverse type i diabetes pathologies in human patients. we are interested in understanding the multicellular mechanisms of islet function, and their role in the regulation of blood glucose under normal and pathological conditions. in many ways, the islet appears to function as a syncytium, which exhibits synchronous behavior of membrane action potentials, ca 2+ oscillations, and pulsatile insulin secretion across all β-cells in the islet. in other ways, the islet works as individual cells, especially in the regulation of gene transcription. using our unique quantitative optical imaging methods and novel microfluidic devices, the dynamics of these molecular mechanisms can be followed quantitatively in living cells within intact islets. these investigations utilize transgenic and tissue-specific knock-out mouse models with demonstrated phenotypes, as well as traditional biochemical and molecular biological approaches. do retinal rod outer segments carry out oxydative phosphorylation? i. panfoli 1 , p. bianchini 2 , d. calzia 1 , s. ravera 1 , a. morelli 1 , a. diaspro 3 1 biology dept., university of genova, italy, 2 lambs-microscobio res.centre, university of genova, italy, 3 neuroscience and brain technology dept., i.i.t. genova, italy visual transduction in vertebrate retinal rod outer segments (os) is very energy demanding. however, atp supply in os, that are devoid of mitochondria, is still puzzling. by a proteomic analysis we identified in purified bovine os disks, proteins involved in vision, as well as the respiratory chain complexes i to iv and f 1 f o -atp synthase, whose activity was comparable to that of mitochondria and sensitive to specific inhibitors. rhodamine 123 fluorescence quenching experiments showed the presence of a proton potential difference across disks. disks consumed oxygen. confocal laser scanning and transmission electron microscopy showed that cytochrome c oxydase and atp synthase are localized on disks. mitochondrial vital dyes stained os ex vivo, and disks. rhodopsin and mitotracker fluorescence co-localized in os. data, suggestive of an aerobic metabolism in os, point to the existence of "mitochondrial inner membrane-like membranes" ectopically producing atp through oxidative phosphorylation, with respect to mitochondria. new scenarios open on the patho-physiology of many retinal diseases associated to mitochondrial dysfunction. -live cell imaging -abstracts 123 unmixing using lifetime-excitation multidimensional confocal fluorescence microscopy data s. schlachter 1 , s. schwedler 2 , a. esposito 1 , a. d. elder 1 , g. s. kaminski schierle 1 , c. f. kaminski 1 1 cambridge university, u.k., 2 universität bielefeld, germany fluorescence imaging provides a powerful tool to probe biological systems, enabling the high resolution investigation of the localization, interaction and biochemical modification of biomolecules. multidimensional imaging, in particular, is a growing application of confocal and other forms of fluorescence microscopy. it seeks to measure not only fluorescence intensity in three spatial dimensions, but also other features of fluorescence emission, such as lifetime and fluorescence spectra. although multi-dimensional fluorescence microscopy has been demonstrated before, little attention has so far been paid to the problem of data interpretation and representation when dealing with such large datasets. we present here an instrument capable of recording fluorescence lifetime and excitation data by combining tcspc for lifetime determination and a supercontinuum excitation source for extracting excitation spectra. these technologies permit the acquisition of 2d and 3d images with lifetime and excitation spectrum information at every pixel. we demonstrate a method whereby the multidimensional datasets acquired with this instrument are processed to yield biologically relevant parameters, (e.g. unmix fluorophores in a multiply labelled sample). our method relies on a global analysis approach and uses ab plots for displaying multidimensional data. we demonstrate the instrument & processing method on dye and multiply labelled biological samples. advanced neuroimaging with diffractive optical elements p. saggau baylor college of medicine, houston, texas, usa studying neural systems is complicated by the large number and small size of its cellular elements. the traditional way of exploring neuronal function by electrical monitoring with micropipettes is increasingly replaced by molecular imaging. fluorescent molecules allow optical monitoring of neuronal signaling with cellular and often subcellular resolution. in addition to monitoring activity, analyzing the functional properties of individual neurons or neuronal populations requires their controlled activation. optical approaches are well-suited, including molecular photolysis of inert precursors and light activation of engineered proteins that control ionic membrane currents. to fully utilize optical techniques for exploring neural systems requires more than adequate spatial resolution to distinguish neuronal elements and sufficient temporal resolution to induce and/or monitor neuronal signaling. because of the non-linear and non-stationary nature of the studied system it is necessary to access many neuronal sites simultaneously. in modern imaging, wide-field illumination and imageformation are often replaced by patterned excitation and nonimaging photon collection. in many instants, diffractive illumination schemes can substitute for conventional reflective or refractive designs. in particular, the availability of programmable diffractive optical elements has made this an attractive alternative, since they permit highly versatile microscope designs and support a significant increase in the overall spatio-temporal resolution. i will present an advanced imaging approach using diffractive optical elements to analyze structure and function of live neurons in brain slices and intact cortex. efficient evaluation of fret in image cytometry with acceptor photobleaching and ratiometric methods j. roszik, d. lisboa, j. szöllősi, g. vereb department of biophysics and cell biology, university of debrecen, debrecen, hungary fluorescence resonance energy transfer (fret) is a powerful technique that can be applied to study nanoscale intra-and intermolecular events and interactions of molecules in situ in biological systems. a robust, easy to use, self-controlled fret method, independent of donor and acceptor concentration and stochiometry, is acceptor photobleaching fret, which requires only simple image mathematics. another approach with more complicated calculations is the intensitybased ratiometric method, which is not based on destroying the acceptor fluorophores, making it applicable for following molecular interactions in live cells. as the need for using fret in image cytometry for evaluating molecular interactions increases, we have undertaken to develop softwares for these methods involving the calculation and usage of all correction factors needed to obtain reliable energy transfer efficiencies. correction possibilities of the acceptor photobleaching method include unwanted photobleaching of the donor, fluorescent photoproduct of the acceptor after photobleaching, cross-talk of unbleached acceptor into the donor channel and partial photobleaching of the acceptor. in the case of intensity-based ratiometric fret, we can correct for all channel cross-talks and calibrate the fret efficiency calculations. both programs provide registration and semiautomatic processing, and they are freely available. the pendrin (slc26a4) gene is responsible, when mutated, for the pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid disfunctions. pendrin is an anion exchanger. the way it works and its role in different tissues, owing to the lack of known isoforms, is matter of wide research and debate. we focused on a still unexplored pendrin function, that is most important in the inner ear: cellular volume control and cl − fluxes regulation. we used hek cells over-expressing wild type pendrin or a mutated isoform together with the eyfp. we challenged cells with hypo-osmolar solutions and followed their volume variations in time. taking advantage of the confocal optical sectioning we independently measured cell volume and fluorescence intensity. in this way, given the dependence of eyfp fluorescence from [cl − ] and ph (measured with snarf5) we could estimate at the same time cl − fluxes, volume and ph variations. the contemporary measurements of the three variables, not yet reported in living cells, allowed to assess the role of pendrin in volume regulation and evidenced its participation to cl − fluxes as compared to the mutated isoform or controls. particle tracking inside the cell largely benefits of the ability to spatially and temporally mark specific structures to follow their "signalling" over a "dark" background as made possible since the advent of the photo-activatable markers. in terms of spatial confinement of the photo-activation process, the use of multiphoton excitation provides several favourable aspects compared to single photon confocal microscopy in photomarking biological structures to be tracked: the confined excitation volumes, of the order of magnitude of subfemtoliter, due to the non-linear requirements provide a unique control of the excitation and consequently photoactivation in the 3d space. in this context photoactivation experiments can be used to assess quantitative information about the binding kinetics of a macromolecule expressed in different cellular compartments. in this work we extended to photoactivation procedures and models originally developed for the quantitative analysis of frap experiments and we evaluated, for different proteins of medical interest (rac-pagfp), the diffusive behaviour in the cytoplasm and the binding kinetics at the large endosomes. the results are compared with standard photobleaching experiments, in order to evidence the gained sensitivity obtained with photo-activatable proteins. motile plaques involved in stress fiber assembly revealed by high-speed spm for living cells k. tamura, t. mizutani, h. haga, k. kawabata division of biological sciences, graduate school of science, hokkaido university, kita-ku, sapporo, japan stress fibers, which are contractile actin cables aligned in a highly-ordered manner, are important for cell migration, mechanical support of plasma membranes and extracellular matrix organization. although stress fibers are dynamically disassembled and assembled again in cells, it is poorly understood how actin filaments are organized into cables with highly-ordered alignment for stress fiber assembly. for investigation of actin cytoskeletal dynamics during actin cable formation, we performed time-lapse observation of actin cytoskeleton in lamella of living fibroblasts by using scanning probe microscopy (spm). high-speed spm observation revealed that motile plaques defined front-side ends of new actin cables and that preexisting mesh-form actin networks were remodeled into new actin cables. directional order analysis of movement of plaques and pharmacological experiments clarified that plaques were driven by myosin-ii-based retrograde actin flow, indicating that plaques are associated with actin cytoskeleton. immunofluorescence experiments showed that plaques were localized on foci of vinculin, a component of cell-substratum adhesions, suggesting that plaques can bind to extracellular substrata via vinculin. based on these results, we propose a model for actin cable formation that motile plaques initiate remodeling of preexisting actin networks into actin cables aligned in a direction of actin flow by associating with extracellular substrata. abscisic acid (aba) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. recently, aba was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells and by human and murine pancreatic β cells. aba autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin (ptx)-sensitive receptor/g-protein complex, cyclic amp, cd38-produced cyclic adp-ribose and intracellular calcium. here, the aba receptor on human granulocytes and on rat insulinoma cells is identified as the lanthionine synthetase c-like protein lancl2. co-expression of lancl2 and cd38 in the human hela cell line reproduces the aba-signaling pathway. the ptx-sensitive g protein coupled to lancl2 is identified as g i by transfection of cd38 + /lancl2 + hela with a chimeric g protein (gα q/i ). identification of the mammalian aba receptor will enable the screening of synthetic aba antagonists as prospective new anti-inflammatory and anti-diabetic agents. investigation of post-thaw damage of s.cerevisiae yeasts using fluorescent dyes 2-dab and 3-dab t. s. dyubko 1 , i. a. buriak 2 , v. d. zinchenko 2 , i. f. kovalenko 2 1 state scientific institution "institute for single crystals", national acadey of sciences of ukraine, kharkov, ukraine, 2 institute for problems of cryobiology and cryomedicine, national academy of sciences of ukraine, kharkov, ukraine we investigate applicability of the fluorescent probes 2-dab and 3-dab available from seta biomedicals (www.setabiomedicals.com) to study the post-thaw damage of saccharomyces cerevisiae yeast cells. the leaving cells stained with these dyes have bright fluorescence in the yellow and red region, respectively. however, the freezing followed by post-thawing causes cell damage, which results in a change of fluorescence intensity. in the native living cells the dyes are preferably localized in the cell membranes and organelles which are highly fluorescent. partially damaged cells have even brighter fluorescence as compared to the living cells. however, their cell ultra-structure is not well-distinguished in the fluorescence mode. ultimately destroyed cells are almost non-fluorescent: the cell membranes are not visible in the fluorescent mode and their organelles are only weakly fluorescent. the number of intensively fluorescing cells with partially destroyed membranes, which were obtained by freezing to -20 • c, is lower compared to those frozen at -196 • c. 2-dab and 3-dab enable not only to distinguish damaged and undamaged cells, but also allow quantitative estimation of the extent of damage in membranes by cryogenic effects. the thylakoid membrane is a structured network in higher plants which is organised into stacked granal thylakoids that are interconnected by single 'stromal' lamellae. we have studied the mobility of a resident protein of the stromal lamellae, hcf106, part of the tat protein translocase. hcf106-green fluorescent protein fusion (gfp) was targeted into thylakoids and studied using photobleaching approaches. we show that small regions fail to recover significant levels of hcf106-gfp fluorescence within 3 minutes after photobleaching. autofluorescence from the photosystem ii light-harvesting complex (lhcii) in granal stacks likewise fails to recover over this time scale. although the thylakoid membrane is a single continuous entity, these data show that both hcf106-gfp and lhcii are constrained within this network. since the hcf106 homologue, tatb, is highly mobile in the escherichia coli plasma membrane, we believe that the stromal lamellae take the form of distinct domains that are effectively constrained by boundaries within the thylakoid network. cytomechanical modifications induced by drugloaded carriers uptake by breast cancer cells the novel opportunities offered by the nanotechnologies have attracted great interest in the development of novel biomaterials for targeted drug delivery in cancer research. the desired features of pharmaceutical drug delivery for intravenous administration are their small size, biodegradability, high drug content, prolonged circulation in the blood and the ability to target required areas. in this work we have compared efficacy of different type of carriers having complementary properties for pharmaceutical delivery in cancer therapy. drug nano-colloids encapsulated by combination of layer by layer (lbl) techniques and ultrasonication, phytochemical encapsulated artificial oleosomes and drug-loading clay/carbon nanotubes have been used for uptake into breast cancer cells. analysis of viscoelastic response of neoplastic cells induced by cargo-carriers uptake has been carried out by a combination of high resolution optical and scanning force microscopy techniques. furthermore, the effects of drug-reservoir carriers on the cytoskeleton (re)organisation of neoplastic cells were further investigated by confocal microscopy using different fluorescent probes. mapping diffusion by raster image correlation spectroscopy (rics) with analog detection time lapse and flow cytometry data integrated in a common cell cycle proliferation model p. ubezio, v. colombo, m. lupi, f. falcetta istituto di ricerche farmacologiche "mario negri", milano, italy we present a cell cycle simulation tool, connecting the basic proliferation process to the cell population data obtained by time lapse and flow cytometry. the computer program is a general framework within which cell cycle progression can be interactively modeled at the desired level of complexity, including g1/s/g2m cell cycle phases with variable duration, g0 phase, cell subpopulations belonging to different generations or differentiation stages, distinct block and cell death parameters for each phase. in this way, we achieved a detailed rendering of cell proliferation of normal or tumor cell populations, additionally including cytostatic and cytotoxic effects of treatments. the program gives as output simulated measures, reproducing those obtained by absolute cell counting, growth inhibition tests, flow cytometric dna histograms and cell cycle percentages, pulse continuous-labeling studies, time-lapse intermitotic times and generation-wise analyses. each technique provides a piece of information related to the underlying proliferation process, catching only some of the phenomena in play, and the measures often cannot be univocally interpreted. we used the cell cycle simulator to fit together time lapse and flow cytometry time courses measures, to achieve a detailed reconstruction of the cell cycle proliferation of an ovarian cancer cell line in vitro after x-ray exposure. -live cell imaging detection of functional modes in protein dynamics j. s. hub 1 , b. l. de groot 2 1 deptartment of cell & molecular biology, uppsala university, sweden, 2 computational biomolecular dynamics group, max-planck-institute for biophysical chemistry, göttingen, germany proteins frequently accomplish their biological function by collective atomic motions. yet the identification of a collective motions related to a specific protein function from, e.g. a molecular dynamics trajectory, is often non-trivial. here, we propose a novel technique termed`functional mode analysis' that aims to detect the collective motion that is directly related to a particular protein function. based on an ensemble of structures, together with an arbitrary`functional quantity' that quantifies the functional state of the protein, the method detects the collective motion that is maximally correlated to the functional quantity. the functional quantity could, e.g., correspond to a geometric, electrostatic, or chemical observable, or any other variable that is relevant to the function of the protein. two different correlation measures are applied. first, the pearson correlation coefficient that measures linear correlation only; and second, the mutual information that can assess any kind of interdependence. detecting the maximally correlated motion allows one to derive a model for the functional state in terms of a single collective coordinate. the new method is illustrated using various biomolecules, including a polyalanine-helix, t4 lysozyme, trp-cage, and leucine-binding protein. logic estimation of the optimum source neutron energy for bnct of brain tumors boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron-10. a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding conditions on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating 65 different source neutron energies, between 0.01 ev and 13.2 mev. mcnp4b monte carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about 30 kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. the results were discussed. v. l. cruz, j. ramos, j. martinez-salazar instituto de estructura de la materia. csic cannabinoid receptors are an important class of g protein coupled receptors. in particular cb1 and cb2 have received considerable interest because they mediate a variety of physiological responses in the central nervous and immune systems. their tertiary structure is still unknown. experimental information suggests the presence of both, an active and an inactive conformation. cb1 and cb2 share a common structural framework consisting in a seven transmembrane α-helix bundle connected by three extracellular and three intracellular loops. however, the knowledge of structural differences between both receptors may serve to design new ligands to activate/deactivate selectively only one of those receptors. in the present research, we report a multi-nanosecond molecular dynamics simulation of both receptors in solution, starting from a structure obtained by homology modelling using the x-ray determined bovine rhodopsin protein. we look for differences in the behaviour of cb1 and cb2 during the simulation process to shed light about those structural features which can be important for ligand selectivity. in this sense, we observed that cb1 tend to present a more flexible and opened helix bundle than cb2. thus, it is expected than the former receptors would present less steric hindrance for ligand binding. we expect these results will be useful to design more selective ligands. self-assembly and equilibration of bola-lipids membranes studied by molecular dynamics m. bulacu, s. j. marrink groningen university, the netherlands bola-lipids consist of two monopolar, twin-tailed lipids that are held together by chemical linkage between one or both ends of the tails from one lipid and the corresponding ends from the other one. membranes formed by these lipids or by their mixtures with monopolar lipids are known to have additional mechanical stability while retaining membrane fluidity. this is traditionally attributed to the fact that, in the membrane phase, the bola-lipids have predominantly spanning configuration (the two polar heads are positioned at opposite membrane-water interfaces) in detriment to looping configuration (both head groups are located in the same membrane-water interface). we perform molecular dynamics simulations, using the coarse grained martini forcefield. we start with self-assembly simulations of bola-lipids followed by bilayer equilibration. an artificial pore is created in the membrane that significantly increases the flip-flop mobility of the lipids and hasten equilibration. the membrane properties are characterized (area per lipid, thickness, order parameter, pressure profile) with emphasis on the spanning/looping ratio. our study can help designing new artificial membranes, with higher stability under extreme conditions. -multiscale simulation evidence for proton shuffling in a thioredoxinlike protein during catalysis d. narzi 1 , s. w. siu 1 , c. u. stirnimann 3 , j. p. grimshaw 2 , r. glockshuber 2 , g. capitani 4 , r. a. böckmann 1 1 theoretical and computational membrane biology, center for bioinformatics, saarland university, germany, 2 institute of molecular biology and biophysics, eth zürich, switzerland, 3 structural and computational unit, embl, heidelberg, germany, 4 paul scherrer institute, villigen psi, switzerland proteins of the thioredoxin (trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. all members of the trx family with thioldisulfide oxidoreductase activity contain the characteristic cys-x-x-cys motif in their active site. here, using poisson-boltzmann-based protonation-state calculations based on 100-ns molecular dynamics simulations, we investigated the catalytic mechanism of dsbl, the most oxidizing protein known to date. we observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. these results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. a consistent catalytic mechanism for dsbl, probably conferrable to other proteins of the same class, is presented. our results suggest a functional role of hydration entropy of active-site groups . the role of water in zn(ii)-abeta(1-16) complexes beta-amyloid (aβ) peptides are the main component of amyloid fibrils detected in the brain of alzheimer patients. fibrils display an abnormal content of cu and zn ions whose binding to aβ-peptides has been recently studied by x-ray absorption spectroscopy [1] and interpreted in terms of ab initio simulations. in order to perform such simulations it is of the utmost importance to find a compromise between the need of having a realistic description of the actual physical system and the difficulty of dealing with too many atoms and electrons. what is usually done is removing solvent (water molecules), thus studying the system in the so called "gasphase". in this work we investigate the relevance of water in the zn-aβ 1−16 coordination mode. relying on a combination of classical [2] and quantum chemistry [3] methods we find a significant difference in the zn coordination geometry depending on whether water is present or not. this information is exploited in building a full model system for subsequent car-parrinello simulations where two aβ peptides in water are in interaction in the presence of zn. [ although experiments which can directly probe the mechanical properties of proteins have only been performed recently, it is clear that the complex folds of polypeptide backbones lead to very heterogeneous mechanical behavior. this heterogeneity is likely to play an important role in protein function and interaction and it would be useful to be able to predict what mechanical (and dynamical) properties will result from a given structure. we have been using coarse-grain elastic network models to investigate this question and have found that these models are able to link specific mechanical properties to a number of functional features including enzymatic active sites, folding nuclei and changes in behavior due to point mutations. these models are also adapted to looking at complex formation between proteins and how specific recognition is achieved, while being fast enough to be applied to very large numbers of interactions. refinement of protein model structures using biasing potential replica exchange simulations s. kannan, m. zacharias school of engineering and science, jacobs university bremen, d-28759 bremen, germany comparative protein modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. frequently, the quality of the target-template sequence alignment is non-uniform along the sequence: parts can be modeled with a high confidence, whereas other parts differ strongly from the template. in principle, molecular dynamics (md) simulations can be used to refine protein model structures but it is limited by the currently accessible simulation time scales. we have used a recently developed biasing potential replica exchange (bp-rex) md method (kannan, s. zacharias, m. proteins 2007, 66, 697-70) to refine homology modeled protein structure at atomic resolution including explicit solvent. in standard rex-md simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. in a bp-rexmd simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. the method requires much fewer replicas for efficient sampling compared with standard temperature rexmd. bp-rexmd simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional md simulations. m. s. p. sansom, p. j. stansfeld, c. l. wee, k. balali-mood department of biochemistry, university of oxford, u.k. coarse-grained molecular dynamics simulations may be used to probe the interactions of membrane proteins with bilayers and their component lipids on an extended (∼1 µs) timescale (1 ) . conversion to atomistic resolution allows more detailed protein/lipid interactions to be examined. this multiscale approach will be examined via three examples: (i) interactions of a small ion channel toxin (vstx1) we present a new competitive approach for the treatment of biomolecular flexibility to provide an alternative to the limitations of current methodologies such as molecular dynamics and normal mode analysis. this method, called static mode method, is based on the "induced-fit" concept and is aimed at mapping the intrinsic deformations of a biomolecule subject to any external excitations: direct mono or multi-site contact, electrical etc... the algorithm allows obtaining a set of deformations, each one corresponding to a specific interaction on a specific molecular site, in terms of force constants contained in the energy model. such a process can be used to explore the properties of single molecular intrinsic flexibility, as well as to predict molecular docking or molecule/surface interactions. from a modelling point of view, the interaction problem can be expressed in terms of reactive sites between the interacting entities, the molecular deformations being extracted from the pre-calculated static modes of each separated ones. the first applications of our method have focused on the intrinsic flexibility of biomolecules like nucleic acids and proteins. more recently, this new methodology allowed us to investigate the folding of the region 1-16 of the amyloid β-peptide, via the docking of a zinc ion on the reactive sites of the molecule. conformational study on a myelin basic protein fragment: molecular dynamics simulations in membrane e. polverini 1 , g. harauz 2 1 dipartimento di fisica, università di parma, parma, italy, 2 department of molecular and cellular biology, university of guelph, guelph, ontario, canada myelin basic protein (mbp) is a multifunctional protein of the central nervous system whose principal role is in maintaining the compactness and integrity of the myelin sheath, the multilamellar membrane wrapped around nerve axons. however, mbp also interacts with other proteins such as cytoskeletal and signalling proteins, adapting its structure to the different roles. mbp is a candidate autoantigen in the human demyelinating disease multiple sclerosis. this study investigated at atomic detail the conformation of a highly conserved central fragment of mbp, constisting of two consecutive regions with different relevant functionalities. the first one is associated with the membrane and comprises the primary immunodominant epitope in multiple sclerosis; the second one was predicted to be a ligand for sh3-domains of signalling proteins. molecular dynamics simulations were perfomed in the presence of dodecylphosphocholine micelle, starting from a structure extrapolated from experimental data (harauz and libich, curr. protein pept. sci., 2009). the results confirm the experimetal hypothesis, showing, in the micelle, a stable alpha-helix anchored to the membrane for the first region and, for the proline-rich second one, a poly-proline type ii helix pointing outwards, ready to interact with the signalling proteins. multiresolution modelling of drug and hormone permeability through a lipid bilayer traditional atomic-level (al) modelling of biomembranes is time-consuming, and hence limited in the range of systems and phenomena that can be simulated. to alleviate this problem, we designed a coarse-grain (cg) representation where each lipid molecule, in reality consisting of more than 100 atoms, is modelled with only 10 cg sites. our cg technique proves two orders of magnitude less demanding of computational resources than traditional al methodology. a unique feature of our approach is that the cg potentials are directly compatible with standard al models, thus facilitating the simulation of multiresolution systems, where the "chemically-sensitive" components (e.g., the solutes in membrane permeation studies) are modelled atomistically, while the surrounding environment is coarse-grained. in this contribution, we present a summary of our multiscale methodology, together with its application to the permeation of large molecules -drugs and steroid hormones. the calculated permeabilities are compared to the available experimental measurements and al simulation data. molecularlevel insights regarding the permeation mechanism are obtained and rationalised. -multiscale simulation the processes of life involve a variety of events that occur on different scales, ranging from a fewå/ps of the triggering steps of the biochemical reactions, up to their macroscopic effects in cells and organs. intermediate steps involve the structural rearrangement of the bio-molecules (∼nm/10-100ns), their aggregation, folding (∼10nm-µm/ µs-ms), internal cell diffusion and dynamics (µm-mm/ms-hours), evidently requiring a multi-scale modeling approach. here the multi-scale approaches are first briefly illustrated with a particular attention to the issue of matching the different resolutions, which is essential to achieve a coherent descripition. the focus is then fixed on the coarse grained (cg) models, typically spanning the nm-µm and µs-ms scale, and in particular on their minimalist -simplest and computationally cheapest -versions [1, 2] . a parameterization strategy that combines accuracy and predictive power within these models is presented here and applications are shown to relevant cases including the proteins involved in the hiv replication, the green fluorescent proteins and examples of macromolecular complexes. [1] controlled degradation of collagen is an important process in tissue remodeling and wound healing. collagenase cleaves fibrillar collagens about three quarters of the distance from the amino-terminus. even though the determination of the cleavage site and the collagenase structure took place decades ago, the mode of action of collagenase on collagen is not clear. to understand the mechanism of collagenase activity on collagen, the structure, stability and dynamics of collagen, its conformation around the cleavage site and the possibilities of conformational rearrangements between the two domains in collagenase was explored using md simulation. the results of principal component (pca) and normal mode (nm) analysis of the collagen and collagenase suggests that the c-terminal domain of collagenase recognizes the collagen, and then the n-terminal catalytic domain undergoes rearrangement on the substrate (with the help of linker regions). the sda software for carrying out brownian dynamics simulations of protein-protein diffusional association with the help of biochemical constraints is being used to predict the mode of interaction of collagen and collagenase. different conformations obtained from pca and nm analysis are being used for the docking. the predicted structures of the complex will help us to understand how collagenase recognizes and binds collagen specifically. acknowledgment: daad, germany, csir-srf india, and the klaus tschira foundation. membrane aoration by antimicrobial peptides combining atomistic and coarse-grained descriptions d. sengupta, a. j. rzepiela, n. goga, s. j. marrink university of groningen, the netherlands antimicrobial peptides (amps) comprise a large family of peptides that include small cationic peptides, such as magainins, which permeabilize lipid membranes. previous atomistic level simulations of magainin-h2 peptides show that they act by forming toroidal transmembrane pores. however, due to the atomistic level of description, these simulations were necessarily limited to small system sizes and sub-microsecond time scales. here, we study the long-time relaxation properties of these pores by evolving the systems using a coarse-grain (cg) description. the disordered nature and the topology of the atomistic pores are maintained at the cg level. the peptides sample different orientations but at any given time, only a few peptides insert into the pore. key states observed at the cg level are subsequently back-transformed to the atomistic level using a resolutionexchange protocol. the configurations sampled at the cg level are stable in the atomistic simulation. the effect of helicity on pore stability is investigated at the cg level and we find that partial helicity is required to form stable pores. we also show that the current cg scheme can be used to study spontaneous poration by magainin-h2 peptides. over-all, our simulations provide a multi-scale view of a fundamental biophysical membrane process involving a complex interplay between peptides and lipids. the varkud satellite (vs) ribozyme is the largest of the nucleolytic ribozymes, and the only one for which there is no crystal structure. we have determined the overall architecture of the complete vs ribozyme using small-angle x-ray scattering in solution. the substrate stem-loop docks into the tertiary fold of the ribozyme, allowing an intimate loop-loop interaction to occur. this brings two key nucleobases a756 and g638 into close proximity of the scissile phosphate, and we believe that these nucleobases are involved in general acid-base catalysis of the phosphoryl transfer reactions. this is supported by functional group substitution, and the ph dependence of the reaction rate for the natural and modified rna. although possessing totally different folds, the functional elements of the vs and hairpin ribozymes are topologically and mechanistically very similar if not identical. this has probably arisen by convergent evolution. other nucleolytic ribozymes have diversified to employ hydrated metal ions and even small molecules to participate in genera acid-base catalysis. by contrast, the larger ribozymes seem to have adopted a different, metalloenzyme, catalytic strategy. why these different groups have evolved different catalytic chemistries is an interesting challenge to our understanding of biocatalysis. comparison of dna and sirna binding and nuclease protection by non-viral vectors for gene delivery j. lam, k. witt, a. j. mason, l. kudsiova, m. j. lawrence pharmaceutical science division, king's college london, uk the biggest obstacle for the success of gene therapy is delivery. with the discovery of rnai, the use of sirna to regulate gene expression as potential therapy has attracted much attention recently. in contrast to dna, sirna delivery does not require nuclear entry. with one less barrier to overcome, the delivery of sirna seems to be easier. it is frequently assumed that comparable delivery strategy could be used for both dna and sirna. in fact the two types of nucleic acids are fundamentally different with distinct properties, which impact their delivery strategies. in the present study it was found that most non-viral delivery vectors including polymers, peptides and lipids were generally more efficient in binding with dna than sirna as shown in gel retardation assay. the inferior sirna binding is possibly due to the rigid structure of sirna, resulting in weaker electrostatic interaction with the cationic vectors. surprisingly, it was observed that all the vectors studied offered better nuclease protection for sirna than dna despite poorer sirna binding. either the nuclease protection for sirna is easier to achieve due to its small size, or the gel retardation assay did not truly reflect the binding efficiency as the weaker sirna complexes may dissociate during electrophoresis. not only the delivery strategy, the results between dna and sirna study must also be carefully adapted and interpreted. single molecule studies of spliceosomal rnas u2 and u6 z. guo, d. rueda department of chemistry, wayne state university, detroit, michigan, u.s.a. splicing is an essential step in the maturation reaction of mrna, in which intervening introns from exons. the spliceosome is a dynamic assembly of 5 small nuclear rnas and >150 proteins that catalyzes splicing. u2 and u6 are the only 2 snrnas strictly required for splicing. major conformational changes are expected to take place during spliceosomal assembly and catalysis. we have developed a single-molecule fluorescence assay to study the structural dynamics of a protein-free u2-u6 complex from yeast. our previous data have revealed a 2-step large amplitude conformational change of the u2-u6 complex. the 1 st step is a mg 2+ -induced conformational change where helix iii and the u6-isl are in close proximity in low mg 2+ and separated in high mg 2+ . the 2 nd step corresponds to the formation of the highly conserved helix ib. we now examine the role of the highly conserved bases in the folding dynamics of the u2-u6 complex. the data show that u80 and the acagag loop play an important role in stabilizing the interaction between helix iii and the u6-isl. we hypothesize that u80 flips out of the u6-isl and interacts with the acagag loop to bring them in close proximity. our results raise the interesting possibility that this interaction plays an important role in bringing the 5' splice site and the branched a into close proximity of u80, which may be critical for catalysis. a structure-based approach for targeting hiv-1 genomic rna dimerization initiation site s. freisz, s. bernacchi, p. dumas, e. ennifar ibmc -cnrs, strasbourg, france all retroviral genomes consist in two homologous single stranded rnas. hiv-1 dimerization initiation site (dis) is a conserved hairpin in the 5' non-coding region of the genomic rna and essential for viral infectivity. the dis initiates genome dimerization by forming a kissing-loop complex, further stabilized into an extended duplex upon interaction with the ncp7 nucleocapsid protein. x-ray structures of the dis kissing-loop and extended duplex revealed similarities with the bacterial 16s ribosomal rna a-site, which is the target of aminoglycoside antibiotics. as a result, aminoglycosides also bind the hiv-1 dis as shown by our x-ray structures of the dis kissingloop bound to aminoglycosides. using fluorescence, uvspectroscopy and microcalorimetry, we further characterized hiv-1 dis/aminoglycosides interactions. we found that the affinity of aminoglycosides for the dis was higher than for their natural target, the 16s a site. they strongly stabilize the dis kissing-loop, blocking its conversion into the duplex form. finally, we also solved x-ray structures of the dis duplex bound to aminoglycosides, revealing an important conformational change following drug binding. these structures show that it is possible to target the hiv-1 dis dimer before and after the ncp7-assisted rna maturation with the same molecule. altogether, our studies create the basis for a rationally driven design of novel potential drugs targeting the hiv-1 genome. the core protein of hepatitis c virus is a multifunctional protein of 179 aa, consisting of a hydrophilic n-terminal domain with three basic domains (bd1-bd3) responsible for the interactions with rna and a hydrophobic c-terminal domain. the n-terminal domain exhibits nucleic acid chaperone properties similar to those of the nucleocapsid protein from hiv. here, we characterized the mechanism of the chaperone properties of a peptide e corresponding to the bd2 and bd3 clusters of the n-terminal domain. to this end, we monitored the promotion by this peptide of the annealing of dtar, the dna analogue of the transactivation response element to its complementary sequence, ctar dna, taken as models. the annealing involves two second-order kinetic components that are activated by at least three orders of magnitude by peptide e. this activation was correlated with the ability of peptide e to destabilize the lower half of dtar stem. using, ctar and dtar mutants, the two kinetic components were assigned to two pathways which are connected with the fast annealing of the terminal bases of ctar to dtar and slow extended duplex formation, limited kinetically by the nucleation of central segments of ctar and dtar stems. structure and conformational dynamics of a unique dead box helicase m. rudolph 1 , m. linden 2 , r. hartmann 3 , d. klostermeier 2 1 hoffmann-la roche, basel, switzerland, 2 biozentrum, univ. of basel, switzerland, 3 univ. of marburg, germany dead box helicases couple atp hydrolysis to rna structural rearrangements. t. thermophilus hera consists of a helicase core and a c-terminal extension (cte) with a putative rnase p motif. crystal structures show that the cte is bipartite, forming a highly flexible dimerization motif with a novel fold and an additional rna-binding module. we provide a first glimpse on the orientation of an rna-binding domain outside the helicase core. in a structure-based model for the complete hera dimer, the rna-binding sites of the helicase cores face each other, allowing for inter-subunit communication. the plasticity of the dimerization motif allows for drastic changes in the juxtaposition of the helicase cores within the dimer. in single molecule fret experiments we identified fragments of the 23s rrna and rnase p rna as substrates for hera. both substrates switch the hera core to the closed conformation and stimulate the intrinsic atpase activity. rna binding is mediated by the cte, but does not require the putative rnase p motif. atp-dependent unwinding of a short helix in 23s rrna suggests a specific role for hera in ribosome assembly, in analogy to the e. coli and b. subtilis helicases dbpa and yxin. in addition, the specificity of hera for rnase p rna may be required for rnase p rna folding or rnase p assembly. simultaneous action of two hera subunits on the same rna molecule may be important for efficient rna remodeling in vivo. structural basis for the encapsidation process of turnip yellow mosaic virus m. petersen 1 , j. hansen 1 , s. s. wijmenga 2 1 nucleic acid center, department of physics and chemistry, university of southern denmark, odense, denmark, 2 physical chemistry/biophysical chemistry, radboud university, nijmegen, the netherlands formation of hairpins with internal loops with c c and c a mismatches in the 5' untranslated region is a common characteristic among the plant viruses belonging to the tymovirus genus. turnip yellow mosaic virus possesses two such hairpins, hp1 and hp2. hp1, and in particular the presence of the c c and c a mismatches in its internal loop, is important for initiation of the encapsidation of the viral genome. the encapsidation occurs under acidic conditions at the neck of invaginations of the chloroplast membrane. we have now determined the 3d structures of revertants involved in the evolutionary pathway using nmr spectroscopy. these structures reveal how the grooves in the hairpin become increasingly positively charged in successive generations of evolution. the similarity between the ccca mutant and the wild-type hairpin (hp1) is striking and explains why this mutant gives rise to a viable virus. in addition, a characteristic tilt of the backbone is observed upon occurrence of a protonated c c base pair. both the positively charged major groove and the kink in the rna backbone appear to be crucial for interactions with the viral capsid. at neutral ph, the structure of hp1 resembles the watson-crick base paired mutant which explains why encapsidation only occurs under acidic conditions. a. percot 1 , j. vergne 2 , m.-c. maurel 2 , s. lecomte 3 1 ladir, umr7075, 94320 thiais, france, 2 lbeam, upmc, 75005 paris, france, 3 cbmn, umr5248, 33607 pessac, france the existence of "rna world" as an early step in the history of life increases the interest for the characterization of these biomolecules. the studied hairpin ribozyme is a self-cleaving/ligating motif found in the minus strand of the satellite rna associated with tobacco ringspot virus. surface-enhanced raman spectroscopy (sers) was successfully used to detect sub-picomole quantities of nucleic acids. sers takes advantage of the strongly increased raman signals generated by local field enhancement near metallic (typically au and ag) nanostructures. sers spectra of dna or rna are strongly dominated by stockes modes of adenine. through an interaction of adenyl residues with silver colloid, adenyl raman signal is 10 6 times increased compared to raman scattering. in controlled conditions, sers signal is proportional to the amount of free residues adsorbed on the metal surface. upon rna cleavage, residues are unpaired and free to interact with metal. in the present study, we proposed to follow the cleavage reaction of hairpin ribozyme (hpr85) using the sers signal of the liberate adenyl residues. as the sers signal is proportional to the adenyl residues, reactivity of hr85 was monitored by measuring the raman intensity of the fragment liberated during the cleavage of hairpin. the results obtained were compared with the electrophoresis method performed on the same sample and similar results were obtained. -rna world eur biophys j (2009) the development of arrays for biomolecular recognition for a broad range of applications in biomedical diagnostics is receiving a constantly increasing attention. the design of efficient protein biochips, however, requires the optimization of protein immobilization protocols for improving the device sensitivity. moreover, innovative platforms for in-vitro detection in highly diluted, small volume samples need to be developed, for which standard micro-fabrication techniques are not suitable. therefore, the development of nano-scale platforms for protein and antibody detection is essential. we report here on a novel approach for the fabrication of multiple protein nanosensors using atomic force microscopy based nanografting and dna-directed immobilization (ddi), which takes advantage of the specific watson-crick hybridization of oligonucleotide-modified proteins to surfacebound complementary oligomers. using nanografting, single-stranded dna nano-structures with well defined local environments were fabricated on a flat surface. dna-protein conjugates were then anchored on the engineered binding sites by ddi in a single chemical operation and detected by the corresponding topographic height increase of the relevant patches. immunological assay were used to demonstrate the biochemical functionality of the immobilized proteins, proving the specificity of biomolecular recognition of our nanodevices, in the micro-molar to pico-molar concentration range. dna accessible surface area and indirect protein-dna recognition: study by bioinformatical approach o. p. boryskina 1 , m. y. tkachenko 1 , a. v. shestopalova 1 , m. y. tolstorukov 2 1 institute of radiophysics and electronics nas of ukraine, 2 harvard-partners center for genetics and genomics, usa revealing the mechanisms of protein-dna recognition is essential for understanding the regulation of many cellular processes. there is growing evidence that recognition through sequence-specific contacts (direct readout) can be enhanced by recognition via dna sequence-dependent deformability (indirect readout). the role of changes in dna accessible surface area (asa) in distorted dna configurations in complexes with proteins is not fully understood yet, even though such changes and related changes in polarity of dna surface are among key factors of indirect readout. to fill this gap we have developed a publicly-available internet database of protein-dna complexes, which integrates the data on dna conformational parameters with information on asa of dna atoms in the minor and major grooves, protna-asa. the database has been used to analyze the effect of changes in dna backbone configuration on asa of dna atoms in major and minor grooves. we observe that sugar puckering and conformation of torsion angle γ affect the accessibility of dna atoms in both grooves to a noticeable extent. we also report sequence specific preferences of the nucleotides for structural domains of γ. these results can shed new light on the mechanisms of indirect protein-dna recognition. a. arakelyan biology dpt., yerevan state university, yerevan, armenia the influence of ligand, irreversibly binding with dna, on the isotherm of adsorption of ligand binding with dna reversibly has been modeled theoretically. the isotherm of adsorption of etbr on dna in presence of cis-ddpt has been considered at low concentrations of cis-ddpt and etbr.. the isotherm of adsorption of etbr on dna has linear form in scatchard coordinates at low degrees of occupation. the comparison with experimental isotherm permits to estimate the parameters of etbr binding with dna. with taking into account the pseudo-ring structures formation with partially molten regions we consider two types of binding regions, linear and ring at low degrees of occupation. the isotherm of adsorption of etbr on dna and also variation of the number of bounded etbr molecules are calculated with taking into account these two types of binding regions. it was shown that at low concentrations cis-ddp, bounding with dna, changes the isotherm of ligands adsorption. the linear in scathcard coordinates isotherm of adsorption transforms into non-linear isotherm. the degree of transformation depends on the fraction of dna in the ring regions, on the ratio between number of etbr binding cites in the ring and linear regions, and also on the binding constants for these regions. it was shown that low concentrations of cis-ddp affect the dependence of dispersion on the concentration of ligands, changing both the magnitude of maximum and its position. tunable nanoconfinement structures for dna manipulation e. angeli, l. repetto, g. firpo, c. boragno, u. valbusa nanomed labs, advanced biotechnology center and physics department, university of genoa, italy nanostructures, such as nanochannels [1] or nanoslits [2] , have been successfully used to confine and stretch dna molecules, offering interesting opportunities of investigation on conformational changes induced by confinement, physical and biological properties, etc. the integration of these nanostructures on lab-on-chip systems has shown their great potential for applications such as dna sieving or single molecule manipulation [3] , [4] . arrays of nanochannels fabricated, using a focused ion beam (fib) on a silicon master, are replicated using elastomeric materials, such as poly(dimethylsiloxane) (pdms), and soft-lithography techniques. the cross-section of these flexible polymeric nanoconfinement structures can be reversibly and dynamically tuned, in order to vary biomolecules transport characteristics and confinement conditions of trapped dna molecules. moreover, these nanochannels, with tunable cross-section, are used to study and exploit the sieving mechanism of "entropic recoil" [5] for the separation of long dna chains. [1] mannion jt, et al., (2006 ) biophys. j., 90, 12:4538-4545. [2] jo k, et al., (2007 ) pnas, 104, 8: 2673 -2678 . [3] fu j, et al., (2007 ) nat. nanotechnol., 2: 121-128 [4] huh d, et al., (2007 nat. mater., 6: 424-428. the nucleocapsid protein ncp7 of hiv-1 is characterized by two conserved zinc fingers and plays crucial roles in the virus, through its binding to nucleic acids. ncp7 is required for efficient proviral dna synthesis, by promoting the initiation of reverse transcription and the two obligatory strand transfers. using fluorescence techniques as well as fcs and nmr, we investigated the chaperone properties of ncp7 on the primer binding sites (pbs) and transactivation response elements (tar) sequences involved in the two obligatory strand transfers. we showed that ncp7 binds mainly to the (-)pbs loop, which results in an extension of the loop and a destabilization of the upper base pair of the stem. these structural changes chaperone a kissing complex with the complementary (+)pbs loop and its further conversion into an extended duplex. in contrast, the ncp7-promoted annealing of ctar-tar results from the destabilization of the bottom of ctar stem, which favors the invasion of the tar stem. by developing new fluorescence methodologies to site-specifically characterize these interactions, we further showed that ncp7 slows down the ps to ns conformational fluctuations of its nucleic acid targets and freezes the local mobility of the bases contacted by the zinc fingers. exploring dna orientation in flow e. l. gilroy, m. r. hicks, a. rodger department of chemistry, university of warwick, uk dna is one of the most important biomolecules. in order to undertake its biological role the dna needs to fold and unfold for which its structure and flexibility are crucially important. we have studied the characteristics of dna in flow to probe its structure. flow aligned linear dichroism (ld) is a technique that uses light polarised parallel and perpendicular to an orientated sample. it can be used to measure how aligned a sample is, and the orientation of any interacting molecules. to create this alignment, the sample is placed between two concentric cylinders where one is spun to create a shear flow. the longer and more rigid the molecule is the better the orientation and the signal. as the sample is in flow there are other factors than need to be considered when using ld. these include viscosity and temperature. there are many methods to measure the viscosity of a sample of dna at varying temperatures. these include viscometer, rheometer and dynamic light scattering. the effects temperature has on viscosity and the sample itself need also to be considered. the findings of all these studies have been reported and show the significance that viscosity and temperature have on dna ld measurements. possible applications of using ld to study dna have also been discussed, showing the importance of the use of ld and in the results shown. a.-m. florescu, m. joyeux laboratoire de spectrométrie physique, université joseph fourier grenoble 1, france we present a dynamical model for simulating non-specific dna protein interactions, which is based on the "beadspring" model for dna with elastic, bending and debye-hückel electrostatic interactions, and where the protein interacts with the dna chain through electrostatic and excluded-volume forces. we study the properties of this model using a brownian dynamics algorithm that takes hydrodynamic interactions into account and obtain results that partially agree with experiments and predictions of kinetic models. for example, we show that the protein samples dna by a combination of three-dimensional diffusion in the solvent and one-dimensional sliding along the dna chain. this model evidences the presence, in a certain range of values of the effective protein charge, of facilitated diffusion, i.e. a combination of the two types of diffusion that leads to faster than 3-dimensional diffusion sampling of dna. moreover, the analysis of single sliding events shows that the number of base pairs visited during sliding is comparable to those deduced from single molecule experiments. in contrast to kinetic models, which predict an increase of the number of different base pairs visited by the protein proportional to the square root of time, our model however suggests that this number increases linearly with time until it reaches a value that is close to the total number of dna base pairs in the cell (published in j. chem. phys. 130, 015103 (2009)). use of md simulation to identify the critical radiation-induced lesions of a dna binding protein n. chalabi, s. goffinont, n. garnier, d. genest, 45071 orléans cedex 2, france a key step in the regulation of gene expression, dna structuring and dna repair is the binding of some proteins to specific dna sequences. we have shown previously that ionizing radiation destabilizes such dna-protein complexes mainly through damage to the protein. for the typical lactose operator -repressor system we have shown by fluorescence measurement and mass spectrometry that upon irradiation, all the four tyrosine residues of the dna binding domain (called headpiece) are oxidized into 3,4dihydroxyphenylalanine (dopa). a circular dichroism study revealed a global conformational change and the destabilization of the headpiece. to decide which lesion is critical for the induction of these effects, a molecular dynamics simulation study was undertaken in parallel with a site-directed mutagenesis one. each tyrosine residue of the headpiece was replaced by another amino-acid that mimics the damaged tyrosine. the most common amino-acid used in site-directed mutagenesis being alanine, we have replaced one tyrosine (7, 12, 17 or 47) in the nmr-based structure of the headpiece from pdb databank (1lqc) by an alanine. the conformational stability of each tyr→ala mutant has been studied by molecular dynamics simulation (md) and compared to that of the native sequence and of the different tyr→dopa mutants. the mammalian high mobility group protein at-hook 2 (hmga2) is a nuclear protein associated with mensenchymal cell development and differentiation. disruption of its normal expression pattern is directly linked to oncogenesis and obesity. our laboratory has utilized a variety of biochemical and biophysical methods to investigate the molecular mechanisms of hmga2 recognizing at-rich dna. using a pcr-based selex procedure, we discovered that hmga2 is a sequence-specific dna-binding protein and recognizes the following two sequences: 5'-atattcgcgawwatt-3' and 5'atattgcgcawwatt-3', where w is a or t. using a double-label emsa assay, we found that hmga2 binds to at-rich dna as a monomer. using isothermal-titrationcalorimetry, we demonstrated that hmga2 binds to at-rich dna with very high binding affinity whereby the binding of hmga2 to a-tracts is entropy-driven and to alternate at sequences is enthalpy-driven. this is a typical example of enthalpy-entropy compensation in which the hydration difference between hmga2-dna complexes is a main reason for the compensation. interestingly, the binding of hmga2 to different at-rich sequences is accompanied with a large negative heat capacity change indicating an important role of solvent displacement and charge-charge interaction in the linked folding/binding processes. partition of gibb´s free energy of drug-dna complexation we report a computation methodology, which leads to the ability to partition the gibb's free energy for the complexation reaction of aromatic drug molecules with dna. using this approach it is now possible to calculate the absolute values of the energy contributions of various physical factors to the dna binding process, whose summation gives a value that is reasonably close to the experimentallymeasured gibb's free energy of binding. application of the methodology to binding of various aromatic drugs with dna provides an answer to the question 'what forces are the main contributors to the stabilization of aromatic ligand-dna complexes' ? we report the effects of 22 khz ultrasound irradiation of double-stranded dna solutions under conditions of transient cavitation. a new method was developed for studying these effects which is based on combination of two procedures: ultrasound irradiation of the solutions of double-stranded dna fragments and subsequent analysis of the high resolution denaturing gel electrophoresis data. statistical treatment of the data on the mobility of 3'-end-labelled restriction fragments with known sequence allowed us to discover the phenomenon of sequence specific ultrasonic cleavage of dna. our analysis results in the following conclusions: 1) all steps with 5'-ward cytosine [5'-d(cpn)-3'] have significantly higher cleavage rates than others; the intensity of cleavage diminishes in the order cpg > cpa ≈ cpt > cpc; 2) the cleavage rates of all 16 steps depend on the type of flanking base pairs; 3) the cleavage rates of the complementary base pair steps are not identical. thus, subtle sequence specific conformational and physical-chemical variations modulate the reaction of sugar-phosphate single bonds on the ultrasound exposure. a theory of the mechanisms on the simultaneous binding of two aromatic drugs to dna it has long been recognized that certain combinations of dna-binding aromatic drugs, x +y, lead to synergistic biological effects. considering x as a basic ligand and y as an added ligand, the change of the integral biological response of x in the presence of y has been interpreted in terms of two mechanisms: the interceptor and protector action of y on x. this mechanisms have been characterized by two criteria, r d and a d , reflecting the removal of x from dna by y (biophys. chem., 2008, vol.132, pages 148-158 ) . in this work we develop the theory of the interceptor-protector action of a mixture of two biologically-active dna-binding aromatic drugs. the theory is based on solution of a general system of mass balance equations in the three-component system x -y -dna with respect to the two factors, r d and a d . the outcome is a set of expressions enabling estimation of the change in biological response of x on addition of y as a function of equilibrium parameters under different restrictions. the results are in good agreement with known in vitro data on caffeine+antibiotic action in leukemia cell lines. the influence of mn 2+ ions on the structure of natural calf thymus dna was studied by raman spectroscopy. measurements were done at room temperature and ph 6.2 ± 0.2, in the presence of the physiological concentration of 150 mm na + ions, and in the presence of mn 2+ concentrations that varied between 0 and 600 mm. no condensation of dna was observed. dna backbone conformational changes were not detected in the whole concentration range of mn 2+ ions as judging from the raman spectra. no evidence for dna melting was identified. binding of manganese(ii) ions to the charged phosphate groups of dna, stabilizing the double helical structure, is indicated in the spectra. as judged from the marker band of dc near 785 cm −1 , altered nucleoside conformations in dc residues are supposed to occur, in the mn 2+ concentration range of 400-600 mm. binding of divalent ions to n7 of guanine and, possibly, in a lesser extent to adenine was observed as judging from the raman marker bands at 1336, 1376, 1490 and 1578 cm −1 . toward rapid dna sequencing -ab initio study of nucleotide sandwiched between au(111) plates c. morari, d. bogdan, i. turcu national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania recently a new technique for dna sequencing based on measurement of transversal conductive properties of a single strained dna molecule has been proposed. such a method would allow single-base resolution by measuring the electrical current perpendicular to the dna backbone. until now, it is still not clear if the electrical signals obtained for the four nucleotide can be clearly distinguished by a hypothetical experimental setup. several factors -like the influence of the pentose group or the presence of water -may influence quite strongly the electrical signatures of the four bases. therefore, in order to obtain a working device, the understanding and detailed description of the conduction mechanisms through dna bases connected to a metallic electrode is essential. the goal of theoretical studies in this field is to describe the electric signatures of dna bases from a theoretical point of view. our study is focused on the detailed description of the electronic structure of dna's base pairs "squeezed" between two au plates. while such a geometrical model closely mimic the sequencing devices proposed in the literature, it allows us to compute meaningful physical properties such as density of states, charge transfer and orbital localizationby using "ab initio" methods. the results allow us to give qualitative prediction over the potential use of such a device in the dna's sequencing technology. multinuclear platinum complexes represent a new class of anticancer agents, distinct in dna binding and antitumor activity from their mononuclear counterparts. bbr3464 as a first representative of this class has undergone phase ii clinical trials for treatment of ovarian and lung cancers. the structure of this trinuclear pt drug consists of two trans-ptcl(nh 3 ) 2 units bridged by a transthe main lesions formed by multinuclear pt complexes in dna are long-range intra-and interstrand cross-links (cls) bridging two guanines separated by up to four base pairs. since interstrand cls can prevent dna strand separation interfering with critical cellular events they represent a serious obstacle in cell survival. in order to contribute to understanding the biological effects of dna interstrand cls of bbr3464, we analyzed the effect of the single, site-specific 1,4-interstrand cl formed by this metallodrug between two guanine bases on opposite strands in the 3'-3' and 5'-5' direction on the thermal stability and energetics of short dna duplexes. the results demonstrate that 1,4-interstrand cls of bbr3464 in both 3'-3' and 5'-5' directions exist as two distinct conformers that are not interconvertible and affect thermodynamic stability of the dna differently. side-by-side and end-to-end attraction of doublestranded dna c. maffeo 1 , b. luan 2 , a. aksimentiev 1 1 university of illinois at urbana-champaign, urbana, usa, 2 ibm watson research center, yorktown heights, usa genomic dna is densely packed inside cell nuclei and viral capsids. such close packing suggests that electrostatic repulsion between negatively charged dna in the condensed states is balanced by counterion-induced attraction. several theoretical models have been proposed to explain dna attraction, however, specific microscopic mechanisms are not known. here, we report all-atom molecular dynamics simulations of the effective force between double-stranded dna in side-by-side and end-to-end orientations. in the side-by-side orientation, the dna molecules were found to form a bond state in the presence of magnesium ions. in the bond state, the dna molecules contact each other via their negatively charged phosphate groups, bridged by magnesium ions. the maximum attractive force in the side-by-side orientation is about 40 pn per dna turn. in the end-to-end orientation, a strong ( > 60 pn) attractive force was observed at short (< 0.8 nm) end-to-end distances regardless of the electrolyte concentration. the presence of a phosphate group at the 5'ends of the fragments was found to direct dna end-to-end self-assembly and produce bound states resembling a continuous dna molecule. the computed potentials of the mean force suggest that the end-to-end attraction, rather than being mediated by counterions, is likely caused by hydrophobic and van der waals interactions between terminal nucleobases of the fragments. doxorubicin (dox) is an anticancer antibiotics with a four-membered ring system containing an anthraquinone chromophore, and an aminoglycoside. it has good anticancer activity against a wide spectrum of tumors and is one of the most extensively used antitumor chemotherapeutic compounds currently in clinical use. interestingly, conversion of dox to 2pyrrolinodoxorubicin analog (p-dox) exhibits 500-1000 times higher toxic effects in human breast cancer cell line (nagy, a. et al., pnas, 93 (1996 ) 2464 . molecular mechanisms responsible for this enormously enhanced cytotoxicity have not been entirely clarified. there is good evidence that a key component of the mechanism of action of dox is its intercalation into dna and the formation of dox-dna adducts. therefore, we have examined in detail, using the methods of molecular biophysics, dna interactions of p-dox in cell-free media and compared these results with the same studies focused on the parental dox. we find distinct differences between dna interactions of dox and p-dox and suggest that these differences are responsible at least in part for different biological effects of these two anticancer drugs. design of a microfluidic devices for the detection of oligonucléotides by sers designing fast and efficient analytical tools allowing the detection of free dna or rna at very low concentration within small volumes, without specific molecular labeling, remains a major issue of significance to perform diagnostic or to detect pathogen agents. our strategy is to use surface-enhanced raman scattering (sers) to probe selectively the spectral signature of each base in polynucleotides. sers takes advantage of the strongly increased raman signals of species when adsorbed on adapted silver colloids. we already demonstrated that adenyl raman signal of pa in presence of silver colloids is 10 6 times enhanced compared to bulk signal. we plan to use nanoliter droplets of uniformed size that form spontaneously in microchannels when two immiscible fluid streams merge. these tiny droplets are almost ideal reactors as they create homogeneous control condition. we will design an optimized channels network platform that result in droplets production hosting both nucleotides and silver colloids: internal fluids recirculation provide fast and efficient mixing, favoring base adsorption on silver nanoparticles. sers will be used to determine the chemical composition of the droplets. nicks represent the most common damage in dna which occurs naturally in living cells. structural properties of nicked dna fragments have been an object of numerous studies due to its special role in reparation processes. here we report experimental results covering ultrasound irradiation of nicked dna solutions. several single-stranded nicks were produced into one strand of dsdna fragments by the nicking enzyme bst9i. we have quantitatively estimated the ultrasonic cleavage rates in nicked dna fragments with known sequences using the polyacrylamide gel electrophoresis. computer analysis of the cleavage pattern in the 3'-end labeled and primarily intact strand reveal cleavage enhancement in the regions of about 10 b. p. up and down the nicks which were initially produced into complementary strand. the intensity of cleavage near the nicks is (in average) about 20 times higher than cleavage in the same sites of the intact dsdna fragments. at the same time, the cleavage rates in positions beyond the regions of the nick markedly grow weak even comparing to the sequence-specific cleavage of intact double-stranded dna fragments. thus, the presence of the nick serves as an expressive structural indignation, which exceeds modulation of the structure caused by the base-pair sequence and is capable of absorbing mechanical stresses applied to the nearby sites of the molecule. comparing the native and an irradiated lactose repressor-operateur complex by md simulation g. naudin, n. garnier, d. genest, rue charles sadron, 45071 orléans cedex 02, france the function of the e. coli lactose operon requires the binding of a protein, the tetrameric repressor, to a specific dna sequence, the operator. the formation of this complex involves the interaction of at least one protein dimer with the operator sequence. this occurs via the dna-binding domains (called headpieces) of the two constitutive monomers. we have previously shown that upon irradiation with gamma rays the complex is destabilised mainly because the repressor losses its dna binding ability. radiation-induced lesions were identified that may be responsible for this deleterious effect: all tyrosine residues of the headpieces are oxidized into 3,4-dihydroxyphenylalanine (dopa). in order to unravel the mechanisms leading to the observed destabilization of the operator-repressor complex, we compare by md simulations two complexes: 1-the native complex formed by a dimer of two headpieces and a fragment of dna with the operator sequence and 2-the damaged complex in which all tyrosines are replaced by dopa. analysis of these trajectories shows a loss of stability and binding energy as well as changes in the structure of the damaged complex with respect to the native one. by comparing precisely the evolution of the two complexes we can explain how the oxidation of the tyrosine residues of the headpieces into dopa may trigger the destabilization of the complex. local conformation of confined dna studied using emission polarization anisotropy in nanochannels with dimensions smaller than the dna radius of gyration, dna will extend along the channel. we investigate long dna confined in nanochannels with dimensions down to 50*50 nm, using fluorescence microscopy. studies of the statics and dynamics of the dna extension or position in such confinements as a function of e.g. dna contour length, degree and shape of confinement as well as ionic strength has yielded new insight into the physical properties of dna with relevance for applications in genomics and fundamental understanding of dna packaging in e.g. viruses. our work extends the field by not only studying the location of the emitting dyes along a confined dna molecule but also monitoring the polarization of the emission. we use intercalating dyes whose emission is polarized perpendicular to the dna extension axis, and by measuring the emission polarized parallel and perpendicular to the extension axis of the stretched dna, information on the local spatial distribution of the dna backbone can be obtained. we will discuss results in shallow (60 nm) and deep (180 nm) channels and describe how the technique can be used to investigate non uniform stretching of a single dna molecule. raman spectroscopy of dna modified by new antitumor nonclassical platinum complexes o. vrana, v. kohoutkova, v. brabec institute of biophysics as cr, královopolská 135, cz-61265 brno, czech republic platinum anticancer agents (cisplatin, carboplatin, oxaliplatin) are in widespread clinical use especially against testicular, ovarian and head and neck carcinomas. there is a large body of experimental evidence that dna is the critical target for the cytostatic activity of cisplatin. platinum complexes form several types of adducts, which occur in dna with a different frequency and differently distort the conformation of dna. clinically ineffective trans isomer of cisplatin (transplatin) also covalently binds to dna bases. the trans geometry in dichloridoplatinum(ii) complexes was activated by various ways. the replacement of at least one amine ligand by planar amine ligand represents example of such activation. raman spectroscopy is powerful technique for examining both structural and thermodynamic properties of nucleic acids in solution. interactions of cis-and trans-pt(ii) complexes having nonplanar heterocyclic amine ligand such as piperidine, piperazine and 4-picoline with dna have been investigated by laser raman spectroscopy. raman difference spectra reveal that the pt(ii) complexes induce great structural changes in b-dna and indicate disordering of b-dna backbone, reduction in base stacking and base pairing and specific metal interaction with acceptor sites on purine residues. acknowledgement: this work was supported by grant as cr, iqs500040581. a. v. vargiu 1 , a. magistrato 2 , p. carloni 3 , p. ruggerone 1 1 cnr-infm-slacs and physics dept., university of cagliari, cagliari, italy, 2 cnr-infm-democritos and sissa/isas, trieste, italy, 3 iit and sissa/isas, trieste, italy the minor groove of dna is the target of several anticancer agents, which interfere with replication and translocation processes, leading to cell death. the molecular recognition event is a key step to achieve detailed knowledge of the interactions behind selectivity and affinity of a ligand towards a particular nucleic acids sequence. recognition is a multiroute process which can involve many steps before the formation of the most stable adduct. in particular, many studies have pointed out the importance of events like sliding along the groove and dissociation (which is a relevant step in the translocation among different sequences) for the affinity and the specificity of minor groove binders. despite this, no studies on the dynamics of this event were reported in the literature. in this contribution i present our recent work on the subject. umbrella sampling and metadynamics were used in the framework of classical md to characterize mechanisms andfree energy profiles of molecular recognition routes by the antitumoral agents anthramycin, duocarmycin and distamycin. our results are in very good agreement with the available experimental data, and provide insights on the influence of factors like size, charge and flexibility on the molecular recognition process. amplification of oligonucleotide sequence recognition using bioresponsive hydrogels s. tierney, b. t. stokke biophysics and medical technology, dept. of physics, the norwegian university of science and technology, ntnu, no-7491 trondheim, norway we describe development and characterization of oligonucleotide functionalized hydrogels for amplifying the molecular recognition signal occurring on hybridization between dioligonucleotides. the 50 µm radius hemispherical hydrogels were integrated on a high resolution interferometric fiberoptic readout platform supporting determination changes in the optical length of the hydrogel with 2 nanometer resolution. the hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network as network junctions in addition to the covalent crosslinks or oligonucleotides grafted to the network chains. the probe oligonucleotide destabilizing the junction point by displacement hybridization yielded an optical signal about five times as large as for binding within the hydrogel design with a comblike grafted oligonucleotide. the optical signal was found to be dependent on the concentration of the probe, the sequence and matching length between the probe and sensing oligonucleotide. concentration sensitivity applied as specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. the current design support detection in excess of 1x10 12 sequences. amplification of the molecular recognition employing the developed oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequence designed as aptamers for determination of other types of molecules are discussed. tracing t-cells by paramagentic nanoparticles in the brain of the rat model of als d. bataveljic 1 , g. vanhoute 2 , g. bacic 3 , p. andjus 1 1 inst. for physiology and biochemistry, univ. of belgrade, serbia, 2 bio imaging lab, univ. of antwerpen, belgium, 3 school of physical chemistry, univ. of belgrade, serbia amyotrophic lateral sclerosis (als) is a devastating neurological disorder affecting upper and lower motoneurons. since immune disbalance is known to be an important manifestation of the disease we were particularly interested in following the labeled immune cells in the familial als rat model, hsod-1 g93a . a t2-or t1-weighted mri protocol was used with a mini surface coil placed over the skull of the anesthetized animal in a 1.5 t wide bore magnet. in order to compare this approach to standard high field small animal imaging a 7.0 t mri system was also used. there was a congruence of images of lesions in the brainstem at the two field strengths. it was confirmed with gd-dtpa contrast that the blood brain barrier (bbb) is compromised at the interbrain level. in order to study immune cell infiltration rats were i.v. injected with magnetically labeled antibodies against helper cd4+ or cytolytic cd8+ killer t cells. by combined t1, t2 and t2* weighted imaging cd4+ lymphocyte infiltration was observed in the brainstem-midbrain region while the cd8+ cells were more confined to the brainstem region. comparison of mri of labelled cd4+ vs. cd8+ lymphocytes reveals the relevant cellular inflammatory mechanism in als. the appearance of the mri signal from the latter t cell type points to the mechanism of bbb disruption as suggested from a recent study on the role of cd8+ t cells in a model of multiple sclerosis. emodin uptake study in u-87mg cells using optically trapped surface-enhanced raman scattering probes s. balint 1 , s. rao 1 , p. miskovsky 2 , d. petrov 1 1 icfo -the institute of photonic sciences, barcelona, spain, 2 department of biophysics, university of p.j.šafárik, košice, slovakia emodin (1,3,8-trihydroxy-6-methylantraquinone) is a photosensitizing pigment present in plants of herbal laxatives. emodin inhibits nuclear transcription factor-κb activation and induces free radical production in human mononuclear cells resulting in its antiviral and anti-cancer activity. the uptake and distribution of emodin inside the u-87 mg cell line is studied by combining optical tweezers and surfaceenhanced raman spectroscopy (sers). sers greatly enhances the spectrum of an otherwise weakly scattering material which is achieved by attaching nano-sized silver colloids to micron-sized dielectric beads. the distribution of emodin in the cell is studied by simultaneously trapping and exciting the sers bead and scanning it across the membrane while recording the emitted light. secondly, the beads are statically placed inside the cell and excited at certain intervals in order to track the migration of emodin through the membrane. the results give new insight in to the metabolic pathways of emodin and demonstrate a new imaging and detection technique that is fast and less invasive than current standards. acknowledgement: miin fis2008-00114 (spain), fundació cellex barcelona, nadacia spp (slovakia), apvv-0449-07 (slovakia) amphotericin b (amb) is a polyene antibiotic that has been widely used for treatment of systemic fungal infections. the main mechanism of biological mode of action of amb is considered to be associated with formation of ionic membrane pores or channels in the lipid membranes. the aim of this work was to study the influence of the k + and na + ions on the aggregation process of amb in aqueous medium. the analysis of electronic absorption and fluorescence spectra of amb shows that the increasing k + concentration have influence on the level of aggregation of the drug much more than the same amount of na + ions. this effect is especially noticed at neutral ph values. the rls technique was used to study aggregation of amb in solution, in the environment of the k + and na + ions. the application of this technique makes it possible to study the electronically coupled chromophores, especially molecular aggregates. the results of the atr-ftir and raman spectroscopic studies also support this conclusion. these results provide a better understanding of the interaction between k + and na + ions and antibiotic which has not been previously considered to be significant for biological action of amb. g-quadruplexes: combining theory with experimental spectroscopic methods guanine-rich oligonucleotides can form unique tetrameric structures with four coplanar guanine bases, known as gquadruplex motifs. the g-quadruplexes have been found in vivo in the terminal parts of telomeres and other genomic regions. ligands, specifically binding to the g-quadruplex regions inhibit telomerase activity and thus can play an important role in the cancer therapy. most recently, the potential use of these structures has been tested in biosensors and nanotechnology industry. in the present work we use the infrared spectroscopy, including the relatively novel technique of the vibrational circular dichroism (vcd), in a combination with molecular dynamics and quantum chemistry computational methods to investigate the structure and spectroscopic response of the quadruplexes formed by the d(g) 8 and selfassociated dgmp. we obtained a good agreement between the computed and experimental spectra, confirming that the proposed geometrical models are realistic. the vcd technique appears especially convenient for the studies as it can detect the liquid-crystalline phases of the g-quadruplexes by an anomalous enhancement of the signal. j. bogdanovic pristov, a. mitrovic, k. radotic, i. spasojevic institute for multidisciplinary research, belgrade, serbia fructose, due to its high antioxidative capacity, represents a significant component of non-enzymatic defense of some plants against cold-provoked oxidative stress. in the present study, we have investigated role of fructose in seasonal adaptation of picea omorika (pančić) purkinye to cold. this endemic coniferous species is exposed to subfreezing temperatures that range from -10 to -30 • c during the autumn/winter and high temperatures exceeding 30 • c during the summer. characteristic epr signal of free or weakly bound mn 2+ was used as an indicator of oxidative status of needles, since coldrelated oxidative damage leads to mn 2+ release from photosystem ii. it was observed that prooxidative conditions developed in the autumn, at the beginning of cold season, which corresponded to significant increase of fructose level. total sod, as well as mnsod activity also rose significantly higher in the autumn. observed activation of antioxidative system (non-enzymatic and enzymatic) led to adaptation of needles to cold, as oxidative status during winter was decreased and similar to the status of needles in cold-free seasons. calyx of held: sted nanoscopy of a glutamatergic terminal p. bingen 1 , t. m. staudt 2 , c. kempf 3 , h. horstmann 3 , j. engelhardt 1 , t. kuner 3 , s. w. hell 2 1 german cancer research center / bioquant, 69120 heidelberg, germany, 2 max planck institute for biophysical chemistry, 37077 göttingen, germany, 3 institute for anatomy and cell biology, university of heidelberg, 69120 heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of <50 nm inside 3 µm thick tissue sections. three-dimensional shim and 2pem to study collagen arrangement and crimping pattern p. bianchini 1 , m. franchi 3 , l. leonardi 3 , a. diaspro 2 1 lambs-ifom microscobio research centre and department of physics, university of genova, genova, italy., 2 italian institute of technology (iit), genova, italy, 3 department of human anatomical sciences and physiopathology of the locomotorapparatus, university of bologna, italy ligaments have been generally described as multifascicular structures with collagen fibre bundles cross-connecting to each other or running straight and parallel with crimps. a different collagen array and crimping pattern in different ligaments may reflect a different mechanical role. aim of this study was to relate the 3d collagen arrangement and crimping pattern by backward and forward second harmonic imaging microscopy (shim) and 2-photon excitation microscopy (2pem). shim on a laser-scanning system is a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of tissue architecture. although it is a coherent process the multiple scattering through the tissue give us the capability to acquire signal in both backward and forward direction [1] . shim and 2pem were combined in a dual-mode nonlinear microscopy to find out collagen fibre arrangement and crimping pattern. both polarization dependence and differences between forward and backward signals allowed to yield information on local structure [2] . currently used tcspc flim systems are characterised by high counting efficiency, high time resolution, and multiwavelength capability. the systems are, however, restricted to count rates on the order of a few mhz. in the majority of applications, such as fret or autofluorescence, the photostability of the samples limits the count rate to much lower values. the limitation of the count rate is therefore no problem. however, if flim is used for ion concentration measurements or imaging of chlorophyll in plants the available count rates can exceed the counting capability of a single tcspc channel. we therefore developed a flim system that uses eight fully parallel tcspc channels. by using a polychromator for spectral dispersion and a multichannel pmt for detection we obtain multi-spectral flim data at a rate of several frames per second. we will demonstrate the application of the system to dyamic changes of the fluorescence lifetime of chlorophyll in living plants. -imaging and spectroscopy scattering effects on non linear imaging of thick biological samples f. cella 1 , z. lavagnino 1 , a. diaspro 2 1 lambs-ifom, microscobio research center, university of genoa, italy, 2 iit, italian institute of technology, genoa, italy non linear optical scanning microscopy became a useful tool for living tissue imaging. biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. while performing imaging of biological scattering tissues in two photon excitation (2pe) regime, the localization of the maximum 2pe intensity was found to shift closer to the surface 1 and the imaging depth limit appears strongly limited by near surface fluorescence 2 . in this work we computed illumination and photobleaching intensity distribution 3 in order to characterize the effects induced by scattering. simulations of 2pe illumination and photobleaching intensity profiles have been performed for different scattering coefficients and at different focus depth. furthermore imaging of fluorescent immobile sample (polyelectrolyte gel) allowed to perform an experimental test on thick turbid media. results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface. [1]ying et al., appl. opt. 38, (1999) . [2] theer p. and denk w, j. opt. soc. am. a. (2006) [3]mazza d. et al., appl. opt. 46 (2007) . biospectroscopic probes for real time measurement of hydrogen-deuterium exchange p. carmona 1 , m. molina 2 1 instituto de estructura de la materia (csic), madrid, spain, 2 escuela universitaria deóptica, madrid, spain isotopic exchange has long been used for the analysis of biomolecular structure and dynamics. hydrogen-deuterium exchange rates depend on ph, temperature and biomolecular environment. this is due to hydrogen bonding, low solvent accessibility, and steric blocking. time resolved measurement of hydrogen-deuterium exchange for subsequent 2d correlation spectroscopic analysis can, then, be very useful to obtain structural information from the said viewpoint. we have developed a microdialysis quartz cell for use in conjunction with raman spectroscopy to investigate hydrogen-isotope exchange reactions of biomolecules. the system requires only 40 µl volumes of the initial substrate and perturbing effluent solutions. we have obtained a d 2 o efflux rate of k d = 0.38 ± 0.008 min −1 with the greatest mwco (18 kda) used here, which involves that an exchange rate of 2.5 min −1 is the limiting rate that could be resolved with the said cell system. analogous results have been obtained using an infrared biospectroscopic microdialysis probe. the use of the method described here has the advantage of avoiding sample dilution (and subsequent signal loss) involved in the known stop flow methods. acknowledgements: the authors gratefully acknowledge financial support from the spanish ministerio de ciencia e innovación (project ctq2006-04161/bqu). polarized transient absorption to resolve electron transfer between tryptophans in dna photolyase photoactivation of dna photolyase comprises electron transfer through the chain fadh • -w382-w359-w306. photo-excited fadh • abstracts an electron from the tryptophan residue w382 in ∼30 ps (monitored by transient absorption spectroscopy). the subsequent electron transfer steps (from w359 to w382 •+ and from w306 to w359 •+ ) are difficult to resolve experimentally, because electron transfer between chemically identical species does not give rise to net absorption changes. to overcome this difficulty, we make use of the fact that polarized excitation (pulse laser) induces a preferential axis (that of the excited flavin transition) in the system (photoselection), and that w359 and w306 form different angles with that axis (known from the crystal structure). thus, polarized detection should allow distinguishing between them. using polarized "classical" transient absorption on a nanosecond time scale and the pump-probe technique on a picosecond scale, we demonstrate the feasibility of the method and provide evidence that electron transfer from w306 to w359 •+ is faster than the 30 ps time constant of the initial electron transfer from w382 to excited fadh • . synchrotron based fourier transform infrared (sr-ftir) microspectroscopy was applied to investigate apoptotic death of u-87 mg cells induced by the photosensitizer hypericin (hyp), in using different transport systems (hyp alone vs. hyp/ldl complexes) and incubation protocols. the differences between ir spectra of non-treated and hyp treated cells are mainly manifested in the positions of amide i and amide ii vibrational bands of proteins. these vibrational shifts are attributed to the protein structure changes from dominantly alfa-helix, in the non-treated cells, to beta-sheets and random coil structures, which prevail 4h and 24h after photodynamic treatment, respectively. the observed conformational changes of proteins can be explained as the consequences of the processes leading to apoptosis as was verified by flow cytometry experiments. the results confirm suggestion that ir spectroscopy can be successfully applied for the detection of early apoptotic processes. a. k. de, d. goswami indian institute of technology kanpur, india molecular fluorescence has been an indispensable tool in modern day optical imaging. one of the state-of the-art challenges in fluorescence microscopy is having better depth resolution as embodied by the confocal and multi-photon laser-scanning microscopic techniques. however, each technique bears its own limitation in having sufficient out-of-focus signal for the former while the low non-linear photon absorption cross-section for the latter. for confocal microscopy using one-photon excitation, we have shown how the clever choice of pulsed illumination instead of continuous-wave excitation leads to a gigantic enhancement in fluorescence that also has immediate applications in microscopy. moreover, single-photon illumination with ultrafast pulses leads to a novel way of achieving axial resolution along with numerous other advantageous applications e.g. reduced photo-bleaching of the chromophore. on the other hand we have thrown new insight demonstrating that the use of mode-locked laser pulses in multi-photon microscopy induces severe solvent-induced photo-thermal damage and prescribed methods to get rid of it. besides, the use of pulse pair excitation in multi-photon microscopy leads to probe and control the dynamics of fluorophores which has crucial role in selective excitation of fluorophores from quantum control perspectives. all these cutting edge research works will be presented in addition to our recent work on application of laser pulse shaping in multi-photon microscopy. interkingdom signalling in pseudomonas aeruginosa b. davis 1 , r. jenson 2 , p. williams 2 , p. o'shea 1 1 institute of biophysics, imaging and optical science, university of nottingham, u.k., 2 institute of infection, immunity and inflamation, university of nottingham, u.k. quorum sensing is the process through which some bacterial species coordinate cell-cell communication. pseudomonas aeruginosa; the pathogen responsible for over 90% of chronic lung infections and the leading cause of mortality in cystic fibrosis patients, expresses two major classes of quorum sensing molecules characterized as n-acyl homoserine lactones and 2-alkyl-4-quinolones. these compounds, in addition to their signalling roles have also been found to possess virulent properties, not only against competing species of bacteria such as staphylococcus aureus but also eukaryotic cells such as t-lymphocytes. the process of bacterial quorum signalling molecules influencing eukaryotic cell activity is termed 'interkingdom signalling'. to date it has been suggested that the quorum sensing molecules outlined above act on eukaryotic cells through interactions with an as yet unidentified plasma membrane or cytosolic receptors. this project is directed towards developing an understanding of how these compounds elicit eukaryotic response through a combination of membrane based interactions at physiologically relevant concentrations. particular emphasis is placed on studies of ligand binding with membrane microdomains in and the consequent downstream signalling are also considered. this work is significant as it will not only lead to a better understanding of pseudomonas infection, but may also lead to the discovery of new classes of agents for the treatment of infective diseases. a xas study of the sulphur environment in human neuromelanin and its synthetic analogues neuromelanin is a complex molecule accumulating in the catecholaminergic neurons that undergo a degenerative process in parkinson's disease. it was shown to play an either protective or toxic role depending on whether it is present in the intraneuronal or extraneuronal milieu. in the present study x-ray absorption spectroscopy is employed to investigate the sulphur binding mode in natural human neuromelanin, synthetic neuromelanins and in certain structurally known model compounds, namely cysteine and trichochrome c. based on comparative fits of human and synthetic neuromelanin spectra in terms of those of model compounds, the occurrence of both cysteine-and trichochrome-like sulphur coordination modes is recognized and the relative abundance of these two types of structural arrangement is determined. data on the amount of cysteine-and trichochrome-like sulphur measured in this way indicate that among the synthetic neuromelanins those produced by enzymatic oxidation are the most similar ones to natural neuromelanin. c. cremer kirchhoff-institute for physics, university of heidelberg, germany here we report on "spectral precision distance/position determination microscopy (spdm) with physically modifiable fluorochromes (spdm phymod ) to analyse the spatial distribution of single nuclear proteins and dna sequences at the macromolecular optical resolution level. like other methods of "spectrally assigned localization microscopy" (salm), spdm phymod is based on labelling 'point like' objects (single molecules) with different spectral signatures, spectrally selective registration and high precision localization monitoring by far field fluorescence microscopy. the intranuclear spatial location of single molecules was determined up to a density up to ca. 1000 molecules/µm 2 of the same type, and distances down to 15 -30 nm were nanoscopically resolved. quantum dots (qds) are semiconductor nanoparticles with increasing application as fluorescent markers in biology.we investigated structure of the cell walls of different species complexed with cdse qds using fluorescence microscopy, fluorescence spectroscopy and ftir techniques. in the experiments we used the cell walls isolated from three distinct plant species: arabidopsis thaliana, acer sp. and picea omorika. we studied both unlabeled and cdse-labeled cell walls. fluorescence spectroscopy and microscopy were used for detection of qds alone or complexed to the cell walls. emission spectra were deconvolved using the nelder-mead algorithm in matlab 6.5. we calculated approximate probability distribution (apd) for positions of spectral component maxima. there was certain difference between unlabeled cell walls and those complexed with qds. the ftir spectra also show some difference between the complexed and pure cell walls. the results show that structure was changed, but not significantly in reaction with cdse qds. these results are promising in context of use of qds as labels in cell wall studies. the characterization of the complex of cell wall structure with qds is a part of the study of nanoparticles application in investigations of plant materials. modulating the response of single neurons and neuronal networks with biophysical stimuli f. difato, a. maccione, l. berdondini, f. benfenati, a. blau italian institute of technology, department of neuroscience and brain technologies, genoa, italy during differentiation, cell processes create connections with other cells to form tissue capable of performing complex tasks. biophysical constraints provide necessary inputs for cellular organization in living organism 1 . to better understand how biophysical conditions influence tissue development, it is necessary to bridge the gap between experiments on single cells and complex tissues 2,3 . to achieve this goal we pair optical tweezers with electrophysiology measurements 4 . by adopting neuronal networks as a biological model, neuronal signal transmission can be recorded either by patchclamp electrophysiology or microelectrode arrays (meas). dissociated neurons will be cultured on meas to record neuronal network activity at different sites of the network while applying spatio-temporally defined biophysical stimuli to individual neurons. a. diaspro 1 , k. cortese 2 , p. bianchini 2 , c. gagliani 2 , c. tacchetti 2 1 iit -italian institute of technology, morego, genova, italy, 2 microscobio, university of genoa, italy correlative light/electron microscopy (clem) is becoming increasingly frequent in molecular and cellular biophysics. we successfully applied the method to analyze the 3d structure of rough and smooth russell bodies used as model systems. the major advantages of this approach are the following: (i) the ability to correlate several hundreds of events at the same time, (ii) the possibilitˆto perform 3d correlation, (iii) the potential to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the effective combination of the high data analysis capability of flm with the high precisionaccuracy of transmission electron microscopy in a clem hybrid morphometry analysis. we have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3d reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach. the relevance of the presented approach lies in two important key elements, namely: the development of optical nanoscopy methods and the potentiality for exploring different correlative frameworks like optical nanoscopy vs. optical microscopy adding scanning force microscopy techniques. multidrug resistance is a well known phenomenon which limits effectiveness in treating malignancy with chemotherapy by modifying the internalization and/or externalization flow of the drugs through the cancerous cells. combined chemotherapies, such as mvac, are therefore currently used in bladder cancer treatment. however, about 30% of patients do not respond this chemotherapy because of inherent or acquired drug resistance. we developed a non invasive predicative test on urinary cells to estimate the chemotherapy effectiveness before treatment, based on the fluorescence emission of mvac. we first studied the mvac photophysical properties in solution and using five cell lines: a drug sensitive cancer cell line mgh-u1s, its multidrug resistant subline mgh-u1r, a not tumorigenic cell line sv-huc-1, its tumorigenic counterpart mc-sv-huc t-2 and a cell line from transitional cell carcinoma t24. the results revealed a penetration and localization of the drug depending of the cell line type, allowing us to find a specific fluorescence signature for the identification of mvac resistant cells. similar data have been obtained for cytospined fixed culture cells and patients urinary cells. -imaging and spectroscopy -abstracts multilayered photoresist system as a ghost model for biological samples in confocal microscopy. l. ferrari, f. cilloco, f. r. bertani, s. selci istituto dei sistemi complessi cnr rome we have realized a bilayer photoresist system as a model to perform optical characterization in the vis-ir range of multilayered complex biological specimen. our goal is to obtain structural and spectroscopic reference parameters from the identification of reflectance pattern features within a novel project granted by miur (skintarget, ideas firb). our model is composed by a 1300 nm thick layer (shipley photoresist 1813) with a refractive index n= 1.6-1.8, wavelength dependent, and a 600 nm tick hsq (hydrogen silsesquioxane) layer with a refractive index n around 1.38, both spin-coated over a glass substrate. we present the analysis of reflectance pattern that can be obtained by a confocal laser reflective system as compared to the expected values as coming from analytical calculations, using a matrix approach, or a microscopic electromagnetic finite element analysis. interfacial roughness and consequent optical scattering are analyzed using a neural network approach. translational biophysics: multimode imaging for preclinical and clinical applications d. l. farkas cedars-sinai medical center, los angeles, usa our focus is where light and patient meet, and improvements yielding better outcomes. surgery is moving towards minimally invasive intervention, where biophotonics represents a major area of hope and growth. the translation of useful laboratory-derived knowledge into clinical practice has been hampered by the difficulty of detecting, characterizing and monitoring molecules and cells in the human body, especially dynamically. advanced bi ophotonic imaging is best suited for studying such entities, but has been lagging in clinical acceptance, in spite of major advances, and a clear need for the kind of resolution (spatial, temporal, spectral) and specificity that it alone can offer. biophysics-based new strategies are needed to address this challenge. the development of biophysical methods for translational medicine will be reviewed, with emphasis on our recent advances. our approach is a multimode one -combining methods to achieve early, quantitative detection of abnormalities. with imaging fulfilling its dual role of better describing anatomy and physiology, intrasurgical histopathologyequivalent molecular and cellular imaging is achievable in vivo, as is a closer spatio-temporal connection between imaging and intervention. some application areas to be covered: cancer (early detection by spectral reflectance/autofluorescence; progression quantitation by oct; nano-and targeted chemotherapy assessment in vivo); neurobiology (imaging fast calcium transients and alzheimer's plaques); hyperspectral mie scattering imaging for in situ displasia; design and use of an advanced multimode imaging endoscope with in vivo delineation of hirschsprung's disease for better intervention; monitoring of stem cell fate in vivo. immobilization of liposomes in a sol-gel matrix: a fluorescence confocal microscopy study r. esquembre 1 , s. n. pinto 2 , j. a. poveda 1 , m. prieto 2 , c. r. mateo 1 1 ibmc, universidad miguel hernández, elche, spain, 2 cqfm, instituto superior técnico, lisbon, portugal immobilization of liposomes shows interesting applications in protein biology, membrane biophysics and biosensor technology. previous fluorescence spectroscopy works revealed that the entrappement of zwitterionic phospholipid liposomes in a silica sol-gel matrix alters the thermodynamic properties as well as the fluidity of the lipid bilayer. interactions between the polar head of phospholipids and the porous surface of the host matrix could be responsible of such behaviour. in order to get more insight into this possibility we have immobilized, for the first time, giant unilamelar vesicles (guvs) and the shape and size of these structures as well as the possible existence of lipid domains have been visualized through fluorescence confocal microscopy. this technique allows for direct observation of the effect of encapsulation on an individual liposome, in contrast to the averaged information given by macroscopic spectroscopic techniques. liposomes composed of pure popc or dopc as well as mixtures dopc/dppc were labelled with the fluorescent probes bodipy, rhd-pe and rhd-dope. preliminary results shows that only the smallest guvs (6-10µm) survive to the encapsulation process but often with a slight loss of its sphericity, probably due to pressures suffered during the matrix gelation. however no change in the gel/fluid phase proportion has been observed for immobilized dopc/dppc guvs, regarding to solution. solvent fluctuations play a key role in controlling protein motions and functions. here, we have studied how the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (por) couple with solvent dynamics (g. durin et al, biophysical j. (2009 ) 96, 1902 . to simultaneously monitor the catalytic cycle of the enzyme and the solvent dynamics, we designed temperature-dependent uv-visible microspectrophotometry experiments, using flash-cooled nano-droplets of por. the temperature-dependant formation of the first two intermediates in the por reaction were measured, together with the solvent glass transition temperature (t g ) and the build-up of crystalline ice. we find that formation of the first intermediate occurs below t g and is not affected by solvent dynamics, whereas formation of the second intermediate occurs above t g and is influenced by solvent dynamics. these results suggest that internal protein motions drive the first step of the por reaction whereas solvent slaved motions control the second step. we propose that the concept of solvent slaving applies to complex enzymes such as por. amphotericin b (amb) is one of the main polyene antibiotics widely used to treat deep-seated fungal infections. the mechanism of biological action of amb is most probably directly related to the ability of the drug to form hydrophilic pores in the membrane core, thus affecting physiological transport of ions. the effects of amb-cu 2+ complexation are demonstrated by the electronic absorption and fluorescence spectra. the absorption spectra of amb in water (ph=7) after the injection of water solution of copper(ii)sulfate display a complex structure with hypsochromic-and bathochromic-shifted bands indicative of formation of molecular aggregates of the drug. formation of the electronically coupled chromophores of amb, especially aggregates, was analyzed at different cu 2+ concentrations by the rls (resonance light scattering) technique. intensity of the fluorescence emission spectrum (characteristic of the dimeric form of amb) decreases after the amb-cu 2+ complex formation. this effect of the formation of the amb aggregated structures by amb-cu 2+ are different from the spontaneous molecular aggregation process, as deduced from the spectroscopic analysis. morfo-functional asymmetry of the olfactory receptors of the honeybee apis mellifera l e. frasnelli 1 , g. anfora 2 , f. trona 2 , f. tessarolo 3 , r. antolini 3 , g. vallortigara 1 1 cimec, centre for mind/brain sciences, university of trento, italy, 2 iasma research and innovation center, fondazione e. mach, s. michele all´adige (tn), italy, 3 biophysics and biosignals lab., dept. of physics, university of trento, italy lateralization, i.e. the different functional specialisation of the left and right side of the brain, has been documented in many vertebrate and, recently, invertebrates species. in the honeybee apis mellifera l. olfactory memory seems to involve at first the use of the right antenna. the present study investigated physiological and anatomical differences between left and right antennae of honeybees. electroantennographic responses (eag) were recorded from the left and right antennae of 16 honeybees to linalool, a floral volatile compound, and isoamyl acetate, an alarm pheromone, at 5 doses (from 0,25 to 2500 µg). the number of sensilla on the left and right antennae was recorded by scanning electron microscopy (sem). each antenna segment, from 14 insects, was observed from 4 different viewpoints in order to image the whole antenna surface and compute the number of sensilla. the tested compounds induced higher eag responses on the right than on the left antenna at every dose. sem showed that the placoidea olfactory sensilla were slightly more abundant on the right antenna surface than on the left one. results suggest an asymmetry in the peripheral odour perception mechanism in the honeybee a. mellifera. super-resolution imaging of dna through single molecule switching of intercalating cyanine dyes c. flors, c. n. j. ravarani, d. t. f. dryden school of chemistry, university of edinburgh, u.k. a growing trend in far-field super resolution fluorescence microscopy involves the replacement of photoactivatable fluorophores by common dyes such as cy, atto or alexa [1] . it has been shown that these dyes can blink in useful timescales for single-molecule based imaging by adding suitable buffers. this strategy greatly simplifies the sample preparation and imaging scheme, enabling its application to a wider range of biological systems. we have explored if a similar approach might be useful to study dna topology using intercalating cyanine dyes such as yoyo-1. there are two main advantages of this approach: i) dna labelling with intercalating dyes is straightforward, and ii) the free dye in solution is essentially non-fluorescent, greatly reducing the fluorescence background. we show that yoyo-1 can blink in the absence of oxygen and in the presence of cysteamine, which allows its application to nanoscale imaging. we exemplify its use by imaging λ-dna and a puc19 plasmid. we also explore the compatibility of several intercalating dyes with biological systems such as enzymes or cells. our results suggest that dna intercalating dyes are a promising option for fluorescence super-resolution studies of dna topology. seeing more in total internal reflection fluorescence (tirf) microscopy r. fiolka, a. stemmer nanotechnology group, eth zürich, zürich, switzerland total internal reflection fluorescence (tirf) microscopy is an effective widefield imaging tool that selectively excites a very thin sample layer within the evanescent excitation field at the glass-water interface. lateral resolution of standard tirf microscopy is limited to approx. 240nm using green emission, which can be insufficient for a large class of biological investigations. additionally, the evanescent excitation field is prone to light scattering, creating out of focus blur in the final image. we present several techniques that address these mentioned shortcomings of standard tirf microscopy. using evanescent standing waves, the lateral resolution in tirf microscopy can be extended by a factor of 2.5, reaching 90nm. we further show techniques to reduce the blur induced by light scattering of the evanescent field. finally, we demonstrate optical sub-sectioning capabilities in tirf microscopy by acquiring several images with different penetration depth of the evanescent field and applying suitable post processing algorithms. thereby the obtainable z-resolution exceeds the classical limit of widefield microscopy, and object structures lying within the evanescent field can be reconstructed. the natural photosentizer hypericine (hyp) exhibits potent properties for tumor diagnosis and photodynamic therapy. evidences of hyp release from ldls prior to passive diffusion within cells are addressed in this study. fluorescent properties of hyp have been used for dynamic studies of its interaction with low-density lipoproteins (ldls) and u87 glioma cells. subsequent non-specific staining of intracellular membranes compartment were observed by mean of colocalization fluorescent imaging studies. it was shown, that monomers of hyp are only redistributive forms. increasing of hyp concentration leads to the formation of non-fluorescent aggregates within ldls as well as within the u87 cells, and can preclude its photosensitizing activities. in all experiments, hydrophobic character of the molecules appears as the driving force of its redistribution process. acknowledgment: this work was supported by the slovak res. and dev. agency contracts no. lpp-0337-06. we also kindly thank the synchrotron soleil for using the detection system of the disco beamline. ledgf/p75 switches from a dynamic to a tight chromatin interaction upon binding to hiv-1 integrase j. hendrix 1 , z. debyser 2 , y. engelborghs 1 1 laboratory of biomolecular dynamics, katholieke universiteit leuven, belgium, 2 laboratory of molecular virology and gene therapy, katholieke universiteit leuven, belgium human transcriptional co-activator ledgf/p75 is hijacked by hiv-1 integrase (in) during the replication of hiv. little is still known about the molecular complex of these two proteins in the living cell. in this work we first studied the cellular chromatin interaction of egfp-tagged ledgf/p75 with tunable-focus fluorescence correlation spectroscopy (tf-fcs) and show that ledgf/p75 is in equilibrium between a free brownian motion and a very slow movement on the chromatin. being dependent on the size of the laser focus, this slow movement represents a continuous associationdissociation-reassociation process that is governed by diffusion. concentration-dependent continuous photobleaching measurements (cp) furthermore revealed the existence of high-affinity chromatin binding sites. next, we co-expressed mrfp-tagged in and confirmed its intracellular interaction with ledgf/p75 by fluorescence cross-correlation spectroscopy (fccs). interestingly, cp and fluorescence recovery after photobleaching (frap) indicated that the affinity of this complex for chromatin is exceptionally high. by twophoton fluorescence lifetime imaging (2p-flim) we verified if the cellular stoichiometry was altered when the proteins were expressed together. we believe that this work is useful for the understanding and targeting of hiv-replication. biophysical identification of orf10 from clavulanic acid biosynthesis cluster as a cyp450 l. s. goto 2 , c. o. hokka 1 , j. f. lima 2 , o. r. nascimento 2 , a. p. u. araújo 2 1 grupo de engenharia bioquímica, ufscar, br, 2 grupo de biofísica molecular "sérgio mascarenhas", usp, br streptomyces clavuligerus produces the clinically important β-lactamase inhibitor clavulanic acid. biosynthesis related genes reside in three gene clusters, one of these, named clavulanic acid gene cluster, includes most of the known clavulanic acid biosynthetic enzymes. the penultimate step along clavulanic acid biosynthesis remains unclear. required transformation involves at least two events: oxidative deamination and double epimerization of (3s , 5s )-clavaminic acid into (3r, 5r)-clavaldehyde. downstream the known part of clavulanic acid cluster lays orf10 , a putative gene encoding a tentative cytochrome p450-like protein which knockout has been proven deleterious to clavulanic acid biosynthesis. should such protein exist, it would be candidate to fulfill the clavulanic acid pathway missing step. in this work, orf10 encoded protein is characterized aiming to place it as a real p450. for this task, molecular cloning and recombinant expression of orf10 were accomplished. purified protein was submitted to spectroscopic measurements such as circular dichroism and electron paramagnetic resonance which indicate p450 features, including catalytic relevant heme iron redox states and homolytic peroxide scission mechanisms. further, peroxide reaction adducts were characterized by spin trapping. this work is supported by fapesp. muscle structure and gabaergic innervations in the limbs of barnacle cyprid l. gallus 1 , s. ferrando 1 , c. gambardella 1 , a. diaspro 2 , p. bianchini 2 , p. ramoino 3 , v. piazza 4 , g. tagliafierro 1 1 libiom, dibio, università di genova, italy, 2 ifom-lambs/microscobio, difi, università di genova, italy, 3 dipteris, università di genova, italy, 4 ismar cnr, venezia, veneto, italy balanus amphitrite is a sessile crustacean that settles at the larval stage of cyprid. in this stage we studied the gabaergic innervation of limb striated muscular fibers, by immunohistochemistry. the second harmonic generation (shg) and 2-photon excitation (2pe) microscopy were used to set out the muscle structure and its relationship with nerve terminal. sections were observed at a multimodal nonlinear microscope composed by the leica clsm. the laser system used is a ti:sapphire chameleon-ultra (coherent inc, santa clara, ca, usa), tunable between 690 nm and 1040 nm and characterized by a pulse width of 140 fs delivered at a repetition rate of 90 mhz by means of a homebuilt set-up (bianchini p. and diaspro a. j biophotonics 2008 1:443-50). the z-stacks were performed in order to obtain the 3-dimensional distribution of the muscular fibers. in the posterior ganglion gad immunoreactive (ir) motor neurons were arranged in 12 clusters near the emergence of the limb nerves. gaba and gababr1 ir neuromuscular junctions (nj) were localized in the limb muscle fibers; vgat ir cells surrounded each limb muscles. these results suggest that gaba plays a key role in the regulation of limbs movement. the shg was very useful to outline the relationship between nerve terminals and limbs muscle fibers. giant unilamellar vesicles (guvs) are very useful model membrane systems to study many aspects of lipid-lipid and lipid protein interactions, particularly employing fluorescence microscopy related techniques (bagatolli 2006) . the use of this model system can be particularly useful to study aspects related with the lateral structure of bacterial membranes using the aforementioned approach (i.e. fluorescence microscopy). bacterial cells have a size close to the resolution limit of optical microscopy and details about the organization of their membranes are not easy to achieve using such technique. recently a new electroformation method to prepare guvs composed of compositionally complex mixtures under physiological conditions was developed in our laboratory (montes 2007). in the present work we further extended this electroformation method to prepare guvs composed of bacterial lipid extracts and lipopolysaccharide (lps). in our experiments we used e.coli lipid extract to prepare small vesicles containing various lps species, from smooth strains and rough mutants. suvs were used as starting point to electroform guvs using various types of buffers with high ionic strength. the successfully obtained bacterial-guvs were used to study the interaction of these membranes with known lps-binging proteins (lung surfactant protein d, sp-d) and peptides (polymyxin b). our results remark the usefulness of this particular bilayer models to perform studies mimicking bacterial membranes. mechanical properties of polymeric membranes probed by afm m. kocun 1 , i. mey 1 , w. müller 2 , m. maskos 2 , a. janshoff 1 1 georg-august universität, institute for physical chemistry, göttingen, germany, 2 johannes gutenberg universität, institute for physical chemistry, mainz, germany many biological cell functions are dependent on the mechanical properties of the membrane. polymeric membranes that mimic native cell membranes are valuable research tools which can be used to better understand the physics of biological membranes. we have investigated free standing artificial membranes prepared from polybutadiene-b-polyethylene oxide (pb-b-peo). the membranes were prepared from vesicles ruptured on porous silicon substrates. these polymeric membranes were studied by confocal laser scanning microscopy (clsm) and atomic force microscopy (afm). force indentation curves were performed on the membranes and theoretical models were used to extract elasticity constants from the results. the study of polymeric membranes can give insight to the function of biological membranes, furthermore polymeric membranes can be used to create new hybrid systems by incorporating biological (lipids, proteins), artificial (polymers, dyes) and inorganic (nanoparticles) components. site-directed spin labeling study of the lightharvesting complex cp29 the topology of the long n-terminal domain of the photosynthetic light-harvesting complex cp29 was studied using electron spin resonance (esr). cp29 is a minor antenna complex of the photosystem ii (psii), a multisubunit protein complex. wild-type cp29 protein containing a single cysteine at position 108 and nine single cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. in all cases the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. the spin label esr spectra were analyzed in terms of a multi-component spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. these results permit to trace the structural organization of the long n-terminal domain of cp29. we took the crystal structure of light-harvesting complex ii (lhcii), major antenna complex of psii available in pdb as a starting point and constructed a model for cp29 based on esr data. present opportunities and future developments in soft x-ray transmission and emission microscopy b. kaulich, a. gianoncelli, v. babin, m. kiskinova elettra -sincrotrone trieste, s.s. 14 km 163.5 in area science park, i-34012 trieste-basovizza, italy soft x-ray transmission and emission imaging and spectromicroscopy are bridging the gap to other microscopy techniques in terms of lateral resolution, penetration depth and chemical sensitivity. the novel soft x-ray spectromicroscopy approach of the twinmic instrument at the elettra synchrotron radiation facility is combining several imaging modes for morphology characterization, such as scanning, projection and full-field imaging with several contrast techniques including brightfield, darkfield, differential phase and interference contrast at sub-100 nm lateral resolution. complementary chemical information is provided by chemical imaging and micro-spectroscopy using the photon-in/photon-out xray absorption and x-ray fluorescence. unique for twinmic is the low-energy x-ray fluorescence setup operated at 280 -2200 ev photon energies, which allows simultaneous analysis of the morphology, and the distribution of light elements on cellular and sub-cellular level. in the presentation the principles of the methods used in the twinmic instruments the performance and potentials of the instrument will be demonstrated by several examples of applications in the field of human, animal and plant biology, biophysics and chemistry, physiology and genetics. the potential impact of microscopy techniques using free-electron x-ray lasers on biophysics will be illustrated by the diproi project at fermi@elettra. -imaging and spectroscopy fluorescence anisotropy and afm used as tools to characterized porin´s reconstituion in luv´s s. c. lopes, i. sousa, p. eaton, p. gameiro requimte, faculdade de ciências, universidade do porto, rua do campo alegre, 4169-007 porto, portugal a major requirement to perform structural studies with membrane proteins is to define efficient reconstitution protocols that assure, not only, a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. for this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. in this study we report the reconstitution of ompf in preformed dmpc and e. coli liposomes using two different techniques for detergent removal: (1) exclusion chromatography and (2) incubation with detergent adsorbing beads. the incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine ompf effect on the structural order of membrane lipids. these results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. moreover more anisotropy and atomic force microscopy studies will allow a better characterization of bacterial model system membranes. the wavelength dependence of the luminescence for different sized aunp by 2-photon clsm k. li, m. schneider pharmaceutical nanotechnology, saarland university, campus a4 1, d-66123 saarbrücken, germany gold nanoparticles (aunp) with different sizes can exhibit original luminescent properties if excited with pulsed nearinfrared laser light which makes them a suitable object to be detected in biological tissues. the main obstacle is to distinguish between the object of interest (emitted light) and the autofluorescence from the sample which limits the scope of application of aunp. therefore, our aim is to characterize the luminescent properties of such nanoparticles regarding their excitation and emission. in this study, the excitation and emission spectra of aunp with different sizes below 50 nm in an excitation range of 720 nm to 900 nm were investigated. our study shows the emission spectra curves of aunp are broadband spectrum and vary with the changing of excitation wavelength from 720 nm to 900 nm. the results also suggest the minimum laser power necessary to trap the aunp depends on particle size and excitation light wavelength and a maximum power above which the particles are destroyed. in all the experiment above, to avoid the simultaneous effects from slide and cover slip is a must. rocking, tumbling, and sliding: real-time nanomotion of a membrane-bound virus p. kukura 1 , h. ewers 2 , c. mueller 1 , a. renn 1 , a. helenius 2 , v. sandoghdar 1 1 laboratory of physical chemistry, eth zurich, ch-8093 zurich, switzerland, 2 institute of biochemistry, eth zurich, ch-8093 zurich, switzerland the interaction of a virus with its receptors in the plasma membrane is decisive for its infection of cells. optical studies have revealed that after binding, virus particles move laterally on the membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the molecular dynamics of early virus-host couplings. here, we examine a model system, in which single simian viruses (sv40) interact with their gm1 ganglioside receptors in supported lipid bilayers. we employed scattering interferometry and single molecule fluorescence localization to visualize the vectorial rotational motion of virions. at low receptor concentration, we observed sliding and tumbling of single virions during rapid lateral diffusion. in contrast, at increased receptor concentration the virions repeatedly underwent periods of standstill, reminiscent of their behavior prior to endocytosis. by an unprecedented combination of millisecond time and nanometer spatial resolutions, we revealed that during these immobile periods, the virions rock back and forth among nanoscopic spots separated by 9 nm. our insights, together with the structure of the viral capsid, suggest aggregation of receptors in nanodomains and recurrent swap of binding between receptor molecules and neighboring viral protein pentamers. herein, we have developed different membrane probes binding spontaneously to the outer plasma membrane leaflet and showing sensitivity to membrane properties such as surface charge and phase state. the first two types of probes were based on 3-hydroxyflavone fluorophore. one of them showed a high sensitivity to the surface charge and to the phase state of lipid bilayers, while the other was only sensitive to the surface charge. the third type, which was based on nile red fluorophore, was sensitive only to the phase state. surprisingly, the probes that were sensitive only to the surface charge did not respond to apoptosis, while the other two types of probes showed significant spectroscopic response to it. moreover, the latter exhibited response to cholesterol depletion, which was similar to that observed on apoptosis. thus, according to our data, the intact living cells present a remarkable fraction of the cholesterol-rich domains, while apoptotic loss of the transmembrane asymmetry decreases it dramatically. these probes represent a new tool for quantification of surface charge and cholesterol-rich domains in cell membranes. -imaging and spectroscopy due to its porosity and unique optical properties porous silicon (psi) is an attractive template to develop biomaterials and biosensors. porous silicon microcavity (psimc) structures were prepared then functionalized for covalent protein attachment of glucose oxidase (gox) or solubilized bacteriorhodopsin (br). functionalization and protein infiltration was monitored by specular reflectometry sensitive to change in refractive index, when a molecule is attached to the large internal surface of psi. protein infiltration into the porous scaffold was confirmed by edx spectroscopy and the structures were imaged by biphoton microscopy. second harmonic generation and enhanced two-photon excited fluorescence emission from porous silicon was observed when resonantly exciting the structures. in addition, when the microcavities were infiltrated with gox or br, the proteins acted as a very efficient internal two-photon-excited fluorescence emitter, hence protein infiltration enabled the in-depth visualization of the porous structure by taking advantage of the optical sectioning capacity inherent to the non linear optical microscopy technique. patterning of bio-molecules: methods for characterization of neuron-substrate interfaces we investigated how to use and improve micro printing techniques to obtain molecules patterns on cell culture substrates. with micro contact printing (µcp), we generated geometrically defined depositions of poly-d-lysine (pdl) using poly-dimethylsiloxane stamps and optimized neuronal culturing conditions. rat and mice hippocampal neurons grown on those patterns showed to be alive and functional. we then applied µcp to study axonal development by combining cell culture assays with atomic force microscopy (afm) to investigate in more detail the molecule deposition on the surface and to measure morphological changes in the growth cone (gc) during the early phases of neurite development. we found distinct shapes of the gcs depending on whether they were growing on l1 adhesion molecule patterned by indirect-µcp or on pdl coated surfaces. we also attempted to transfer such patterns on multi electrode arrays in order to constrain neuronal cell bodies on the electrode area and to improve electrophysiological recordings from neuronal networks. other patterning techniques were therefore explored using a nano-drop printing system. patterned surfaces were analyzed with afm and scanning electron microscopy to combine different approaches aimed to the improvement and characterization of the printing techniques. insights on the mechanism of electron transfer in complex i a. l. maniero 1 , c. bergamini 2 , m. bortolus 1 , r. fato 2 , s. leoni 2 , g. lenaz 2 1 università degli studi di padova, padova, italy, 2 università degli studi di bologna, bologna, italy complex i (nadh dehydrogenase) plays a central role in cellular energy production, transferring two electrons from nadh through a series of iron-sulfur clusters (fes) to ubiquinone (coenzyme q); the electron transfer is coupled to the translocation of protons across the membrane. the fes center n2 is the last acceptor in the electron-transfer chain, but the mechanism through which the enzyme couples the 1e − reduction of the fes centers to the 2e − reduction of ubiquinone (q→sq→qh 2 ) is unclear [1] . in our experiments, submitochondrial particles were treated with different inhibitors, in the absence or in the presence of different quinone analogues, and nadh addition initiated the electron transfer. we assess by epr (electron paramagnetic resonance) spectroscopy the relative abundance of the reduced n2 center and of the semiquinone radical, and coupled epr data to enzymatic activity assays and to fluorescence measurements on the effect of inhibitors on reactive oxygen species (ros) production. we identify two different classes of inhibitors showing different effects on ros production. moreover the redox state of the complex has shown to depend both on the inhibitors and on the quinone analogues. a possible mechanism of the electron transfer, that can explain the experimental findings, will be presented. conventional fluorescence microscopy suffers from a resolution limit imposed by the diffraction of light. stimulated emission depletion (sted) microscopy overcomes this resolution barrier without being limited by the wavelength by taking advantage of the photophysics of the observed sample into the image formation process, and has proven to be a powerful approach for exploring relevant biological issues. the outstanding resolution of sted microscopy is achieved by drastically minimizing the spatial extent of the focal region from which fluorescent molecules can emit signal in the sample. so far, mainly complex and relatively expensive lasers systems providing pulsed beams have been used to inhibit the fluorescence in the outer area of the focal region. here we report on the development of a new setup based on a fast beam scanning confocal microscope using compact turn-key and inexpensive continuous wave lasers. the great potential of this simple configuration is demonstrated for a selection of commonly used fluorescent markers. characterization of hepatitis b antigen particles by atomic force microscopy each year, over one million people die from hepatitis b virusrelated chronic liver disease, including cirrhosis and hepatocellular carcinoma. the major surface antigen of hepatitis b virus (hbsag) is a cysteine-rich, lipid-bound protein with 226 amino acids. recombinant hbsag produced in yeast can self-assemble into 22-nm immunogenic spherical particles that are used in licensed hepatitis b vaccines (protein/lipid ratio is 60/40 in mass). hbsag size and shape have been mainly investigated by transmission electron microscopy after negative staining of the particles. however, under these conditions, no details of the particle surface can be obtained because of the shadowing effect due to the uranium salts. here we describe new structural insights of hbsag particles using atomic force microscopy (afm) performed under physiological conditions. we applied atomic force microscopy to define structural details of the surface organization with a resolution in the nanometer range. as expected, the diameter of hbsag particles is 26,8 ± 2,5 nm in average. the surface of these particles clearly shows the presence of protuberances that most probably correspond to proteins. indeed, reduction and alkylation induces the disappearance of the protuberances. the number of the protuberances estimated from afm micrographs is about 70 per spherical particles. a biophysical study of equine herpesvirus-1 entry into cells g. mckenzie 1 , p. o'shea 2 , j. kydd 1 , c. rauch 1 1 school of veterinary medicine and science, university of nottingham, uk, 2 institute of biophysics, imaging and optical science, university of nottingham, uk equine herpesvirus-1 (ehv-1) can cause respiratory disease in young horses, varying degrees of paralysis and abortion during the later stages of pregnancy. furthermore, there has been a recent increase in the number of outbreaks involving paralysis of thoroughbred horses in training. virus disseminates rapidly after initial infection via cell associated viraemia. controlling this may prove crucial in combating the pathogenicity of the disease. we have investigated the cellular interactions of ehv-1. initially, binding of ehv-1 to fluorescein phosphatidylethanolamine (fpe)-labelled phospholipid vesicles of various compositions was examined. a variety of microscopy techniques were then employed to study the events surrounding the binding of virus to equine peripheral blood mononuclear cell (pbmc) membranes. confocal microscopy images have highlighted possible colocalisation of ehv-1 with membrane 'rafts'. total internal reflection fluorescent microscopy was then used to identify viral binding at the membrane with high contrast images, able to observe single virus particles. establishing the viral entry pathway into pbmcs would allow drugs that target this process to be employed, reducing clinical viraemia. the interpretation of in-vivo binding rate measurements allows inferring the molecular interactions that regulate cellular functions. fluorescence recovery after photobleaching (frap) is a widely used tool to quantify binding reactions in vivo. however the lack of "golden standards" for these measurements requires the measured binding rates to be validated with other techniques. we present a new fluorescence correlation spectroscopy (fcs) method to measure the in vivo bound fractions and residence times for molecules that interact with an immobile substrate. we apply this method to measure binding of mutants of the transcription factor vbp (vitellogenin binding protein) to the dna. comparison of fcs with frap results in comparable estimates of the measured diffusion constants and bound fractions. however, fcs provides an estimate of the vbp residence time at the dna, while frap does not. this limitation in the analysis of vbp is due to the larger photobleaching volume used in frap, if compared to the observation volume of an fcs experiment. in sum, we present a method to measure binding rates with fcs. substantial agreement of this method with frap is shown. however, further validation on tightly bound molecules will be necessary to assess if frap and fcs agree in the measurement of residence times. -imaging and spectroscopy we have investigated the changes in the mechanical properties of the zona pellucida (zp), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. the response of the zp to mechanical stress has been recovered according to a modified hertz model. zp of immature oocyte's shows a pure elastic behaviour. mature and fertilized oocyte's zps evidence, instead, a transition from a purely elastic behaviour, which occurs when low stress forces are applied, towards a plastic behaviour has been observed. the high critical force necessary to induce deformations, that well supports the non-covalent long interactions lifetimes of polymers, increase after the cortical reaction. afm images show that oocytes' zp surface appear to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles more wrinkled surface characterize marure oocytes with respecto to immature and fertilized oocytes from a mechanical point of view, the transition of the mature zp membrane toward fertilized zp, through the hardening process, consists in the recovery of the elasticity of the immature zp, while maintaining a plastic transition that, however, occurs with a much higher force with respect to that required in mature zp. dynamical behavior of ribose and deoxyribose supercooled water solutions s. e. pagnotta 1 , s. cerveny 1 , a. alegria 2 , j. colmenero 3 1 centro de fisica de materiales, centro mixto csic-upv/ehu, san sebastián, spain, 2 departamento de fisica de materiales, universidad del pais vasco (upv/ehu), facultad de quimica, san sebastián, spain, 3 donostia international physics center, san sebastián, spain ribose and 2'-deoxyribose are probably the most widespread monosaccharides in nature. they can be extensively found in ribonucleic acid (rna) and 2'-deoxyribonucleic acid (dna), respectively, where they form, together with a nitrogenous base and a phosphate group, a peculiar building-block structure called nucleotide. in the present work, the relaxation dynamic of ribose and deoxy-ribose water solutions at different concentrations has been studied by broadband dielectric spectroscopy and differential scanning calorimetry in the temperature range of 150-250k. two relaxation processes are observed for all the hydration levels; the slower (process i) is related to the relaxation of the whole solution whereas the faster one (process ii) is associated with the reorientation of water molecules in the mixture. as for other polymeric water solutions, dielectric data for process ii indicate the existence of a critical water concentration above which water mobility is less restricted. moreover, according with these results, atr-ftir measurements of the same sugar solutions showed an increment in the intensity of the oh stratching sub-band close to 3200 cm −1 as water content increases. the regulation of the formation of cytoskeletal protein complexes by actin-binding proteins m. nyitrai, a. vig, t. kupi, z. ujfalusi, s. barkó, g. hild university of pécs, faculty of medicine, department of biophysics, pécs, hungary in living cells various groups of proteins are associated to supramolecular actin filament structures, often in a nucleation factor dependent manner. for example, actin structures associated with formins can bind tropomyosin and profilin, while those polymerised by the nucleation of the arp2/3 complex bind cofilin and myosin i. the molecular mechanisms underlying the regulation of the formation of these protein complexes is still ambiguous. we have shown recently that formins can bind the actin filaments and change their conformational state. subsequent binding of other actin-binding proteins, such as tropomyosin and myosin, can reverse these changes. it appears that the reversal effect assumes that the actin-binding protein binds the filaments in a well-defined and specific binding site. the altered conformational state of the actin filaments observed after the binding of these proteins provides a possible explanation for the modified affinity of the filaments for other-actin binding proteins. based on the results available so far we assume that the affinities are modified differently by different nucleation factors, and the conformational changes introduced to actin by actin nucleation factors can serve as the molecular bases for the regulation of the formation of actin based intracellular protein complexes. experiments are currently in progress to test and further corroborate the existence of such regulatory mechanisms in living cells. correlation between sub-cellular distribution of photoactive drug hypericin (hyp), determined by applied delivery systems (hyp vs hyp/ldl), and mode of the cell death is addressed in this study. co-localization of hyp with mitochondria, lysosomes and golgi apparatus in u-87 mg glioma cells was determined by confocal laser scanning microscopy in using organelle specific fluorescent dyes as well as by time resolved fret experiments. flow cytometry experiments were realized to study a photodynamic effect of hyp (598 nm/4 jcm −1 ) on cells. significant differences in the proportional representation of live, apoptotic and/or necrotic cells were observed for different types of delivery systems of hyp 24 hours after hyp (5x10 −7 m) photoactivation. conclusions: i) sub-cellular distribution of hyp depends on using delivery systems, ii) the mode of cell death depends more on concentration of hyp inside cells, than on different type of delivery systems (for non selective wide-field cell illumination), iii) fluorescence lifetime is sensitive parameter to study sub-cellular distribution of hyp. novel time-resolved spectroscopic methods have been used to investigate the interactions between a fluorescently-labelled mutant of the peptide melittin and supported lipid bilayers, formed by self-assembly at a silica-water interface via vesicle deposition. time-resolved evanescent wave-induced fluorescence spectroscopy (trewifs) is a surface-selective technique in which the evanescent field from a pulsed laser source is used to photoexcite fluorescent species at an interface. the resulting fluorescence decay kinetics, measured using time-correlated single-photon counting, report on the micro-domains experienced by those fluorescent species at an interface. extending trewifs to time-resolved evanescent wave-induced fluorescence anisotropy measurements (ew-trams) provides dynamic rotational information of a fluorescent species, reporting on its mobility at an interface. presented here are trewifs and ew-trams data obtained from the fluorescence of an alexa 430-labelled melittin mutant interacting with a dipalmitoylphosphocholine bilayer at room temperature, physiological ph and ionic strength. the results provide new insights into the conformation, location and motion of cytolytic peptides interacting with cell membranes. optical tweezers are used to controllably apply forces to red blood cells and the resulting chemical and structural changes are monitored using raman spectroscopy. the forces are applied in vivo and mimic that which the cell undergoes mechanically as it passes through vessels and smaller capillaries. the first result presented will be spectroscopic evidence of a transition between the oxygenation and deoxygenation states of hemoglobin that is caused by the stretching of the red blood cell. the transition is due to mechanically induced enhancements of hemoglobin-membrane and hemoglobin neighbor-neighbor interactions. the latter, lesser known effect is further studied by modeling the electrostatic binding of two of the protein structures using molecular dynamics methods. secondly, polarized raman spectroscopy is utilized to study the packing and ordering of the hemoglobin proteins in the red blood cell as it is stretched. depolarization ratios for a number of heme group modes change, indicating that the applied force additionally packs and orders the proteins inside the cell which further demonstrates the role of cell deformation in the oxygen transport kinetics. acknowledgements: this work was supported by miin fis2008-00114 (spain) and fundació cellex barcelona fret microscopy is widely used to study protein-protein interactions in living or fixed specimens. currently, most commonly used visible fluorescent proteins for live-cell fret studies are the cerulean and venus variants of the cyan and yellow fluorescent proteins. even though this fret pair appears to be ideal for monitoring protein-protein interactions, the most commonly used fixed laser wavelengths do not excite cerulean at peak absorption. recently, we characterized an ideal donor, the monomeric teal fluorescent protein (mtfp), which is excitable using the commonly available 457 (8) opt. june/july, 2008). we used teal as a donor for various red fluorescent proteins as acceptors including tdtomato, mko2, morange2, mtagrfp, mkate. we have employed a "fret standard" genetic construct to minimize variability in separation distances and positioning of the fused donor and acceptor fps. using spectral fret imaging and fluorescence lifetime measurements in living cells expressing the fused proteins, we have characterized both sensitized acceptor emission and the change in the donor lifetime distribution as a result of quenching for each of the fused fp pairings. our results indicate that some red fps are better acceptors than others in terms of quenching the teal donor and sensitizing the emission of the acceptor indicating a fret event. s. m. perepelytsya, s. n. volkov bogolyubov institute for theoretical physics, nasu, 14-b metrologichna str., kiev, 03680, ukraine stability of the dna double helix is determined by na + counterions, neutralizing the negatively charged phosphate groups of the macromolecule backbone. but under spatial conditions they may be replaced by much heavier ions, for example cs + . to determine the influence of heavy counterions on internal dynamics of the double helix the conformational vibrations of na-and cs-dna are studied. for this purpose the model of conformational vibrations of dna with counterions is used [perepelytsya s.m., volkov s.n., eur.phys.j. e. 24, 261, 2007] . as the result the frequencies and amplitudes of vibrations for b -dna with na + and cs + counerions are calculated. the frequencies of internal modes of the double helix are about 110, 79, 58 and 15 cm −1 . the frequencies of ion-phosphate modes are about 180 and 110 cm −1 for na-and cs-dna respectively. the calculated amplitudes of vibrations show that light counterions not disturb the dna internal dynamics, but heavy counterions make move all structure elements of the double helix. using the valence-optic approach the intensities of the dna vibrations in raman spectra are calculated. the calculations show that the ion-phosphate mode in cs-dna spectra is prominent, in contrast to na-dna spectra, where it has very low intensity. obtained results describe the intensity increase of the band 100 cm −1 in cs-dna spectra that was observed in [bulavin l.a., et al., arxiv:0805.0696v1]. s. soria 1 , f. quercioli 3 , r. mercatelli 3 , f. bianco 2 , i. cacciari 2 , g. righini 2 1 centro studi e ricerche "e. fermi", p. del viminale 2, 00184 rome, italy, 2 ifac-cnr, istituto di fisica applicata "n. carrara", via madonna del piano 10, 50019 sesto fiorentino (fi), italy, 3 isc-cnr, istituto dei sistemi complessi, via madonna del piano 10, 50019 sesto fiorentino (fi), italy we report on the application of a simple white light source based on the supercontinuum generation from commercial photonic crystal fibres to confocal fluorescence microscopy and fluorescence lifetime imaging (flim) microscopy. the coherent white light can be tuned by varying the wavelength and intensity of the pump, a ti:sapphire laser. there are several advantages jn the use of sc sources: spatially coherent white radiation, tuning ranges of approximately 400 nm, high brightness, a robust compact system (potentially all-fibre) and relatively low cost. being pulsed, sc sources are suitable for flim. we have used this system for measuring foerster resonance energy transfer (fret) in order to study interactions between ions channels and proteins of membrane within lives cells resolving the quaternary structure of plague f1 capsular antigen a. soliakov 1 , j. r. harris 1 , m. r. hicks 2 , a. rodger 2 , r. woody 3 , a. watkinson 4 , j. h. lakey 1 1 cell and molecular biosciences inst., newcastle univ., uk, 2 chemistry dept., univ. of warwick, coventry, uk, 3 biochemistry and molecular biology dept., colorado state univ., usa, 4 pharmathene uk, billingham, cleveland, uk most gram-negative pathogens express multi-subunit fibres on their surfaces that mediate host cell attachment, biofilm formation, invasion of host defenses and protection against phagocytosis. here we have studied capsular antigen fraction 1 (or caf1), secreted through the conserved chaperone/usher pathway by the plague agent yersinia pestis [1] . caf1 is highly immunogenic and is used in a recombinant subunit vaccine against plague. recent immunological studies indicated vaccines containing polymeric caf1 have higher protective efficacy than vaccines containing its monomeric variant. this difference in protective efficacy was attributed to the quaternary structure. however the quaternary structure of caf1 has not been characterized [2] . in our study we have used transmission electron microscopy of negatively stained specimen and linear dichroism spectroscopy to determine the quaternary structure of recombinant caf1. whereas electron microscopy revealed morphology of caf1 fibres bound to the surface of a carbon coated grid, linear dichroism gave the orientation of subunits in flow aligned caf1 fibres in solution. our results show recombinant caf1 comprises extended linear fibres and circularized fibres, both of which have high degree of conformational freedom. despite the vast application of lambert-beer law in biology, this empirical law cannot accurately describe the light absorption process of molecules with a long-lived excited state. this family of molecules includes most fluorescent molecules, which are very important in biology. lambert-beer law is in fact only valid at very low intensity of incident light and very low concentration of chromophore. if the system doesn't meet these conditions, it falls into a nonlinear regime when it is affected by a phenomenon which we call "dynamic photobleaching": the depletion of chromophores from the first layers, due to their transition to the excited state, leads to a sub-exponential propagation of light in the medium [1] . this phenomenon leads to the necessity of a new formula for the light absorption dynamics which depends on the lifetime of the excited state of the chromophore. the predictions of the theory were successful in describing the absorption dynamics of azobenzene [2] , but now they have been tested also on a biologically relevant molecule like chlorophyll. the results indicate that the absorbance is affected by the intensity of the incident light, and it is therefore a non reliable way of determining, for example, the concentration of the molecule. annular pupil filter to improve spatial highfrequency signal to noise ratio in linear and non linear microscopy e. ronzitti 2 , v. caorsi 1 , a. diaspro 3 1 lambs, microscobio, department of physics, university of genoa, genoa, italy, 2 semm, ifom-ieo, university of milan, milan, italy, 3 neuroscience and brain technologies department, iit, genoa, italy shot-noise significantly affects and deteriorates the imaging capabilities of typical two-photon excitation and confocal laser scanning microscope, especially in biological applications where the detected signal can be remarkable slight [1] . in particular, shot-noise substantially influences the spatial high-frequency range inducing a remarkable reduction of the optical transfer bandwidth. the insertion of an annular filter on the microscope objective lens in the illumination light pathway is here proposed to retrieve the high frequencies information loss [2] .the electromagnetic interference effect induced by the filter insertion, gives a redistribution of the optical transfer function. in particular, the microscope frequency response in filter scheme exhibits an enhancement of signal to noise ratio at the high frequencies able to recover the high frequencies hampered by shot-noise [3] . the goal of biomarker studies is to develop simple noninvasive tests that identify disease states. focus is beginning to shift from identification of individual biomarkers to identification of biomarker panels comprising multiple targets of different molecular species. there are, however, no current technologies available that allow for a comprehensive and simultaneous analysis of the expression levels of multiple cellular components, i.e. proteins and rna. surface plasmon resonance (spr) polaritons are surface electromagnetic waves that propagate in a direction parallel to the interface between a metal surface and an external medium e.g., liquid. these oscillations are very sensitive to any change of this boundary, a phenomenon that has been exploited to facilitate label-free, real-time detection of biological interactions, e.g. protein binding interactions. we are utilising the power of spr to develop technologies that facilitate diagnostic procedures for complex diseases such as alzheimer's disease and chronic obstructive pulmonary disease, through identification and detection of patterns of biomolecules indicative of disease. our approach will facilitate better disease characterisation, improve early detection strategies and aid drug discovery. surface generated fluorescence detection by supercritical angle confocal microscopy d. verdes, m. rabe, s. seeger institute of physical chemistry, university of zürich, winterthurerstrasse 190, zürich, switzerland a two channel confocal microscope for surface and simultaneously in solution fluorescence detection is presented. the microscope's core element is a parabolic mirror objective that collects the fluorescence above the critical angles for total internal reflection (tirf) of the water/glass interface. an aspheric lens incorporated into the parabolic mirror is used for diffraction limited focusing and collecting the fluorescence at low angles with respect to the optical axis. this low angles excitation approach is technically straightforward and gives an advantage over high numerical objectives that require very high angles for tirf illumination. by separating the collection of the fluorescence into supercritical and subcritical angles, two detection volumes highly differing in their axial resolution are generated at the interface. the surface selectivity of the detection volume is obtained on the basis of the dipole emission profile near a dielectric interface. its angular distribution is highly anisotropic and consists of a superposition of traveling and evanescent waves, which both are detected using the parabolic mirror objective. unlike with objective tirf microscopy, the parabolic mirror objective achieves easily diffraction limited excitation/detection volume at the water/glass interface. the objective optical performance is shown by measuring the actin cytoskeleton of cultured cells, fret energy transfer within adsorbed clustered proteins and single molecules detection. differential polarization laser scanning microscopy. anisotropy in biological samples g. steinbach 1 , i. pomozi 2 , g. garab 1 1 biological research center, hungarian academy of sciences, szeged, hungary, 2 pi vision bt., budapest, hungary differential polarization (dp) spectroscopy provides unique information on the anisotropic organization of biological samples [tinoco et al. 1987 ann rev biophys biophys chem 16:319]. however, anisotropy is often a microscopic property. the dp-lsm, constructed in our laboratory enables us to image the main dp quantities: linear and circular dichroisms (ld&cd), also in fluorescence detection ( . further applications include: periodic structure of isolated amyloids, anisotropy variations in cell walls related to drought resistance, and strong anisotropy of 'artificial chlorosomes', nanorods of synthetic porphyrins. dp-lsm might thus represent a novel tool in the better understanding of highly organized molecular macroassemblies. t. m. staudt 2 , p. bingen 1 , c. kempf 3 , h. horstmann 3 , j. engelhardt 1 , t. kuner 3 , s. w. hell 2 1 german cancer research center/ bioquant, 69120 heidelberg, germany, 2 max planck institute for biophysical chemistry, 37077 göttingen, germany, 3 institute for anatomy and cell biology, university of heidelberg, 69120 heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of <50 nm inside 3 µm thick tissue sections. -imaging and spectroscopy r. worch, t. weidemann, p. schwille biotec, biophysics group, technische universität dresden, germany interleukin-4 (il-4), a small soluble protein, is a principal regulatory cytokine, playing an important role during the maturation and clonal expansion of antigen specific b-cells in mammals. at the plasma membrane, il-4 is recognized by a receptor that consists of two single spanning transmembrane proteins: a high affinity il-4r alpha chain, and a low affinity il-2r gamma chain. it is still controversial by which molecular mechanism the signaling complex is fully activated: dimerization of chains, large conformational change upon il-4 binding or a combination of both. moreover, the influence of the lipid environment in which the activation takes place is poorly characterized. to address these issues we aim to reconstitute the receptor component in artificial membrane systems to study the various mutual interactions by means of fluorescence-based techniques, mainly fluorescence correlation spectroscopy. due to reduced background in a chemically defined system this may provide details not yet accessible in the living cell. k. wicker, s. sindbert, r. heintzmann randall division of cell and molecular biophysics, king's college london, se1 1ul london, u.k. fluorescence confocal microscopy, an indispensable tool of modern biology, allows the imaging of live fluorescent specimen with high lateral as well as axial resolution. through the introduction of a sufficiently small confocal pinhole, the lateral resolution can be enhanced compared to that of a wide field microscope. however, this gain in resolution comes at the cost of a decrease in detection efficiency, as light blocked by the pinhole is lost. we present a method for improving the lateral resolution (extending work of sandeau et al. [1] ) and detection efficiency of scanning microscopes by adding an interferometer with image inversion in one of its arms to the detection pathway [2] . this surpasses the lateral resolution achievable in a conventional confocal microscope (closed pinhole) while increasing the detection efficiency substantially. point spread function measurements for a uz-interferometer (uzi) are shown. the light in this setup follows threedimensional u-and z-shaped paths and relies on reflections off planar surfaces only in order to achieve image inversion. we achieved an interference contrast of 91% for white light, and excellent agreement with theoretical predictions. g. vicidomini, a. schönle, j. keller, r. schmidt, c. von middendorff, s. w. hell max planck institute for biophysical chemistry, department of nanobiophotonics, göttingen, germany fluorescence far-field microscopy is a indispensable tool in modern life science. however, the resolution of its standard variants is limited by diffraction to ∼200nm in the focal plane and ∼600nm along the optical axis. to overcome this limit, a new class of super-resolution microscopy techniques has been designed. one example is 4pi microscopy. to obtain isotropic resolution, 4pi uses interference of the wave fronts from two opposing lenses. the point spread function (psf) of 4pi system is characterized by multiple maxima/sidelobes, which replicate the object in the image. therefore image restoration is mandatory to render unambiguous imaging. the situation is further complicated because the positions and the relative heights of the multiple maxima/sidelobes of the psf depend on the phase difference (pd) between the two wave fronts at the focus. if the refractive index of the sample varies in space, this pd becomes a function of position and 4pi image formation process looses its shift invariance. the pd function (pdf) may not even be known a-priori and must then be estimated from the image, leading to a blind image restoration problem. here, we propose a maximum a-posteriori based method to solve the problem. we either assumed a mathematical model for the pdf that depends on a small number of parameters or allowed for an arbitrary pdf but introduced a smoothing constraint. designer multicomponent lipoplexes have recently emerged as especially promising transfection candidates, since they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes [1] [2] [3] . here, we show, for the first time, that after internalization binary complexes of lower transfection potency remain in compact perinuclear endosomes, while multicomponent systems have intrinsic endosomal rupture properties that allow plasmid dna to escape from endosomes with extremely high efficiency [4] . endosomal rupture results in an extraordinarily homogeneous distribution of unbound plasmid dna throughout the cytoplasm and in the nucleus [4] . fluorescence spectroscopy and stopped-flow technique were utilized for the study of the kinetics of incorporation of hypericin (hyp), a natural photosensitizing pigment, into lowdensity lipoproteins (ldl). triphasic kinetics of hyp association with ldl was observed when solutions of hyp and ldl were mixed together. the most rapid phase of hyp incorporation is completed within tens of msec, while the slowest one lasts 10-20 min. the most of hyp molecules are incorporated into ldl in the slowest phase. the kinetics of the incorporation of hyp into ldl particles pre-loaded with hyp were also investigated. the observed decrease of the lifetime and total intensity of hyp fluorescence with the increase of the incubation time of hyp with hyp/ldl complex is a sign of the formation of aggregates and the dynamic quenching of singlet excitation state of hyp inside ldl. to study the kinetics of a transfer of hyp molecules between ldl particles, the time evolution of the stopped-flow and time-resolved fluorescence experiments were investigated after the mixing of the complex hyp/ldl=200:1 with appropriate amounts of free ldl. for each final hyp/ldl ratio the increase of the lifetime and total intensity of hyp fluorescence was observed. the half-time of this process is similar to that one of the slowest phase of hyp incorporation into free ldl. a. bosca 1 , r. magrassi 2 , g. firpo 2 , l. repetto 2 , c. boragno 2 , u. valbusa 2 1 italian institute of technology, genova, italy, 2 nanomed labs (difi-cba), genova, italy the technique of choice to measure the electrophysiological activity of neuronal cells is the so called patch-clamp method because of its precision and sensitivity; this procedure could play a major role also in the investigation of the behavior of a biological neuronal network and so represents an important tool for understanding its functionality and for screening the effects of drugs and compounds on it. the final aim of this project is the development of a planar patch clamp device suited to measure simultaneously the electrical activity of cultured neurons associated in a network. this device will be made of polymeric disposable material and will include a microfluidic perfusion system. in order to create a smooth micro sized pore in a thin polymeric membrane we exploited the prototyping capabilities of focused ion beam etching and we extended the air moulding technique by combining it with soft moulding, obtaining micro structured substrates with the requested features. we also subjected our substrates to a chemical treatment capable of rendering its surface stable and hydrophilic and we verified that it makes them suitable for neuron culturing. use of lipoamino acids for nasal delivery of therapeutic proteins c. bijani 1 , s. sice 1 , j. elezgaray 2 , c. degert 1 , o. broussaud 1 , e. j. dufourc 2 1 physica pharma, 33600 pessac, france, 2 umr 5248 cbmn, cnrs-université bordeaux1, iecb, 33607 pessac, france most of the therapeutic proteins are clinically administered through an intravenous injection several times a day / week. because the repeated injections are not convenient and cause pain in patients, an alternative route of administration is desirable such as oral or nasal. unfortunately, proteins are easily degraded by proteolytic enzymes in the gastrointestinal tract and, therefore, have a low bioavailability when administered via oral route. physica pharma has gained experience in forming sprayable solutions combining lipoamino acids (laa) and small therapeutic organic compounds with the aim to improve their intranasal absorption. in the present work we investigated the ability of laa to complex large molecule such as human erythropoietin, human growth hormone and salmon calcitonin in order to form easily sprayable colloids. these three proteins are used respectively to treat anaemia, growth problem and hypercalcemy. circular dichroism and dynamic light scattering were used to further characterize the laa-protein colloids. a specific molar ratio of laa versus protein was found for wich the proteins keep their secondary structure and have an overall isotropic size slightly increased. molecular dynamics show that proteins are indeed coated with laa. such a complex is shown to pass very easily through a culture of nasal cells growth at confluence. in this study we describe two systems based on soft matter designed for the drug delivery and for the replacement of synovial fluid in osteoarticular pathologies. (i) a new class of temperature sensitive hydrogels pva/poly(ma m nipaam n ) shaped as microspheres obtained with a water-in-water emulsion photocrosslinking reaction. microparticles of pva/poly(ma m nipaam n ) with m:n theoretical molar ratios equal to 1:0; 1:4, 1:8 have been studied in terms of average size and responsiveness to temperature characterized by confocal laser scanning microscopy (clsm), dynamic light scattering (dls) and differential scanning microcalorimetry (dsc). (ii) a physical network based on hyaluronic acid with a small extent (degree of substitution: 1%) of hydrophobic moiety grafted on the backbone, hyadd4, has been characterized in order to account for the influence of thermal treatment on the stability of the hydrogel. dynamic light scattering (dls) and small angle neutron scattering (sans) have been used for dynamic-structural characterization of hyadd4 hydrogels. diffusion of macromolecular probes has been studied by fluorescence recovery after photobleaching (frap) to study the mesoscopic texture of the hydrogel and molecular dynamic (md) simulations were used to approach the time evolution of the physical junction points and of chains clusters. multi-scale estimation of water soluble diffusivity in polysaccharide gels g. eisele 1 , s. bertini 1 , d. paganini 1 , l. piazza 2 1 ronzoni institute, milan, italy, 2 distam, university of milan, italy diffusion properties in gels play important role in food and biotechnological applications. an attractive goal is to design gels in such a way that the active molecules are delivered by the material according to specific release sequences. the transport of macromolecules within polymeric gels depends: on the obstruction effects of the surrounding gel strands, on the molecular interactions between the gel and the solute, on the interactions between the solute molecules themselves and the interactions between the solute and the solvent. physical polysaccharide gels were evaluated in this work to probe diffusion over both microscopic and macroscopic distance scales. physical gels from agar and starch were investigated by high and low resolution nmr techniques in order to characterize their structures. obstruction effects of the surrounding gel strands were considered by studying diffusion of glucose. local diffusion, due to brownian motion, was quantified by low resolution nmr spectroscopy. the fickian diffusion coefficient was measured by modelling experimental concentration-distance curves obtained by means of a two-compartment diffusion-cell. diffusion coefficients depend on the viscoelastic properties of the gel matrix and on water-polysaccharide interactions. j. l. cuellar, g. koehler, m. fischlechner, e. donath medizinische institut für physik und biophysik, leipzig, germany rather than being just pathogens, from the view point of biotechnology and materials science viruses can be regarded as nanocontainers in which nucleic acids have been enclosed in a protective self assembled protein cage. in some viruses an additional lipid-protein envelope wraps the capsid shell. the capsid is constituted of several copies of one single protein subunit or just a few, arranged in a regular fashion showing icosahedral symmetry. the mechanical properties of the cage are important for understanding the infection mechanism involving the release of the encaged genome. by means of atomic force microscopy, it becomes possible to probe such material properties by nanoindentations of single viral particles. it is very interesting to learn how strong or brittle a virus can be. here we have studied the mechanical properties of empty rubella virus particles (rlps) due response of external applied forces. we found that rlps are extremely soft comparable to that of some rubber materials. a peculiarity of the rubella virus is that the capsid is considerably smaller than the surrounding shell filling only a fraction of the lumen provided by the envelope. the envelope in rubella has a distinguishable response on the material properties of the virus. deformation and fracture of the capsid requires comparatively larger forces. our results indicate that the ph is a major factor influencing on rubella particle material properties. this can be related to the infection mechanism. pentavalent antimony (pa) (glucantime, sanofi-aventis or glaxo) are the mainstream agents of choice for leishmaniasis treatment. in therapeutic doses the pa treatment has cardiac side effects like electrocardiographic (ecg) alterations, that include qt segment prolongation, t wave flattening or inversion, inversion of st segment, p, r and t waves amplitude reductions, torsade de pointes arrhythmias and sudden death by cardiac arrest. the objective of this study was to characterize the arrhythmogenic potential of pa. we used 30 guinea-pig to assess the chronic effects of pa therapeutic dose on corrected qt interval, qt dispersion, ventricular action potential (ap) amplitude and duration at 30% and 90% of maximal repolarization and survival rate. guinea-pig received daily 30mg/kg pa or saline for 15 days. eight lead ecg were recorded before and in the last treatment day. at the end the animals were killed and the left ventricle papillary muscles excised for ap recording with the intracellular microelectrode technique. our results of chronic pa treatment showed significant increase of qrs complex duration, qt interval duration, qt dispersion and incidence of t wave flattening or inversion and arrhythmias. the ap analysis demonstrated prolongation at 90% duration. the treatment was lethal in 30% of the animals. we concluded that pa is a proarrhythmic drug that upon chronic use may causes arrhythmias and mortality by disturbances in the ventricular repolarization process. m. m. khvedelidze 1 , t. j. mdzinarashvili 2 , t. partskhaladze 1 , n. nafee 3 , m. schneider 4 1 ivane javakhishvili tbilisi state university, tbilisi, georgia, 2 institute of molecular biology and biological physics, tbilisi, georgia, 3 biopharmaceutics and pharmaceutical technology, saarbrücken, germany, 4 pharmaceutical nanotechnology, saarbrücken, germany we have studied the thermodynamical properties of chitosan-coated nanoparticles (cnp) and non-coated nanoparticles (np) and have gained some insights about plga nanoparticles' properties using supersensitive differential microcalorimetry. the experiments show that in a wide ph interval the changes in transition temperature did not take place. it was shown that such nanoparticles could be used in acidic surrounding for drug transfer. stability and their other properties are less depended on either the particles were in bidistilled or deionized water, or the suspension of particles were located in buffer. to determine the interaction of plga nanoparticles with dna. in the case of dna presence in cnp solution the calorimetric experiments show that the heat absorption peak is constricted, what biophysically means that interaction between them takes place. for more exact determination the contribution of cnp in spectrum, we have compared the spectra of pure dna with the spectrum of the same concentration dna plus cnp. the optimal ratio for dna loading onto the cnp was found to be 7:1. protein-based "epoxy-like" physical hydrogels for stem cell transplantation s. c. heilshorn, c. wong po foo, j. s. lee materials science and engineering, stanford univ., u.s.a. stem cell transplantation has emerged as a promising therapy for multiple injuries and diseases; however, cell survival after transplantation is often poor and unpredictable. we hypothesize that co-injection of stem cells encapsulated within an optimized physical hydrogel will enhance viability. whereas, current physical hydrogels require a shift in environmental conditions (e.g., ph, temperature) to initiate the sol-gel phase transition during encapsulation, our newly designed molecular-recognition gels do not require environmental triggers. instead, these "physical epoxy-like" gels consist of two components that undergo a sol-gel transition upon mixing due to specific hydrogen bonding. the gel viscoelasticity is predictably tuned through precise variation in the molecular-level design of the two components, created using recombinant protein techniques. the design of the two components is based on simple polymer physics considerations and utilizes bio-mimimcry. adult neural stem cells or mesenchymal stem cells are encapsulated within these gels with high viability at constant physiological conditions. the gels promote the growth and differentiation of neural progenitors into neuronal phenotypes, which adopt a 3d-branched morphology. the gels are further optimized for use in the central nervous system by tethering neuroprotective peptides to the gel through molecular-recognition sites. these peptides are released-on-demand by cells through the action of proteolytic enzymes. we consider the integration of the portal protein of the bacteriophage virus φ29 into lipid bilayers of giant unilamellar vesicles (guvs) membranes with the aim of constructing a functional cargo-device able to transport dna and later translocate it outwards. our nano-engineering plan consists of growing guvs from bilayer membranes built up from proteoliposomes previously prepared by extrusion. we have designed two alternative chemical routes for integrating the portal protein in the lipid bilayer, the first considering the native protein and the second a mutant modified with a hydrophobic belt made of histidine-tags. in our contribution we will present details on the different nano-engineering strategies and experimental evidence about the integration of the portal protein in the membrane with an orientation adequate to allow for functional dna translocation. p.-h. guelluy 1 , m.-p. fontaine-aupart 2 , m. hoebeke 1 1 biomedical spectroscopy, ulg, belgium, 2 laboratory of molecular photophysics, univ. of paris-sud, france ppme is a second generation photosensitizer (ps), and a promising candidate for photodynamic therapy (pdt) treatment. we have previously demonstrated that ppme can be easily and efficiently encapsulated in dmpc liposomes, used as ps-vector. we therefore compared the photophysical and photochemical properties of free and encapsulated ppme incubated with human carcinoma cells. absorption and fluorescence microspectroscopy as well as flim analysis allow evaluating the aggregation state of ppme inside the different cellular organelles and the extracellular medium. confocal microscopy established undoubtedly the colocalization of ppme, by robust probabilistic exclusion method, within mitochondrion (central siege of apoptosis). after ps activation (4h and 24h), the balance apoptosis-necrosis was double-estimated by facs device and fluorescence confocal microscopy. quantification of hydroxyl radical was purchased by spin trapping-esr spectroscopy and quenching technique. all these techniques required peculiar settings because of the fluorescence activity of ppme. these results allow to ascertain that the vectorization of ppme affords a better cellular penetration and a monomeric state of ppme. the presence of ppme inside mitochondrion orientated the cellular death in apoptosis 4h following ps-activation. but necrosis is the major actor 24h after treatment. active substrates to study mechanotransduction j. le digabel, p. hersen, b. ladoux laboratoire matière et systèmes complexes, université paris diderot -cnrs, paris, france cellular processes imply an important coordination of interactions with the extracellular medium. accumulating evidences demonstrate that cell functions can be modulated by physical factors such as the mechanical forces acting on the cells and the extracellular matrix, as well as the topography or rigidity of the matrix. these extracellular signals can be sensed by mechanosensors on the cell surface or in the cell interior to induce various cell responses. we have developed an original approach based on micro-fabricated substrates of polydimethylsiloxane (pdms) to study cell migration. we used a closely spaced array of flexible micropillars (diameter∼1?m) to map the forces exerted by cells on their substrates. in this case, the micropillars act as passive force sensors. here, we propose to analyze the cell response to an external applied stress by a well-controlled actuation of the substrate. to do so, we used magnetic pillars. such substrates allow us to modify dynamic adhesion conditions of cells and to better understand the coupling phenomena between mechanical sensing and biochemical activity of a living cell. using polyacrylamide hydrogels doped with ferromagnetic iron oxide particles or ferrofluids, we can make magnetic pillars with diameters of 5 to 10 microns while a magnetic field can be locally applied with a magnetic needle. such a technique will be helpful to study the mechanical response of cells to an external force or to local changes in their microenvironment. glucose scavenging activities of pamam dendrimers m. labieniec 1 , t. gabryelak 1 , c. watala 2 1 university of lodz, deptartment of general biophisics, lodz, poland, 2 glycation is a spontaneous, non-enzymatic modification of biomacromolecules with hexoses, mainly glucose. in terms of its pathophysiological relevance, it targets predominantly proteins, but also nucleotides and phospholipids, and is of major importance to both physiology (ageing) and pathology (diseases with a metabolic background, like diabetes). earlier we demonstrated in vivo that the administration of pamam g4 to diabetic rats resulted in a significantly reduced blood glucose, as well as the early (hba 1c ) and late (ages) protein glycation products. in this study we investigated the ability of pamam g2 (16 surface nh 2 groups) and g4 (64 nh 2 ) to inhibit glycation of proteins in plasma, and a model protein -bsa. pamam g2 and g4 competed chemically with protein nh 2 for the binding of glucose, and hampered protein glycation. in a high-glucose medium they underwent an excessive glycation themselves. this modification was more effective in pamam g2, in which surface nh 2 were more mobile and accessible. pamam modification with glucose rendered these dendrimers less polycationic in aqueous solutions. pamams neither affected bsa conformation nor formed stable complexes with a protein. we conclude that pamams are very effective glucose scavengers. thus, even less toxic pamams of lower generations, like g2, may appear useful in further medical applications as the agents attenuating the detrimental effects of sever hyperglycaemia on biomacromolecules. selective drug delivery and novel drug approaches by polyelectrolytes s. krol cbm-cluster in biomedicine, area science park, s.s. 14, 163.5 km; trieste, italy the use of polyelectrolytes in the past was mainly related to targeted drug delivery and nanoparticle preparations for medical applications. but rarely the polyelectrolytes were investigated for the own features as a drug. in our present work we use a special physical feature of cancer cell membranes as a target for a specific polycation. we found that the polycation is selectively up-taken by cancer cells (leukemia, hepatocarcinoma, cancer stem cells) while normal cells remain unaffected. another interesting application of polyelectrolytes are in form of a multilayer coating on the surface of nanogold particles. for this topic, two new approaches were developed for a drug delivery through the blood brain barrier. in the first case, high amounts of creatine were bound to the gold particles and delivered as protective agents for ischemic stroke (viota et al., j colloid interface sci. 2009, 332 (1):215-23). in the second case coated multifunctional gold particles were prepared as a drug for neurodegenerative disease on the basis of protein aggregates. the use of gold as core for coated nanoparticles offers the possibility to study our systems as "theranostics", system which are modified to recognize selectively diseased cells and carry the moieties to treat or destroy the malfunctioning cells. possible treatments can be local hyperthermal therapy by using the particles as amplifier and enhancer or photodynamic therapy with gold as a drug. m. koneracka 1 , v. zavisova 1 , m. muckova 2 , p. kopcansky 1 , a. jurikova 1 , n. tomasovicova 1 , g. lancz 1 , m. timko 1 , m. fabian 3 1 institute of experimental physics, slovak academy of sciences, košice, slovakia, 2 hameln rds a.s., modra, slovakia, 3 institute of geotechnics, slovak academy of sciences, košice, slovakia the aim of this study was to develop biodegradable and biocompatible paclitaxel loaded magnetic plga nanospheres (nps) suitable for biomedical applications. biodegradable poly(d,l-lactic-co-glycolic acid) (plga) was used as a capsulation material and the magnetic fluid was used as a magnetic carrier. incorporation of magnetic particles and drug in the plga polymer matrix was confirmed by infrared spectroscopy (ftir) and differential scanning calorimetry (dsc). the release of the drug from the prepared nps to the surroundings under the different conditions was studied also. the prepared magnetic plga nps with encapsulated paclitaxel (spherical shape, size 200 -250 nm) have good stability in the presence of high nacl concentration at 25 • c, the toxicity of prepared samples declared 3 times higher value of lethal dose ld 50 in comparison with pure paclitaxel (ld 50 = 33mg/kg) and showed the significant response to external magnetic field which is useful from the point of view to achieve pharmaceutically acceptable drug delivery systems for tumour treatment. success of human gene therapy depends upon the development of delivery vehicles or vectors, which can selectively deliver therapeutic genes to target cells with efficiency and safety. many cationic polymers have been used to condense dna by electrostatic interaction into small particles (polyplex), for protecting the dna from degradation and enhancing uptake via endocytosis. polyethylenimine (pei) appears to be one of the most advanced delivery system that can condense dna efficiently forming pei-dna polyplex complex. the physicochemical properties of different molecular weights of pei, such as condensation ability, buffer capacity, time kinetics, ftir and surface charges of the pei-dna complexes may be important factors to obtain a higher transfection efficiency of the polycation vectors. our intent in this study was to characterize pei-dna complexes to see whether these physicochemical properties have any influence on their disposition characteristics and cellular uptake process. we found that pei-dna complexes, obtained by the 25 k pei at n/p ratio > 4, were more stable in the presence of tissue culture medium & serum, and did not dissociate in nacl easily. key words: polyethylenimine, polyplex, polycations, dna, transfection, gene delivery. effect of nanopatterned substrate on neuronal growth cones activity in the last20years an increasing interest has been address to explore, at the level of the single cell, the physical interaction between neurons and micro-and nano-patterned surfaces which mimic the biological environment and can induce specific biological behavior. a consistent number of substrates have been tested (nano-grooves, pillars, gap nanowires) but the effect of nano-topographical features at subcellular level e.g. branching and pathfinding of growth cones(gc)is still unexplored. using nanoimprinting lithography we fabricated gratings on glass with grooves of variable pitch, depth and width all in the nm range. embryonic stem cell derived neurons were seeded on nanostructured and on flat control glasses. we investigate gc morphology with nm resolution by afm. a significant effect on sub-cellular architecture was observed.on nanopatterned substrates72% of gc were branched with a large number of long and thin filopodia(average length and height4.3µm and 75nm)while on control only34%of gc were branched with an higher percentage of long and thick filopodia (average length and height 6,5µm and 300nm). on the contrary we did not observe a significant influence of the nanopatterning on the alignment and elongation of neurites. in both cases the distribution of angles between axon and filopodia showed a preferential direction at30 • . in conclusion, the tested nanopatterns do not influence the neurite directions but do enhance the gc morphology and explorative activities. growth enhancement and adhesion control of pc12 on micropatterned ns-tio 2 thin film c. lenardi 1 , a. v. singh 2 , p. milani 3 1 c.i.ma.i.na., dip. di scienze molecolari applicate ai biosistemi, università di milano, italy, 2 c.i.ma.i.na., semm, european school of molecular medicine, fondazione ifom, milano, italy, 3 c.i.ma.i.na., dip. di fisica, università di milano, italy cluster assembled nanostructured titanium dioxide (ns-tio 2 ) has been explored as novel substrate for in vitro cell culture. in this work, we report micropatterned ns-tio 2 thin film as putative microdevice for neuron culture and growth. in additions, we show a simple scheme of molecular patterning of bovine serum albumin (bsa) as cell anti-adherent substrate complementary to ns-tio 2 micropattern which favors a selective spatially confined adhesion of neurons. bsa was drop coated and physisorbed over glass coverslip covered with a thin conducting layer of indium tin oxide (ito) and then pmma was spin coated over it using standard protocols. later, using combinations of e-beam lithography and pulsed microplasma cluster source (pmcs), a thin layer of ns-tio 2 was deposited over micropatterned pmma. further, lift off process enabled us to generate complementary micropatterns of hydrophobic bsa (cell repellent) and hydrophilic ns-tio 2 (cell adhesive). cell culture studies have confirmed that pc12 cells like to grow on ns-tio 2 substrate and not on bsa layer. the technique offers a novel approach for neuronal cell assay applications. gene delivery with chitosan: influence of chain length on intracellular trafficking and dissociation s. lélu 1 , c. d. l. davies 1 , n. reitan 1 , s. p. strand 2 1 department of physics, ntnu, trondheim, norway, 2 department of biotechnology, ntnu, trondheim, norway chitosan, a cationic polysaccharide presenting low cytotoxicity, is a promising nonviral gene delivery vector. chitosan complexes dna into nanoparticles, and the complexation and cell transfection efficacy are function of the chitosan/dna ratio and of the intrinsic properties of chitosan, i.e. degree of polymerization dpn, charge density, and molecular structure. nanoparticles formed by shorter chitosan (dpn31-41) mediated higher transgene expression than nanoparticles based on longer chitosans. the purpose of this work is to relate the dpn of linear chitosan with the cellular uptake, intracellular trafficking and dissociation of chitosan-pdna complexes, measured by confocal laser scanning microscopy and fluorescence correlation spectroscopy (fcs). cells were incubated with oligomers (dpn 31 and 88) either free or complexed with plasmid-dna. both chitosan oligomers seem to penetrate cell nucleus in association with or free from pdna. 24 hours after incubation, accumulation of oligomers in cell nucleus is similar for free and complexed dpn31, whereas it increases for complexed dpn88 compared to free, indicating a possible delayed dissociation of the complexes based on dpn88 in the nucleus and suggesting a dissociation of pdna-dpn31 in cytoplasm. these data are consistent with previous studies which suggested that longer chitosan chains led to tighter complexes, and hence to a delayed dissociation process and lower transfection efficiencies compared to shorter chitosans. material surface properties greatly influence dna purification and pcr yield in microsystems c. potrich 1 , l. lunelli 1 , l. marocchi 1 , l. pasquardini 1 , g. guella 2 , d. vozzi 3 , l. vanzetti 1 , p. gasparini 3 , c. pederzolli 1 1 fbk, trento, italy, 2 univ.trento, italy, 3 univ.trieste, italy modern microchip platforms integrate dna purification, target amplification by pcr and dna detection in a single device. combination of these processes minimizes sample loss and contamination problems as well as reduces analysis time and costs. different strategies are available to perform dna extraction on a chip. here we exploited amino-coated materials as a tool for specific binding of dna through the electrostatic interaction between amine groups and nucleic acids. we analyzed the ability of different treated substrates to selectively absorb/desorb the genomic dna with the aim to purify dna from unwanted components. amino-coated substrates were characterized by afm, xps, fluorescence microscopy and absorption spectroscopy to define the surface chemical and morphological properties. the distribution of the dna adsorbed on materials was homogeneous and the eluted dna was tested for pcr. same materials were analyzed for their compatibility with pcr and the use of different enzymes and reagents or proper surface treatments was employed. we established the best conditions for dna amplification in silicon/pyrex microdevices depending on the type and the fabrication method used and on the quality of reagents more than on the passivation treatment or increment in standard taq polymerase concentration. nanoporous alumina fabricated using unconventional acids for enhanced biomolecular physisorption n. patra, m. salerno, r. losso, r. cingolani italian institute of technology, genova, italy in the last decades the available porecell sizes of porous alumina have been extended, but operating conditions to obtain some pore diameters still have to be optimized, particularly below the 50 nm limit. in this range the use of biocompatible porous alumina thin films could find applications in biosensors, after functionalization by appropriate physisorbed biomolecules. while pore ordering and film growth rate are mainly influenced by the electrical parameters of voltage and current, respectively, the typical pore size primarily depends on the electrolyte. in the search for alternative anodization conditions, we have investigated several acids that have never been used before, namely gallic, lactic, propionic, and glycolic acid. the anodizations were carried out in galvanostatic conditions at fixed concentration and durations, by varying the current. atomic force microscopy was used to test the oxidized surface morphology. in particular, lactic and propionic acids demonstrated feasible. lactic acid gave best results for ∼30 ma/cm 2 current density, corresponding to roughly constant ∼15 v potential, with resulting pore diameter in the 4060 nm range, whereas propionic acid performed best for ∼5 ma/cm 2 , corresponding to ∼43 v, resulting in 7090 nm pore diameter. both kind of films looked lightgray, different from the yellowish oxalic acid porous alumina, which can be a hint of lower ion contamination. chitosan-arabic gum nanoparticles as potential vehicles for peptide delivery l. moutinho, s. rocha, m. coelho, m. c. pereira lepae, chemical engineering department, faculty of engineering, university of porto, portugal chitosan (cs) -arabic gum (ga) nanoparticles were produced by an ion-ion interaction process, using different weight ratios of polysaccharides. according to zeta potential (zp) measurements, particles are ionically stable. zp values ranged from 30 to 70 mv for cs:ga ratios of 1:3.3 and 1:0.8, respectively. cs:ga 1:5 yielded uncharged nanoparticles and aggregates, showing that, at this ratio, the negative charges of ga neutralize the positive charges of cs. the particle diameters ranged from 300 to 500 nm, as measured by dynamic light scattering (dls), and displayed a slight tendency for decreasing as the ga concentration increased. transmission electron microscopy (tem) images showed numerous spherical nanoparticles. peptides were encapsulated within the nanoparticles, by mixing them with chitosan solution prior to adding ga. this system can protect short peptides from rapid metabolization, prolonging their blood half-life. physical characterization of nanocarriers for drug delivery s. motta 1 , y. gerelli 2 , g. sandri 3 , e. ricci 3 , p. brocca 1 1 dept. of chem, biochem and biotech for medicine -university of milan, italy, 2 dept. of physics -university of parma, italy, 3 dept. of pharmaceutical chem -university of pavia, italy nanoparticles used as nanocarriers for pharmaceuticals can improve solubility and bioavailability of problematic drugs and protect labile or toxic molecules. fundamental parameters like drug encapsulation efficiency and release kinetics are tuned by the physico-chemical features of the drug/carrier complex. a challenging aspect of the pharmaceuticallyoriented issues resides in the relatively low number of molecules allowed to build up nanosystems with different properties and morphology, according to the specific drug and to the intended therapy. we have studied: 1) soybeanlecithine/chitosan nanoparticles for progesterone and tamoxifen delivery; 2) solid-lipid nanoparticles (compritol 888 ato, poloxamer 188, tween 80 and chitosan) for cyclosporine-a delivery via ophthalmic formulation. both void carriers and drug-loaded nanoparticles have been studied, to understand how the structure of the carriers can be modified by the active molecules. several non-invasive physical techniques have been used to achieve a detailed knowledge of the systems: a) dynamic light scattering for the dimensional distribution of the nanocarriers, b) zeta potential to determine surface charge, c) cryo-tem for morphological analysis d) x-ray scattering (in small angle configuration) and dsc to access information on the inner structure. . a. schollier 1 , a. halperin 3 , m. sferrazza 2 , g. fragneto 1 1 institut laue-langevin, grenoble, france, 2 universite libre de bruxelles, belgium, 3 cnrs-ujf grenoble, france protein adsorption on surfaces is responsible of several unwanted effects in technological and pharmaceutical applications: fouling of contact lenses, clotting in blood containing devices, inflammation around artificial organs for instance. those phenomena can be repressed with certain types of polymer brushes, in particular peg (polyethylene glycol) brushes, and a better understanding of the mechanism of adsorption could lead to improvements in the design of biomaterials. we have developed a theory describing this mechanism and carried out measurements to confirm the theoretical predictions [langmuir 2007, 23, 10603] . three cases are predicted: primary adsorption at the grafting surface, secondary adsorption at the outer edge of the brush, and ternary adsorption within the brush itself. we prepared samples with different grafting densities and different degrees of polymerization and used different protein size and concentrations. neutron reflectivity experiments, able to determine the structure and composition of material interfaces with a fraction of nanometer resolution, were carried out and, by using deuterated proteins (anti-freeze protein, dihydrofolate reductase and myoglobin) on different peg compositions and grafting densities, it was possible to locate the proteins in the brush and to distinguish between the different kinds of adsorption : primary adsorption is dominant for short brushes and ternary adsorption for long brushes. effect of powder air polishing on nanocomposite dental materials measured by atomic force microscopy m. salerno 1 , g. derchi 2 , a. m. genovesi 2 , l. giacomelli 2 1 italian institute of technology, genova, italy, 2 istituto stomatologico tirreno, lido di camaiore, italy in dentistry, airpolishing with glycine and bicarbonate powders is widely used to remove accumulated plaque. however, the resulting teeth and gingival surface roughness, which is a risk factor for further plaque accumulation, has to be considered. in this in vitro study the effect of the above mentioned technique on the surface of a novel nanocomposite dental material is evaluated by means of atomic force microscopy (afm). square specimens (0.5×0.5 cm 2 size) were airpolished either with glycine or bicarbonate, using different application times (2, 8, 30 sec) and distances (2, 4, 6 mm) . four specimens were evaluated for each timedistance combination, and checked versus untreated specimens (controls). the specimens were imaged with tappingmode afm at two different scan sizes (3×3 and 30×30 µm 2 ) in two different regions for each sample. the surface roughness was measured as the rms of the feature heights. for the 2 sec2 mm group airpolishing resulted in an increased surface roughness as compared to the controls; however, glycine was associated with a lower roughness than bicarbonate (glycine: 238±91 nm; bicarbonate: 436±75 nm; controls: 14±14 nm; p<0.0001 for treated specimens vs controls, p<0.001 for glycine vs bicarbonate, anova test). similar results were obtained for all the other timedistance combinations. the work comprises the design of polysaccharide based nanoparticles for drug delivery. chitosan/arabic gum and arabic gum/maltodextrin nanoparticles were prepared by polyelectrolyte complexation and spray drying [1] , respectively. dynamic light scattering characterization established that arabic gum/maltodextrin nanoparticles are more polydisperse (diameters ranging from 8 to 400 nm) than chitosan/arabic gum particles (diameters of approximately 500 nm). scanning electron microscopy measurements demonstrated that the particles are spherical and have a smooth surface. the systems are highly stable to forces up to 80 nn as observed by atomic force microscopy. due to their nature they are hydrophilic and biodegradable. the nanoparticles were used to entrap short peptide sequences and antioxidants and were proved to be efficient in maintaining the biological activity of the molecules. kyotorphin (ktp) was found in 1979 in bovine brain. despite revealing remarkable analgesic properties, analgesia was only induced after central delivery. this limited ability to cross the blood-brain barrier (bbb) combined with the unknown mechanism of action largely confines its pharmacological use. to surpass these problems, we designed new ktp derivatives: ktp-rc and ktp-rc-lipogen. biophysical studies were carried out using fluorescence methodologies to characterize the peptides' interaction with biomembrane model systems. partition coefficient quantification showed a clear preference of the derivatives towards fluid zwitterionic and anionic membranes. moreover, a relationship between anionic lipid percentage and in-depth insertion in membrane was established. additionally, studies with fluorescent probe di-8-anepps revealed different membrane-interaction profiles of morphine and ktp-derivatives, suggesting distinct actions between them. the analgesic efficacy of the compounds was studied in vivo after systemic administration in models of acute and tonic pain. unlike ktp, both ktp-rc and ktp-rc-lipogen displayed high efficacy from doses as low as 1.67 and 0.85 mg/100g, respectively, indicating bbb crossing. the observed correlation between higher partition/insertion in the membrane and enhanced analgesic action proved the biophysical rationale to be a powerful strategy for early screening in cns drug development. metabolic syndrome (ms) is now regarded as a major risk factor for cardiovascular disease. as prooxidant/antioxidant balance is disturbed in the course of this disorder, it is possible that melatonin may exhibit protective effect against oxidative damage of blood cells. ms was diagnosed according to international diabetes federation definition (2005) . the investigated group consisted of patients before and after the melatonin supplementation (5 mg per day for two months) and was compared to control group of healthy individuals on normal diet. our experiments show that erythrocytes from patient group exhibit significantly higher tbars and total cholesterol levels whereas the protein thiol concentration, na + k + atpase and glutathione peroxidase activities were decreased in comparison to those of healthy volunteers. after two month melatonin supplementation, tbars and cholesterol concentrations significantly decreased, whereas the na + k + atpase, catalase and glutathione peroxidase activities increased. the glutathione concentration was also higher. these results show that melatonin supplementation has a protective effect on erythrocytes of ms patients. action of ferritin, a nanoparticle model, on ros formation and glutamate uptake in synaptosomes t. waseem, a. alekseenko, s. fedorovich institute of biophysics and cell engineering, minsk, republic of belarus nanoparticles are currently used in medicine as agents for targeted drug delivery and imaging. however it has been demonstrated that nanoparticles induce neurodegeneration in vivo and kill neurons in vitro. the cellular and molecular bases of this phenomenon are still unclear. we have used the protein ferritin as a nanoparticle model. ferritin contains iron particles (fe 3+ ) with size 7 nm and a protein shell. we investigated how ferritin influences uptake and release of [ 14 c]glutamate and free radical formation as monitored by fluorescent dye dcfda in rat brain synaptosomes. we found that even a high concentration of ferritin (800 µg/ml) did not induce spontaneous [ 14 c]glutamate release. in contrast the same concentration of this protein inhibited [ 14 c]glutamate uptake two fold. furthermore ferritin induced intrasynaptosomal ros (reactive oxygen species) formation in a dose-dependent manner. this process was insensitive to 30 µm dpi, an inhibitor of nadph oxidase and to 10 µm cccp, a mitochondrial uncoupler. these results indicate that iron-based nanoparticles can cause ros synthesis and decrease glutamate uptake, potentially leading to neurodegeneration. functionalized carbon nanotubes 1 (cf-cnts) have a promising future as vectors for drug delivery 2 and for neuronal cell growth and communications 3 . it has been demonstrated that cnts can be employed in drug delivery systems. the toxicological properties of cnts result strictly correlated with nanotube solubility 4 . in this study we focused our attention on multi-walled cnts (mwcnts) because they are easy to manipulate and we covalently functionalize them in different ways to increase their solubility.. we grew different derivatives of a dendrimer on mwcnts surface with positive charges at the periphery, in order to study the different solubilisation properties and correlate them to the biological activity in gene therapy 5 . slowdown of 1-40 β-peptide aggregation by addition of two synthetic biocompatible polymers p. sciortino 1 , r. carrotta 1 , g. cavallaro 2 , d. bulone 1 , p. l. san biagio 1 1 ibf-cnr, palermo, italy, 2 ctf dept., university of palermo, palermo, italy fibril deposit formation of amyloid β-protein (aβ) in the brain is a hallmark of alzheimer disease (ad). fibrils formation is triggered by molecular conformational changes and protein-protein interactions involving partially unfolded regions of different aβ peptide molecules. increasing evidence suggests that toxicity is linked to diffusible aβ oligomers, which have been found in soluble brain extracts of ad patients, rather than to the insoluble fibres. new therapeutical approach, based on searching molecules capable of regulating the peptide aggregation, is currently developing. here, we study the effects on the aggregation of aβ 1-40 peptide of two different synthetic polymers with structure similar to that of a protein (α,β-polyasparthylhydrazide -pahy and α,β-polyasparthylhydrazide-polyethyleneglycol -pahy-peg). static and dynamic light scattering measurements showed that the aggregation kinetic is slowed down by the presence of both polymers. optical microscopy revealed the presence of aggregates of different dimension in all samples. transmission electron microscopy allowed to establish that all aggregates are made of fibers, as confirmed by fluorescence spectroscopy measurements on thioflavine binding. a. smorodchenko 1 , d. sittner 1 , a. rupprecht 1 , j. goyn 1 , a. seiler 2 , a. u. bräuer 1 , e. e. pohl 1 1 institute of cell biology and neurobiology, charité-universitaetsmedizin, berlin, germany, 2 german federal institute for risk assessment, zebet, berlin, germany ucp4 is a member of the mitochondrial anion transporter family and one of the three ucps (ucp2, ucp4 and ucp5) associated with the nervous system. in our previous work we have shown that ucp4 appears in brain at embryonic day 14 (e14). we hypothesized that the ucp4 expression can be related to neuronal differentiation. to prove this idea we now investigated protein and mrna levels in two different systems: (i) mouse embryonic stem cells of line d3 (mesc) and (ii) brains from pre-and postnatal mice. ucp4 was not present in undifferentiated mesc. during differentiation of mesc in neurons the expression of neuronal marker map2 and ucp4 started at the same time -as early as on day 7 in culture -and were increasing simultaneously. (ii) the comparative analysis of gene transcripts prepared from whole embryos, brains and different brain regions (neocortex, hippocampus, cerebellum) demonstrated that levels of ucp4 mrna were increasing from e8 till e19, reached an expression peak between e19 and postnatal day 0 (p0) and remained constant in adult animals. in contrast, expression of ucp5 was increasing permanently until birth, whereas ucp2 expression was invariant in time. our results suggest that ucp4 contributes to specific neuronal functions. the plant hormone abscisic acid stimulates the proliferation of human hemopoietic progenitors through the second messenger cyclic adp-ribose s. scarfì 1 , c. fresia 2 , c. ferraris 1 , s. bruzzone 2 , f. fruscione 1 , c. usai 3 , m. magnone 2 , m. podestà 4 , l. sturla 2 , l. guida 2 , g. damonte 2 , a. salis 2 , a. de flora 2 , e. zocchi 2 1 advanced biotechnology center, genova, italy, 2 dept of experimental medicine, biochemistry section, and cebr, university of genova, italy;, 3 institute of biophysics, cnr, genova, italy, 4 stem cell center, s. martino hospital, genova, italy abscisic acid (aba) is a hormone involved in pivotal physiological functions in higher plants. recently, aba was demonstrated to be produced by human granulocytes, β pancreatic cells and mesenchymal stem cells (msc) and to stimulate cell-specific functions. here we show that aba expands human hemopoietic progenitors in vitro, through a cadpr-mediated increase of the intracellular calcium concentration. incubation of cd34 + cells with micromolar aba also induces transcriptional effects, which include nf-κb nuclear translocation and transcription of cytokines encoding genes. stimulated human msc produce and release aba at concentrations sufficient to exert growth-stimulatory effects on co-cultured cd34 + cells, as demonstrated by the inhibition of colony growth in the presence of an anti-aba monoclonal antibody. these results provide a remarkable example of conservation of a stress-hormone from plants to humans and identify aba as a new hemopoietic growth factor involved in the cross-talk between hp and msc. t. golovanova, g. b. belostotskaya sechenov institute of evolutionary physiology and biochemistry, russian academy of sciences, saint petersburg, russia a subpopulation of small cells (volume 141±6 µm 3 ) have been found in the neonatal cardiac myocytes culture whereas the same parameter of the remaining cells was 819±68 µm 3 on the 1 st day. in contrast to main part of hypetrofied cardiomyocytes, the small cells were able to proliferate, form colonies and differentiate spontaneously into cardiac myocytes in the culture. on the 8 th day there were slight, slow (2-3 beats/min), arrhythmic contractions in the centre of colonies. pulsing cells were not united by common contraction and had individual beating profile. on the 11-12 th days, the colonies displayed comprehensive contractile activity with the pulsation rate 25-28 beats/min and reached 46 beats/min on the 23 d -25 th day and 58 beats/min on the 26-30 th days in the culture. it has been shown that receptors of the surface membrane and sarcoplasmic reticulum of colony cells gradually mature. by estimating the ca 2+ transition under the specific agonists action: acetylcholine, kcl and caffeine, we have detected the activity of basic structural and functional elements of excitation-contraction coupling in contractile cells inside colonies. the small cells ability to proliferate and differentiate under the influence of near mature cardiomyocytes allows us to put forward a hypothesis, that they belong to a category of resident stem cells. changes in the gating properties of na + channels were studied in es-derived neural stem (ns) cells during in vitro neuronal differentiation with the use of the whole-cell and cell-attached variants of patch-clamp technique. ns cells represent a novel stem cell population remaining stable and highly neurogenic over multiple passages.voltage-clamp recordings during neuronal differentiation of ns cells indicated significant changes in the key properties of na + currents. a voltage-gated and tetrodotoxine-sensitive na + current,absent under self-renewal conditions, was first recorded following application of differentiative agents. current density increased with time of exposure to differentiating conditions. whole-cell and single channel analysis revealed that the observed increase in current density was due at least in part to changes in steady-state activation and inactivation properties. namely, half activation potential shifted from -34 mv to -45 mv, while half-inactivation potential shifted from -94 mv to -78 mv. furthermore, a contribution to the increase in na + current density could also be given by an enhancement in channel expression, as suggested by an augmentation in the number of single channels per patch area, with increasing neuronal differentiation. interestingly, those changes in na + channel activity well correlate with the capability of ns cells to generate action potentials during in vitro neuronal differentiation. a. viale, g. bonizzi, a. cicalese, f. de franco, c. pasi, s. pece, a. orleth, p. di fiore, p. pelicci european institute of oncology, milan, italy we are characterizing the biological differences between normal and transformed scs. scs are defined by their ability to generate more scs, termed self-renewal, and to produce cells that differentiate (asymmetric cell division). scs, however, possess the ability to expand in number (i.e. during development, in adulthood after injury/disease); this increase is not accounted by asymmetric divisions, in which only one daughter cell maintains sc identity. recent findings in invertebrates indicate that scs can also generate daughter cells destined to acquire the same fate (symmetric cell division). on the other hand, sc quiescence is critical to maintain tissue homeostasis after injury. here we show increased symmetric divisions of cscs in breast tumors (due to inactivation of the p53 tumor suppressor) and dependency of leukemia development on quiescent leukemia scs (due to transcriptional up-regulation of the cell cycle inhibitor p21 by leukemia-associated fusion proteins). we suggest that sc asymmetric divisions function as a mechanism of tumor suppression, that sc quiescence is critical to the maintenance of the transformed clone and that symmetric divisions of scs permit their geometric expansion. a. uccelli department of neurosciences ophthalmology and genetics, genoa, italy stem cells are considered as a possible source of cells for tissue repair. in this scenario damaged tissues will be reconstructed by newly formed cells with the result of a recovery of lost functions. thus, the rationale for utilizing stem cells for the treatment of neurological diseases such as multiple sclerosis (ms) stemmed from the idea that they differentiate in neural cells regenerating the damaged tissues at the basis of irreversibile disability. however, while multipotential embryonic stem cells may provide in the future an optimal source of cells competent for myelin and axons repair in ms, their use is still far from being exploitable in a clinical setting. moreover, it is widely accepted that adult stem cells may in vitro transdifferentiate in neural cells but recent experimental data have challenged the biological importance of this event in vivo. nevertheless, several experimental studies have provided new evidences supporting the use of adult stem cells derived form different human tissues for the treatment of ms. in the cases of mesenchymal stem cells and neural stem cells current experimental data support an unexpected therapeutic plasticity mediated by diverse paracrine mechanisms including neuroprotection, induction of local neurogenesis and modulation of the immune response. therefore, current experimental and clinical studies support the use of stem cells for the treatment of neurological diseases such as ms. unravelling the structure of the water splitting site of photosynthesis and implications for mechanism of catalysis j. barber imperial college london, u.k. photosystem ii (psii) is a multi-subunit membrane protein complex which catalyses the oxidation of water to molecular oxygen and reducing equivalents. the reaction occurs at a catalytic centre composed of 4 mn ions and a ca ion, is thermodynamically demanding and generates highly oxidised species. unavoidable side reactions cause detrimental effects on the protein environment leading to the rapid turnover of the reaction centre d1 protein. to understand the mechanisms of water oxidation and d1 turnover structural information is required. initially the positioning of various protein subunits and their transmembrane helices were determined by electron microscopy. more recently a refined structure of the cyanobacterial psii unit has been elucidated by x-ray crystallography giving details of specific environments of the redox active cofactors. the implications of these structural studies will be discussed in relation to the unique facets of psii function, particularly the water splitting reaction. importantly this new knowledge is providing a blue print for the design of photochemical catalysts which can mimic the photosynthetic water splitting reaction and thus give hope that new technologies will emerge to provide humankind with a sustainable energy supply. photochemically controlled molecular devices and machines v. balzani department of chemistry "g. ciamician", university of bologna, italy the macroscopic concepts of a device and a machine can be extended to the molecular level. molecular-level devices and machines operate via electronic and/or nuclear rearrangements and, like macroscopic devices and machines, they need energy to operate and signals to communicate with the operator. the extension of the concepts of a device and a machine to the molecular level is of interest not only for basic research, but also for the growth of nanoscience and the development of nanotechnology. if a molecular device or machine has to work by inputs of chemical energy, it will need addition of fresh reactants ("fuel") at any step of its working cycle, with the concomitant formation of waste products that compromise the operation. currently there is an increasing interest in the use of light to power molecular devices and machines. the lecture will illustrate examples of recent achievements [1, 2, 3] , which include molecular wires, switches, plug-socket systems, extension cables, antennas, and light powered nanomotors. biophysics laboratory, institute of botany, azerbaijan national academy of sciences, baku, azerbaijan it is well known that fast component of induction curves of millisecond delayed chlorophyll fluorescence (ms-df) originates via radiative recombination of reaction center with product on the donor side of psii. in view of our previous data (j. photochemistry and photobiology b: biology 86, 2007) it was shown that partners for radiative recombination of p 680 q a − localized as a hole on the donor side of psii. depending on ph this hole might be on the camn 4 -cluster or on y z and their recombination with p 680 q a − can be monitored by the ms-df fast component. for analysis of site of damages, photoinhibition of psii particles from spinach at different ph was monitored by ms-df. during photoinhibition of psii (4000 µmol photons m −2 s −1 ) the fast component of ms-df was shown to be more stable at ph 5.5 and ratio of the fast component to the steady-state level of ms-df was approximately constant, while at ph 8.5 the fast component essentially decreased, the steady-state level increased and ratio of these components rapidly went down. it is possible concluded that strong light damaged of recombination reaction with in the presence ppbq -artificial electron acceptor -the protection of psii from photoinhibition has been observed only at acidic condition. stress factors such as heavy metals, strong light and low temperature were investigated by measurement of transient pictures of ms-df in intact plants, isolated chloroplasts and pure psii particles. the heavy metals action on psii was investigated on wheat seedlings. the targets for toxic action of al, mn and co ions were found to be a q a -q b acceptor side of psii. the damage site for cd 2+ may be partners for recombination with p680 + -depend on of medium ph -either y z or camn 4 -cluster. investigated ions (mn 2+ , al 3+ , cd 2+ and co 2+ ) have lead to reduction of chlorophyllprotein complexes pigment fund and cd 2+ and co 2+ destroyed also an apoproteins, especially of psii. the photoinhibition of psii was investigated in intact leaves of barley and maize seedlings at low and normal temperature. a very sharp reduction of intensity of ms-df second fast component, possibly y z * p680ÿq a − radiative recombination of maize seedlings was observed even after short illumination by strong light at 4 • c. ph dependence photoinhibition of chloroplasts and pure psii particles have shown that strong light damaged of camn 4 -cluster in great degree than y z . hydrogenases are considered as potential energy sources. in particular, the ability of the green alga c . reinhardtii to reduce protons to hydrogen gas upon illumination by means of a [fefe]-hydrogenase is a phenomenon of great scientific interest, as it would need only light and water to generate energy. however, the catalytic activity is strongly inhibited by the o 2 produced during photosynthesis; furthermore, the protein is expressed at very low levels, only in conditions of strict anaerobiosis. this mutually exclusive nature of o 2 and h 2 photoproduction represents a crucial problem in the development of h 2 bio-production. the study of the structurefunction relationship of [fefe] hydrogenases, which would help to clarify the molecular mechanisms underlying both h 2 production and o 2 sensitivity, requires the characterization of purified native and modified proteins, which can be obtained by site-directed mutagenesis. we expressed the algal hydrogenase in the cyanobacterium synechocystis sp. pcc 6803, which holds a bidirectional [nife]-hydrogenase with a well known maturation system (1) . we obtained two constructs to stably transform synechocystis, enabling it to express the c. reinhardtii hydrogenase in an active form. this suggests that the [nife]-hydrogenase maturation pathway is able to drive the biosynthesis of functional [fefe] enzymes. these data open new perspectives about the indispensable presence of hyde, hydf and hydg auxiliary proteins (2, 3) to obtain a correctly folded [fefe]-hydrogenase. the a2 low energy monomeric chlorophyll of cp29 to investigate the role of low energy spectral forms in energy transfer among chlorophyll protein complexes, we characterized one of the redmost chlorophylls of psii antenna complexes, the a2 of cp29. pigment stoichiometry of the wild type (cp29wt) and mutant lacking the a2 binding residue (cp29a2) reconstituted complexes confirmed the loss of a chlorophyll a. to exclude the possibility of a marked excitonic interaction of the a2 with other chlorophylls, we quantified the decrease in dipole strength after mutagenesis as a function of the frequency and performed a second derivative analysis of the difference absorption spectrum cp29wt minus cp29a2. these data, along with circular dichroism spectra of both complexes, support the idea that the a2 is a monomer in cp29, in disagreement with recent suggestion of involvement in an excitonic dimer (mozzo et al., bba 2008) . chla a2 reorganization energy and inhomogeneous broadening were then determined through a thermal broadening analysis in the 80-285 k temperature range. these parameters allowed us to calculate the förster overlap integral of the homogeneously broadened a2 bandshape, a fundamental factor for energy transfer rate estimations between chromophores. a comparison with foi of chlorophyll in solution suggests that chla a2 may connect cp29 complex to psii core favoring energy transfer from cp29 towards other inner antenna low energy chlorophylls. in oxygenic photosynthetic organisms, reaction centre complexes catalyze electron transport from water to co2 using light energy absorbed by tetrapyrrole pigments bound to so called "antenna proteins". a major challenge in performing this type of photosynthesis consists on the difficulty of operating "one electron" transport through a multi-step pathway in the chloroplast environment which is the source of oxygen of biosphere. in fact, synthesis of ros, mainly singlet oxygen and superoxide anion and consequent photoinhibition is an intrinsically unavoidable consequence of photosynthesis that must be prevented and/or controlled in order to avoid photodestruction and death. mechanisms involved include chlorophyll (chl) triplet quenching, ros scavenging and controlled heat dissipation of excess chl singlet excited states. the latter process has been studied for over 40 years with little success in elucidating its mechanism. reverse genomics and ultrafast-spectroscopy led to the proposal that the transient formation of carotenoid radical cations, followed by charge recombination might be the underlying mechanism of energy dissipation while three proteins belonging to the lhc superfamily could be the site hosting the reaction. probing the dark cycle to reveal regulatory networks controlling photosynthetic efficiency the food, fibre and fuel needs of an ever-increasing population are one of the challenges facing current society. higher plant photosynthetic efficiency is ∼1% whereas the theoretical maximum is thought to be ∼6-8% giving potential for great improvements. it is not however clear where in the photosynthetic process the additional losses are occurring. we are adopting a systems biology approach to reveal the regulatory networks that control photosynthetic efficiency, the challenge being to make quantitative measurements of the input parameters to models of the network of photosynthetic reactions, and also to identify missing physical parameters and processes. the aim being to understand how they interact with other key physiological pathways and what impact they hold over photosynthetic efficiency. we report initial results in this poster from experiments using conventional and novel proteomic methods. e.g. an optical technique based on a non-linear 2dir method is being developed for proteomic and metabolomic analysis. unlike many conventional proteomic methods, which provide information on the relative levels of various proteins and metabolites, the viability of the 2dir method as a quantitative, sensitive and high throughput proteomics platform has recently been demonstrated. in collaboration with klug this project will employ 2dir as a both a proteomic and metabolomic tool. t. morosinotto 1 , p. arnoux 2 , g. saga 1 , g. m. giacometti 1 , r. bassi 3 , d. pignol 2 1 dip. di biologia, padova, italy, 2 cea, cadarache, france, 3 dip. di biotecnologie, verona, italy violaxanthin de-epoxidase (vde) is the enzyme responsible for zeaxanthin (z) production. the synthesis of this carotenoid in plants exposed to high light conditions is an important photoprotection mechanism, enhancing excess energy dissipation and reactive oxygen species scavenging. the inactive enzyme is normally soluble but, upon activation by low ph, it binds to the thylakoids membrane, where its substrate is found. we present here the first structural data on this enzyme obtained at both acidic and neutral ph. at neutral ph, vde is monomeric with its active site occluded in the lipocalin barrel. upon acidification, the barrel opens up resulting in a functional dimerization of the enzyme. the channel linking the two active sites of the dimer can harbour the entire carotenoid substrate and thus allow the parallel de-epoxidation of the two violaxanthin β-ionone rings, making vde an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate. structural data opened the possibility to investigate deeper this enzyme and further work allowed the identification of its active site, the protein domains responsible of its membrane association as well as key residues involved in the ph dependent conformational change. hydrogen is considered the fuel of the future if produced from sun light-driven water splitting. a hydrogen economy based on genetically modified organisms, immobilized enzymes or biomimetic synthetic catalysts requires a profound knowledge of the structure and function of the respective enzymes in nature. 1 light-induced water splitting is performed by a tetranuclear manganese cluster located in photosystem (ps) ii of oxygenic photosynthesis. the protons generated by ps ii can be converted to molecular hydrogen by the enzyme hydrogenase, which is for example found in green algae and cyanobacteria. [nife]-and [fefe]-hydrogenases are the two main classes of this enzyme. they contain bridged binuclear transition metal cores, which are tuned by a special ligand environment to efficiently convert protons to hydrogen -or vice versa -via a heterolytic mechanism. 2 rhodobacter sphaeroides strain a-10 isolated from mineral springs in armenia produces h 2 with high rate in anaerobic conditions under light. in our previous work the inhibition of h 2 production by n,n' -dicyclohexylcarbodiimide (dccd), the f 0 f 1 -atpase inhibitor, was shown, and dccd-inhibited atpase activity was determined. therefore it is possible to admit a role of this atpase in h 2 production by r. sphaeroides; otherwise dccd inhibition of hydrogenase is not ruled out. in order to examine the mediatory role of proton-motive force (pmf) or proton atpase in this process transmembrane electrical potential (∆ψ) and ∆ph are determined and the atpase activity is studied in r. sphaeroides grown under light. pmf was determined under anaerobic conditions in the dark. at ph 7.5 the ∆ψ was of -94 mv and the reversed ∆ph was +30 mv, resulting in the pmf of -64 mv. but ∆ψ was not affected by dccd. moreover, adenine nucleotide phosphates (anp) content is essential for cell functioning and may result with atpase activity. the percentage of atp was calculated from the total quantity of anp in whole cells, it was 46.6 %. a relatively high concentration of adp (∼35 %) and amp (∼18 %) and accordingly low energetic charge were noted; these might be indicative for the atpase activity, a further study is required. relationship between h 2 production and atpase activity by rh. sphaeroides is suggested; possible mechanisms are discussed. identification of the sites of chlorophyll triplet quenching in relation to the structure of lhc-ii from higher plants. evidence the chl a molecules involved in the triplet-triplet energy transfer to the central luteins in trimeric lhc-ii are identified by time-resolved and pulse epr techniques. the concept of spin conservation during triplet-triplet energy transfer is exploited. the sites with the highest probability to form triplet states, which are quenched by the central luteins, are chl603 and chl612. unquenched chl triplet states are also produced by photo-excitation in the lhc-ii complex. putative sites of these triplet states are chl614, chl611, chla604 and chl613, since they do not contribute to the formation of the observed carotenoid triplet states. fluorescence lifetime spectrum of the plant photosystem ii core complex the photosystem ii kinetic model (diffusion or trap-limited) is still much debated. there is discussion about whether energy transfer from the core antenna (cp47 and cp43) to the reaction center complex (d1-d2-cyt b559) is rate-limiting (transfer to trap-limited). this study investigates this problem in isolated core particles by exploiting the different optical properties of the core antenna and the reaction center complex near 680 nm, due to p680 and an isoenergetic pheophytin. this was used as a marker feature for the reaction center complex. if the transfer to the trap-limited model were correct, assuming excited-state thermalization, the specific reaction center fluorescence decay lifetime should be shorter near 680 nm, where there is reaction center complex specificity, than at the other emission wavelengths. such a selective reaction center feature was not observed in fluorescence decay measurements. at the experimental resolution used here, we conclude that the traplimited energy transfer to the reaction center could, at the most, be 20% limiting. thus, the transfer to the trap-limited model is not supported. photosynthetic apparatus response under heat stress n. pshybytko 1 , i. divak 1 , l. kabashnikova 1 , e. lysenko 2 1 institute of biophysics and cell engineering, national academy of sciences of belarus, belarus, 2 institute of plant physiology, russian academy of sciences, russia the mechanisms of psii thermoinactivation and adaptation under high temperature impact and participation of hydrogen peroxide at these processes were studied. the twice suppression of oxygen evolving activity of thylakoids with simultaneous decrease in d1 protein content and the release of extrinsic 33 kda polypeptide from woc after 15-20 min heating at 40 o c were registered. using inhibitor analysis it was shown that thermoinduced degradation of d1 protein after 20 min heating occurred by proteases. the participation of ftsh protease in thermoinduced d1 protein degradation was observed. level of transcription of psba gene in chloroplast was raised after 20 min heating and was decreased through 1 h. the content of hydrogen peroxide was increased three times after 20 min of heating and was decreased to normal level through 1 h and was raised after 2.5 h again. it is interesting that level of peroxidation lipids products was increased after 2.5 h heating only. received data indicated that hydrogen peroxide is signal molecular at the photosynthetic apparatus under heat stress. during heating the inactivation of woc and d1 protein is occurred. as result the h 2 o 2 is generated. hydrogen peroxide as signal molecule activates transcription of psba. turnover of psii is occurred. more long heating induces degradation of proteins and lipids and h 2 o 2 represents as the destructive agent. a. nurisso 1 , m. a. morando 2 , f. j. cañada 2 , j. j. barbero 2 , a. imberty 1 1 cermav-cnrs, grenoble, france, 2 centro de investigaciones biológicas, madrid, spain the mechanism of symbiosis between legume plants and rhizobial bacteria is a relevant topic of interest since it is at the basis of the nitrogen fixation process. this mechanism is strictly related to the production of lipochitooligosaccharides, nod factors (nf), which allow the bacterial invasion into the legume host roots. in medicago truncatula, the recognition of nf from the symbiont sinorhizobium meliloti requires the nfp gene, able to encode a lysm-motif receptor-like kinase. the extracellular part of this protein is characterized by three lysm motifs: only one of them seems to be involved in the nf recognition. herein, we report an in silico investigation of different structural aspects of this process. first of all, the conformational behaviors of a new generation of nf analogues were elucidated by mds simulation. then, a homology model of the lysm2 domain from m. truncatula was proposed and compared with a model of lysm domain from pisum sativum: docking calculations of natural nf identified a common binding site in which the carbohydrate portion is the main responsible of the binding. both studies provide support for the idea that the carbohydrate part of nf plays a key role in the interaction with lysm domains, while the lipid moiety modulates ligand specificity probably interacting with a potential second receptor. interaction between frutalin with biomembrane models and its correlation with cell adhesion property frutalin is a homotetrameric lectin d-galactose (d-gal) binding that activates natural killer cells in vitro and leukocyte migration in vivo, being a potent lymphocyte stimulator. in this study we investigated the interaction of frutalin with different phospho/glyco-lipids using langmuir monolayers as biomembrane mimetic system. the results attest the specificity of the protein for the carbohydrate d-gal when attached to the biomembrane model. the adsorption kinetics for frutalin to the mixed monolayers containing glycolipids showed that the interaction depends on the presence of charged groups and on the position of the d-gal on the polar head of the glycolipid. using brewster angle microscopy (bam), we investigated the morphology of the interface for the binary mixtures containing galcer, where small domains were formed at high lipid packing, suggesting that frutalin can induce the formation of likelipid rafts domains in vitro. the results obtained with the membrane models were associated with those from fibroblast adhesion induction. the cell adhesion promoted by frutalin is in accordance with the results observed in langmuir monolayers, which probes the specificity of the interaction between the lectin and d-gal on cell-membrane surfaces. based on these results, frutalin can be considered as a promise biotechnological tool to actuate in tissue engineering regeneration. supported by fapesp, cnpq and capes aggregation and glycation processes of proteins are of peculiar interest for several scientific fields. serum albumins are widely studied proteins for their ability to self-assemble in aggregates and also to undergo to non enzymatic glycosylation in cases of diabetes. in this work we report a study on thermal aggregation of glycated bovine serum albumin (bsa) prepared with different concentrations of glucose at ph 7.4. increasing concentration of sugar modulates the effect of different glycation levels on the protein aggregation. fluorescence spectroscopy, ftir absorption, static and dynamic light scattering are used to follow the time evolution of the aggregation process and of protein conformational changes. conformational changes of secondary and tertiary structures are measured by ftir absorption; the kinetics of amide i, amide ii and amide ii' bands are monitored. the kinetics of tryptophans fluorescence give complementary information on the tertiary structure changes and on the polarity modification of the fluorophores environment. the aggregates growth is studied by dynamic light scattering measurements and rayleigh scattering peak. the results show that the partial unfolding of the protein is not affected by the glycation, while the presence of increasing amounts of glycated molecules progressively inhibits the aggregates formation. solvent occupancy analysis in ligand structure prediction: the case of galectin-1 s. di lella, m. a. martí, d. a. estrin inquimae-conicet, universidad de buenos aires, argentina formation of protein ligand complexes is a fundamental phenomenum in biochemistry. during the process, significant solvent reorganization is produced along the contact surface. using md simulations in explicit solvent combined with statistical mechanics analysis, thermodynamic properties of water molecules around proteins can be computed and analyzed in a comparative view. based on this idea, we developed a set of analysis tools to link solvation with ligand binding in a key carbohydrate binding protein, human galectin-1 (hgal-1). specifically, we defined water sites (ws) in terms of the thermodynamic properties of water molecules strongly bound to protein surfaces. we then succesfully extended the analysis of the role of the surface associated water molecules in the ligand binding and recognition process to many other carbohydrate binding proteins. our results show that the probability of finding water molecules inside the ws, p(v), with respect to the bulk density is directly correlated to the likeliness of finding an oxhydril group of the ligand in the protein-ligand complex. this information can be used to predict possible complex structures when unavailable, and suggest addition of ohcontaining functional groups to displace water from high p(v) ws to enhance drug, specially glycomimetic-drugs, protein affinity and/or specificity. glyconanoparticles: nano-biomaterials for application in biotechnology and biomedicine s. penadés laboratory of glyconanotecnology, cicbiomagune -ciber-bbn, san sebastian, spain to study and intervene in carbohydrate interactions our laboratory has developed a chemical strategy (glyconanotechnology) to produce sugar functionalized gold nanoclusters with multivalent carbohydrate display (glyconanoparticles, gnps). by combining these tools with biophysical and analytic surface techniques (itc, afm, tem, spr) we have demonstrated and evaluated ca 2+ -mediated carbohydratecarbohydrate interactions involved in cell adhesion processes. the gnp system complements other currently available multivalent systems incorporating carbohydrates and presents some advantages as: 1) exceptionally small core size; 2) high stability in water and physiological buffers; and 3) multivalency and multifunctionality with control over ligands number. specific gnps have been applied to the inhibition of melanoma metastasis in mice and as inhibitors of hiv-trans infection. magnetic gnps have also been used in cellular labelling and imaging as probes for mri. in this lecture, the glyconanotechnology strategy will be presented and some application will be highlighted. -glycobiophysics expanded ataxin-1 induces membrane mechanical instability: evidences from model systems and cells c. canale 1 , s. averaimo 2 , d. pesci 3 , d. paulis 2 , v. fortunati 3 , a. gliozzi 4 , m. mazzanti 2 , c. jodice 3 , a. relini 4 1 iit, genoa, italy, 2 university of milan, italy, 3 university of tor vergata, rome, italy, 4 university of genoa, italy spinocerebellar ataxia type 1 is a neurodegenerative disorder involving the expansion of a polyglutamine stretch beyond a threshold in the protein ataxin-1, with intracellular deposition of amyloid aggregates. ataxin-1 variants with polyglutamine stretches of pathological length are not associated to disease when the stretch is interrupted by histidines. mounting evidence suggests that ataxin-1 aggregates interact with the nuclear membrane. to get insight into the mechanisms leading to neurological disorders, we studied model lipid membranes containing an expanded pathological form of ataxin-1 or expanded normal forms interrupted by histidines. electrical measurements on planar bilayers showed the occurrence of current steps, much larger for the pathological form, indicating the formation of pore-like structures. atomic force microscopy measurements showed that the longer the polyglutamine tract, the smaller was the force required to penetrate the bilayer with the afm tip. the smallest penetration force, and then the strongest membrane destabilization, was observed for membranes containing the expanded pathological form. experiments on cos1 cells provided evidence that pathological protein aggregates damage the nuclear membrane eventually causing cell autophagy. modification by dopamine adducts links αsynuclein to oxidative stress in parkinson disease m. bisaglia 1 , e. greggio 2 , l. tosatto 1 , f. munari 1 , i. tessari 1 , p. polverino de laureto 1 , s. mammi 1 , m. r. cookson 2 , l. bubacco 1 1 university of padova, italy, 2 nih, bethesda, md, usa oxidative stress has been proposed to be involved in the pathogenesis of parkinson disease (pd). a plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine (da) and produce various toxic molecules, i.e., free radicals and quinone species. α-synuclein (αsyn), a small protein found in lewy bodies characteristic of pd, is also thought to be involved in the pathogenesis of pd. to investigate the possibility of a synergistic role of oxidative stress and αsyn in pd, we analyzed the modulation of da toxicity by αsyn overexpression in dopaminergic human neuroblastoma cells. our results indicate that the increased expression of αsyn enhances the cellular toxicity induced by the accumulation of intracellular da. we then correlated our results with the structural modifications induced by the oxidation products of da on αsyn by studying two potential pathways for the oxidative chemistry associated with da. the first one is the auto-oxidation reaction which leads to the concomitant formation of radical and quinone species. the second is the enzymatic oxidation, mediated by tyrosinase, which leads only to the accumulation of quinones. our data suggest a link between da and αsyn in the progression of pd and provide novel insights on both the mechanisms involved in the oxidative chemistry and the aggregation properties of αsyn. dynamic light scattering aggregation studies of aβ (1-40) peptide s. bertini, r. beretta, d. gaudesi, a. naggi, g. torri ronzoni institute, milan, italy heparan sulfate (hs) proteoglycans play a role in formation of amyloid plaques by facilitating formation of amyloid aggregates. heparins and low-molecular weight heparins which mimic hs sequences could interact with amyloid peptides putatively involved in neurodegenerative processes. the aim of this work is to study the influence of chain length and structure of heparin oligosaccharides on the aggregation of amyloid. to this purpose, the interaction with amyloid peptide aβ 1−40 with a number of heparin/heparin oligosaccharides had been investigated using dynamic light scattering (dls) which permits to determine the size and the stability of molecular aggregates in solution. as indexes of aggregation in solution, two parameters were chosen, i.e., the area under the autocorrelation function (af) and the average hydrodynamic ratio (rh). we evaluated the aggregation kinetic of different preparations of aβ and the ph of solubilization. a clear indication was obtained for the trend of aggregation of the peptide aβ 1−40 alone and in the presence of oligosaccharides. the size of the aggregate in the presence of the tetrasaccharide is definitely lower than for the peptide alone. the size of the aggregates increases with increasing size of the oligosaccharides. in fact, the octasaccharide promotes further aggregation. it is crucial for pharmaceutical protein formulations to have low aggregate content. using the monoclonal igg1 antibody rituximab as a model system, we have studied the mechanisms by which antibodies aggregate at physiological ph when incubated at 52-65 • c. light scattering showed two coupled stages: an initial fast stage followed by several hours of exponential growth of the scattered intensity. data analysis showed the fast formation of a large species, which subsequently increased in size. the aggregate number density had a maximum suggesting that aggregates increase in size by coagulation. the analysis also predicted the actual underlying increase in aggregate mass to be linear and reach saturation. this was confirmed by size-exclusion chromatography of incubated samples. in an arrhenius plot the activation energy of the first stage was similar to the unfolding energy of the ch2 domain, suggesting a pivotal role of this domain in the aggregation process. cd and fluorescence showed only minor structural changes in the temperature interval studied. we conclude that coagulation is the main mechanism driving rituximab aggregation and that aggregation is due to small structural changes. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils -abstracts 123 solid-state nmr structural studies of alzheimer's disease amyloid β(1-42) in lipid bilayers j. d. gehman 1 , a. k. mehta 2 , f. separovic 1 1 school of chemistry, bio21 institute, university of melbourne, 3010 vic, australia, 2 department of chemistry, emory university, 1515 dickey dr., 30322 atlanta, ga, usa amyloid-β peptides are believed to cause loss of nerve cell function in individuals suffering from alzheimer's disease, where evidence suggests that interaction with the cell membrane correlates strongly with cytotoxicity. previous studies report a range of different but plausible structures, which depend on the molecular environment of the peptide. this sensitivity to sample preparation suggests that the structure relevant to disease is found in lipid bilayer membranes. while model membrane vesicles are too large to be studied by conventional solution nmr, solid-state nmr is one of the few technologies available to study such systems. we present recent measurements which suggest a novel peptide structure in a lipid bilayer environment: (i) rotational-echo double-resonance (redor) distance measurements between selectively enriched 13 c-carbonyl and 15 n-amide positions constrain dihedral angles of intervening residues and suggest that at least part of the aβ(1-42) peptide folds into a β-sheet like conformation, in contrast to the helical and coiled structures in previous reports; and (ii) double quantum filtered draws measurements indicate that the extended strand does not assemble into an in-register parallel sheet as reported for amyloid fibrils. a mutation in app gene with dominant-negative effect on amyloidogenesis: a light scattering study e. del favero 1 , g. di fede 2 , f. tagliavini 2 , m. salmona 3 , l. cantù 1 1 university of milan, segrate, italy, 2 "carlo besta" national neurological institute, milan, italy, 3 istituto di ricerche farmacologiche "mario negri", milan, italy we studied the effect of a single-point mutation (a673v) on the aggregative properties of alzheimer's peptides aβ1−40. by laser light scattering it was possible to follow the early stages of aggregation of aβ1−40 wt and aβ1−40 mut (within 48 hours after sample preparation), when intermediates in the aggregation pathway, rather than the mature insoluble fibrils, are formed. results show that both the kinetics and the extent of aggregation are strongly enhanced by the mutation. on the reverse, coincubation of mutated and wild-type peptides slows down the aggregation process and results in aβ aggregates that are much more unstable against dilution. it is evident that the interaction between mutant and wild-type aβ1−40 interferes with nucleation or nucleation-dependent polymerization, hindering amyloidogenesis. these results account for the highly amyloidogenic effect of the mutation in vitro, if alone, reversely reduced by the mutated-wild type mix. also a clinical case exists that constitutes the in-vivo evidence of these two opposing effects. this finding may offer grounds for the development of therapeutic strategies, based on modified aβ peptides or peptido-mimetic compounds, for the potential treatment of alzheimer's disease. the collagen i is the major structural protein of the body and it is found organized in a hierarchical manner in the extra-cellular matrix of several tissues (bone, tendon, cornea and sclera, etc..). it is responsible for their specific architecture. it is already known that collagen i is capable of forming cholesteric liquid crystalline phases that once stabilized by a ph increase, lead to collagen matrices that mimic the organization of the organic matrix found in bone [1] . in the present work, we analyze physico-chemical ways to modulate long range organizations obtained in this liquid state. we study the effect of collagen concentration, ph (2.6 / 3.6) and acids (hydrochloride acid / acetic acid) over the liquid crystalline order. in a original approach, correlation of collagen endofluorescence and shg signals has enabled us to quantify cholesteric pitch and phase transition as a function of collagen concentration within a gradient. cholesteric pitch have proved to be very responsive to physico-chemical conditions. indeed, the results allowed us to quantify a i / cholesteric transition as a subsequent transition from cholesteric to a phase hexagonal or colummar. prion diseases are deadly neurodegenerative diseases affecting human and mammalian species. according to the 'protein-only' hypothesis, the key event in the pathogenesis is the conversion of the α-helix-rich monomer (prp c ) into a polymeric β-sheet-rich pathogenic conformer (prp sc ). we have used a combination of biophysical techniques with molecular dynamics simulations (md) to elucidate the molecular mechanisms of prp c unfolding and polymerization. under well established conditions, three β-sheet-rich soluble oligomers were generated from the partial unfolding of the monomer, which were found to form in parallel. to obtain a deeper insight into the molecular events, doublecystein mutants were designed, thus 'locking' different regions of prp. single mutations were also performed, which affected dramatically and selectively the prp oligomerization pathway. furthermore, we have now identified the minimal region that leads to the same oligomerization profile as the full-length prp, namely, h2h3. the existence of at least three distinct oligomerization pathways and the effect of single mutations reveal the conformational diversity of prp and a possible relationship with prion strain phenomena. the identification of domains involved in the conversion process may lead to a better understanding of the effect of mutations or gene polymorphism on the evolution of prion pathology. investigating toxic protein nanoclusters and their interactions with living cells s. nag, b. sahoo, a. bandyopadhyay, c. muralidharan, r. abhyankar, s. gurav, s. maiti tata institute of fundamental research, homi bhabha road, colaba, mumbai 400005, india amyloid protein aggregation is responsible for many neurodegenerative diseases, such as alzheimer's and parkinson's. it appears that quasi-stable small soluble aggregates are the key to amyloid toxicity. though amyloid aggregation appears to be a nucleation mediated process, simple nucleation theory is unable to explain the stability of these intermediate species. we investigate this issue with fluorescence correlation spectroscopy in solution and on cell membranes. our initial data, varying the ph and the ionic strength of the solution, show that a charged colloid model can explain the overall stability of these species. in addition, this understanding suggests possible ways of modulating the stability of these aggregates in solution. some of these strategies successfully decimate the stable aggregate population, and also reduce the toxicity of amyloid beta to cultured neurons. afm studies of artificial amyloid fibrils and filaments p. mesquida 1 , a. kurtossy 1 , c. macphee 2 1 department of mechanical engineering, king's college london, london, u.k., 2 school of physics, university of edinburgh, edinburgh, u.k. amyloid fibrils are beta-sheet-rich superstructures of peptides or proteins. although these aggregates have first been found in connection with protein-misfolding diseases, such as alzheimer's or parkinson's disease, there is evidence that the ability to form fibrils is a generic property of any polypeptide rather than a result of specific, disease-related amino-acid sequences. fibrils can easily be formed in vitro from nondisease-related proteins and even from synthetic peptides. these artificial fibrils can thus serve as model systems to investigate the biophysical properties of amyloid fibrils. the system presented here is built from the artificial peptide ttr 105−115 , which contains 11 amino-acids and which forms well-defined nanorods of ca 10 nm diameter. we present atomic force microscopy (afm) results of their inner morphology by chemically dissecting the rods into their constituent filaments without completely breaking up the aggregates. in contrast to the stiff rods, their constituent filaments of ca 1-2 nm diameter were much more flexible. this shows that the particular way filaments are arranged around each other can massively change the mechanical rigidity of the resulting structures in amyloid fibrils, which could be one cause of the different strains in amyloid diseases. the formation of insulin fibrils is characterized by an initial apparent lag-phase, related to the formation of oligomers, protofibrils and aggregation nuclei. afterwards, the aggregation proceeds via fibril elongation, thickening and/or flocculation, and eventual gelation. here, we focus on the formation of such a gel, made of insulin amyloid fibrils, upon incubation at high temperature and low ph. by light scattering and rheological techniques, we monitor the development of the structural, dynamical and mechanical properties of fibrillar aggregates, up to the dynamic arrest of the sample and to the appearance of a non-ergodic behaviour, which marks the onset of gelation. our experiments were able to reveal the structural details hidden in the apparent lag-phase, displaying the slow fibril nucleation and elongation. this initial stage is followed by the known exponential growth of structures of different sizes. these two kinetic stages of structural growth are mirrored by the kinetics of the viscoelastic properties and, in particular, by the growth of the elastic modulus. our results show that the appearance of a noteworthy elastic network, is associated with the initial fibril nucleation and elongation more than with the formation of large structures, which causes the eventual gelation. capturing the initial events of in-cubo crystallization of membrane proteins c. v. kulkarni 3 , o. ces 1 , s. iwata 2 , r. h. templer 1 1 chemical biology centre and department of chemistry, imperial college london, united kingdom, 2 division of molecular biosciences, imperial college london, united kingdom, 3 institute für chemie, heinrichstrasse-28, university of graz, austria lipid based methods for the crystallization of membrane proteins including in-cubo crystallization are becoming popular in recent times. however, a complete understanding of the basic principles behind these methodologies is still elusive. the crystallization of membrane proteins in the lipid cubic phases involves following major steps: removal of membrane proteins from the native membrane using detergentlike molecules, followed by their insertion and equilibration into a lipid bilayer. the crystallization itself commences with precipitant induced osmotic dehydration which in turn stimulates the nucleation that subsequently leads to a crystal growth. using time-resolved x-ray scattering (saxs) and ultraviolet spectroscopy we have been able to gain new insights into the mechanism behind protein insertion into these complex three dimensional lipid structures including information on the timescales of protein folding relative to crystal growth and the effect of protein insertion on the morphology of the surrounding lipid matrix. several proteins can form amyloid fibrils under given environmental or thermodynamic conditions that affect their native conformation. lysozyme forms amyloid fibrils upon incubation of solutions at acid ph and at about 65 • c for a few days. differential scanning calorimetry experiments confirm that lysozyme is mainly unfolded above 60 • c at ph 2. we monitored the growth of aggregate size by dynamic light scattering for some days at different temperatures. our results show that the fibrillogenesis is characterized by an initial apparent lag phase and a subsequent growth with quadratic dependence upon time of the scattering intensity. this behaviour recalls a simple kinetic model of nucleation and elongation, with nuclei in equilibrium with monomers. at the end of the incubation at high temperature, we collected atomic force microscopy images which show fibrils with a diameter of a few tens of nanometers and a length of a few microns, characterized by a periodicity along the elongation axis. interestingly, the fibrils morphology exhibits no branching or thickening. this is consistent with the non-exponential growth observed in light scattering experiments. in order to elicit the role of repulsive electrostatic interaction in protein unfolding and self-assembly we extended our study at different ph. in this work we used afm to follow the amyloidogenesis pathway of transthyretin (ttr) to form protofilaments. single-molecule force spectroscopy (smfs) of native ttr and protofilaments were also compared in order to evaluate dynamic and structural differences. we observed that this pathway proceeds through the formation of transient amorphous aggregates, followed by the occurrence of annular oligomers. although implicated in cytoxicity, the role of such oligomers within the amyloidogenesis pathway is poorly understood. we show that the annular species display a tendency to stack, forming tubular-like structures that precede the formation of protofilaments. the protofilament height and pitch resemble those of ttr amyloid reported in previous structural studies. upon solvent exchange, we also observed protofilament disassembly that revealed structures reminiscent of the initial ttr annular oligomers. smfs of protofilaments showed a time-dependent increase in the length of the manipulated structures, suggesting that associations between monomers stabilize with time. force spectra of native ttr and protofilaments contained transitions spaced by ∼4 nm, indicative of sequential unfolding of individual β-strands. based on these results a model of ttr protofilament assembly is proposed. ability to undergo amyloid aggregation is affected by protein size c. parrini 1 , h. ramshini 2 , f. chiti 2 , a. relini 1 1 physics department, university of genoa, italy, 2 biochemical sciences department, university of florence, italy short polypeptide chains, typically between 28 and 253 residues, are generally involved with the conversion from the native state into insoluble fibrillar aggregates. the low prevalence of large proteins is disproportionate with their high occurrence in the human proteome. in order to explore the propensity of large proteins to form amyloid-like fibrils, the 486-residue hexokinase-b from saccharomyces cerevisiae (yhkb) has been induced to aggregate under two separate conditions, at low ph in the presence of salts and at ph 5.5 in the presence of trifluoroethanol. such conditions are among the most promising to form amyloid-like fibrils by normally globular proteins. under both conditions yhkb aggregates very rapidly into species with significant β-sheet structure, as detected by circular dichroism, and a weak thioflavin t and congo red binding. atomic force microscopy revealed globular aggregates eventually clustering into large amorphous aggregates at low ph, while in the presence of trifluoroethanol ribbon-like structures with distinct morphology from typical amyloid fibrils were observed. they had irregular width, no twist, and were connected by thinner fibril segments perpendicular to them. in general, yhkb aggregates displayed an unusual softness, as they were very easily perturbed by the afm tip. these results suggest that inability to form amyloid fibrils may prevent large proteins from being associated with protein deposition diseases. the initial stage of proteins aggregation leading to amyloid fibrils: a saxs study m. g. ortore 1 , f. spinozzi 1 , f. carsughi 1 , t. narayanan 2 , g. irace 2 , s. vilasi 3 , p. mariani 3 1 dip. saifet, univ. pol., ancona, italy, 2 esrf, grenoble, france, 3 dip. biochim. biofis., ii univ., napoli, italy under some conditions, a protein converts from its soluble form into highly ordered aggregates called amyloid fibrils, which are associated with many human diseases. in order to tackle the prevention and treatment of these diseases, we need to understand the mechanism of the pathological aggregation of proteins. in vitro amyloid fibril formation is preceded by the formation of metastable non fibrillar forms, which are responsible for cytotoxicity underlying neurodegeneration. the molecular mechanisms leading proteins into prefibrillar aggregates are still unclear. we present a saxs study performed at id02 beamline of esrf on the apomyoglobin mutant w7fw14f, which at physiological ph, firstly aggregates in prefibrillar forms that are cytotoxic and then forms amyloid fibrils. the first stages of w7fw14f oligomerization are induced by a ph jump. data show that big changes in w7fw14f in solution happen in less than 100 ms. singular value decomposition (svd) of the data yields a set of functions, from which all the scattering curves can be reproduced. the major result of our study is the determination of the presence of different oligomers in each step of the process. hence, time-resolved saxs experiments together with the estimation of different oligomers via svd method, can be a new and useful approach to investigate the first stages of amyloidogenesis. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils study of structure/toxicity relationship of amyloids by infrared spectroscopy h. p. ta amyloid diseases (alzheimer, parkinson, type ii diabetes, prion) correlate with protein aggregation. all the proteins associated with these pathologies aggregate into amyloid fibrils with a common ß-cross structure. according to many in vitro studies, the toxicity of various amyloids seems to be linked to some intermediates formed during the aggregation pathway and to their interaction with membranes. our aim is to understand the link between structure and toxicity of amyloids by studying their interaction with model membranes. for this, we use as an amyloid model, the prion-forming domain pfd218-289 (wild type wt) of het-s, a prion protein of the fungal p. anserina. when expressed in yeast, wt is not toxic whereas one of these mutants, m8 is toxic. in vitro, m8 forms very unusual short amyloid fibers contrary to wt which polymerizes as long fibers. furthermore, atr spectra have shown that m8 is essentially assembled into mixed parallel and anti-parallel ß-sheets whereas wt displays a predominant parallel organization. we are currently studying the interaction between wt or m8 with lipid langmuir film by polarized modulation-infrared reflexion absorption spectroscopy (pm-irras). this technique allows determining the secondary structure of proteins and their orientation in the membrane. the study of interactions with different charged phospholipids is following. a. stirpe, m. pantusa, r. bartucci, l. sportelli, r. guzzi dipartimento di fisica, laboratorio di biofisica molecolare & udr cnism, università della calabria, rende (cs), italy an increasing number of experimental studies is demonstrating that the propensity of forming amyloid fibrils is not limited to proteins related to neurodegenerative diseases but it is a more general phenomenon. we present data on human serum albumin (hsa) aggregation induced by a combination of thermal and metal ions effects investigated by optical density, fluorescence and electron paramagnetic resonance (epr). the turbidity experiments as a function of temperature show that the hsa aggregation starts at about 70 • c which is higher than the denaturation temperature (∼ 60 • c). in the presence of copper and zinc metal ions the onset temperature for protein aggregation is markedly reduced as the metal:protein molar ratio is increased from 1:1 to 10:1. moreover, the copper is more effective in inducing protein aggregation compared to zinc ion. the hsa aggregation analyzed by tht fluorescence at different incubation temperatures lacks a lag phase and the kinetic traces can be fitted by double exponential functions. the tht fluorescence increase evidences the formation of protein aggregates with fibrillar features. epr experiments for the cu(ii)-hsa complex show the binding of cu(ii) in the protein native state in a square planar coordination with 4 equatorial n atoms, and is not influenced by the heat treatment of the protein. the overall results suggest that hsa aggregation is compatible with a downhill process that does not require the formation of an aggregation nucleus. protein condensation diseases -a colloid physicists viewpoint p. schurtenberger adolphe merkle institute, university of fribourg, ch-1723 marly 1, switzerland a broad class of diseases, such as cataract, alzheimer's disease and sickle-cell disease, involve protein association phenomena as an essential aspect. the basic element common to all members of this class of molecular condensation diseases is the subtle interplay between protein interactions that produces condensation into dense, frequently insoluble mesoscopic phases. among this class of diseases, cataract is particularly important as the world's leading cause of blindness. this disease is most often the consequence of an uncontrolled aggregation (or phase separation) of the proteins in the eye lens that results in a loss of its transparency. the high concentration protein mixtures present in the eye lens are normally stable and produce a high refractive index that aids the eye in adaptive focusing of light. moreover, the proteins themselves exhibit a rich variety of repulsive interactions, attractive interactions, sizes, phase transitions and self-association. these mixtures also exhibit the pathological aggregation and opacification of the cataract disease that has inspired their study. in my presentation i will illustrate how we can use a combination of small-angle neutron and xray scattering experiments combined with molecular dynamics computer simulations to identify, measure and model the molecular interactions and emergent optical and viscoelastic properties and the phase behavior of the relevant, complex cytoplasmic mixtures. fluorescence microscopy studies of iapp fibrillation at model and cellular membranes d. c. radovan 1 , n. opitz 2 , r. winter 1 1 department of physical chemistry i -biophysical chemistry, tu dortmund university, dortmund, germany, 2 max-planck institute for molecular physiology, dortmund, germany type 2 diabetes mellitus (t2dm) is characterized by islet amyloid deposition and beta cell death, the main culprit being a small 37 a.a. peptide hormone, islet amyloid polypeptide (iapp), which forms fibrils under pathological conditions. we studied the interaction of iapp with giant (guvs) and large (luvs) unilamellar vesicles as well as with ins-1e cells. by using confocal / two-photon excitation fluorescence microscopy and guvs, we tested the influence of charge (dopc:dopg 7:3) and model raft systems, displaying liquid-ordered (l o ) / liquid-disordered (l d ) phase coexistence (such as dopc:dppc:cholesterol 1:2:1), respectively, on the kinetics of iapp fibril formation. a preferential partitioning into the l d phase was observed and fibrils grew along with lipid uptake. fluorescence spectroscopy leakage experiments with carboxyfluorescein-filled luvs and the corresponding tht kinetics of iapp fibril formation were carried out as well. moreover, using the wst-1 reduction assay and fluorescence microscopy, we could show that the red wine compound resveratrol is a potent inhibitor of iapp fibrillation and its cellular toxicity on ins-1e cells, these findings highlighting the potential role of resveratrol in future clinical applications, i.e., in the treatment of t2dm. a remarkably high viscosity has been induced in aqueous solutions of lysozyme by the addition of certain structurally related organic solvents, such as tetramethylurea (tmu), dimethylsulfoxide (dmso), dimethylformamide (dmf), and hexamethylphosphortriamide. dmso-induced gelation is observed in samples fulfilling the two following requirements: (1.) lysozyme concentration in excess of 5 mm, and (2.) volume fractions of dmso exceeding 0.7. based on spectroscopic data, the whole process was characterized as consisting of two mutually independent stages. the first involves an extensive transition of the polypeptide backbone, from a predominantly helical to increased random coiled and beta-sheet structures, with the occurrence of non-orthodox protein secondary structures at regions above the solvent critical point. the second stage consists of short-lived interchain contacts leading to an entanglement of the macromolecular system as a whole. here we present a set of scattering and microrheology experiments that investigate both the structural and dynamic properties of the gels under various experimental conditions. studying alpha-synuclein aggregation by fluorescence polarization based kinetics l. tosatto, f. munari, i. tessari, g. de franceschi, m. pivato, p. polverino de laureto, m. bisaglia, l. bubacco università degli studi di padova, padova, italy alpha-synuclein (syn) is linked to parkinson's disease (pd) by two evidences: the accumulation of amyloid fibrils of the protein and the autosomal dominant forms of the disease (a53t, a30p and e46k mutants). protein oligomers seem to be the most toxic species, causing dopaminergic neuronal death, probably disrupting cell membrane (volles & lansbury, 2002) . the aggregation of syn follows a nucleation dependent mechanism, i. e., aggregation is favoured only after the formation of an oligomer composed of a critical number of monomers (wood et al., 1999) . as the constitution of the nucleus is a rare event, early aggregation stages are difficult to study. to explore the early stages of the aggregation process a fluorescence polarization (fp) based method (luk et al., 2007) was applied to study syn oligomerization. fp depends on the size of the fluorescent molecule, therefore it is suitable for the detection of oligomers formation. we measured aggregation kinetic properties under several different conditions. a 100 to 1 mixture of syn and oregon green labelled syn was used to analyze the aggregation behaviour of wild type (wt) and pd mutants. the wt protein shows the fastest aggregation rate. this aggregation process is slowed down by the presence of the chaperone 14-3-3eta in wt syn samples. finally, dimers formed by a disulfide bond at the n-terminus or c-terminus of syn, when tested for their aggregation behaviour, show a different propensity to aggregate. a. pardini 1 , r. bizzarri 2 , a. diaspro 3 , p. bianchini 3 , c. usai 1 , p. ramoino 3 , g. checcucci 1 , g. colombetti 1 1 istituto di biofisica, cnr, italy, 2 a) nest, sns, iit udr, pisa, italy; b) nest, cnr-infm, pisa, italy, 3 univiversity of genova, genova, italy the colored ciliates blepharisma japonicum and fabrea salina show photomotile responses triggered by endogenous pigments of the hypericin family. b. japonicum exists in two forms: the wild one contains red blepharismin; the other one, generated by irradiating the cells with dim visible light, blue blepharismin. b. japonicum shows step-up photophobic response, whereas f. salina, that contains fabrein, shows step-down photophobic responses and positive phototaxis. we showed by confocal microscopy that the pigments are localized not only in pigment granules, but also in the cilia. this fact implies that the structure of the pigment in the cilia differs from that in the cell body, (pigment granules are too big to fit in the axoneme) and suggests that ciliary pigments might play a decisive role in photoreception. fluorescence lifetime imaging microscopy (flim) shows that there is a spatial distribution of lifetimes, which are shorter for the pigment in the cilia. this might indicate a functional role for these pigments. furthermore, lifetimes for f. salina are always longer than those of blepharisma. in order to further characterize the structure of ciliary pigments by means of spectroscopic methods, we have also performed spatially resolved static and time-resolved fluorescence anisotropy measurements. biosensing applications of micro/nano structured silicon several topics related to porous silicon (ps) biosensing properties were carefully considered in this study. ps allows the increasing of the immobilised biomolecule number on its surface and the creation of stable covalent bonds due to its controllable chemistry. beside this, ps is suitable for electrical (conductance, impedance), electrochemical and optical amplification of the detected signal. the experimental results on the fabrication of the ps microstructures such as: (i) protein immobilization and detection using microarray technique; (ii) dna biomolecule detection by impedance or by fluorescence spectroscopy; (iii) very sensitive sers biosensors (raman signal of 11-mercaptoundecanoic acid); (iv) sensitive element for neurons in nutmix culture are in detail presented. various characterisation techniques have been used, optical and scanning electron microscopy (sem), x-ray diffraction, raman, laser fluorescence and impedance spectroscopy for investigation molecule attachment on the au/ps structures. we have demonstrated that different morphologies of ps as-prepared or coated with gold nanoparticles have an important role in biomolecule/cell detection, due to its large internal surface combined with specific optical properties, being in the same time sensing element/support for immobilization of sensing biomolecules as well as transducer for biochemical interactions. heterotrimeric g-proteins interact with their g-protein coupled receptors (gpcrs) via key binding elements comprising the c-terminal segment of the α-subunit and the two lipid anchors at the α− and γ−subunit. direct information about diffusion and interaction of gpcrs and their g-proteins is mandatory, as these properties will affect the timing of events in the complex signal transduction cascade. in the case of the photoreceptor rhodopsin, receptor packing in the membrane and the related diffusion coefficients are discussed controversially (1, 2). by using single particle tracking we show that the encounters of rhodopsin with the fluorescently labeled cterminus of the α-subunit as well as with the holo-g-protein transducin change upon rhodopsin light-activation. our results indicate confined areas of interaction for the c-terminal segment of the α-subunit with inactive rhodopsin disk membranes and less restricted diffusion of the receptor-bound cterminal segment after light-activation. this suggests dynamic short-range order in rhodopsin packing and specific structures for efficient interaction (3) in vivo, skeletal muscle actively shortens and also resists lengthening, for example when landing after a jump. we study the energy during shortening and during lengthening by measuring the rate of pi release which results from atp hydrolysis. we show here the rate of pi releases in an in vitro protocol that involves muscle stretched followed by muscle shortening. it is hypothesised that the magnitude of deceleration of pi release in the stretch phase is less than the acceleration during the release due the energy input of the motor that is used to apply the length changes. therefore the end point of pi release is similar to isometric conditions. pi release responses to a ramped stretch (5%) followed 100 ms later by a symmetrical release at low velocities (0.5 l 0 /s −1 ) were measured in permeabilised fibre bundles of rabbit psoas at 20 • c. laser diffraction and high speed video were also recorded to confirm length change. we show that the rate of pi release drops during the stretch phase, returns to isometric levels during the length hold phase, and finally accelerates during the ramped release. tracking of sarcomere length change using video analysis demonstrated that laser diffraction is unreliable at times during stretches due to lack of uniformity of the sarcomere spacing. microbial rhodopsins: receptors, channels, and pumps from a single design j. l. spudich, e. negri-spudich center for membrane biology, university of texas, houston, texas, u.s.a. the microbial rhodopsin family is comprised of ∼5000 homologous proteins containing 7 transmembrane helices forming a pocket for the chromophore retinal. most are lightdriven ion pumps ("transport rhodopsins") and others are photosensory receptors ("sensory rhodopsins"). phylogenetic analysis indicates frequent lateral gene transfer of proton pumps among prokaryotic and unicellular eukaryotic species, followed by coupling of the pump's mechanism to the cell's existing signal transduction machinery to create photosensors. this evolutionary path is strongly supported by our studies of sensory rhodopsins in various organisms which demonstrate remarkably diverse signaling mechanisms with diverse transducer partners. the best studied are the phototaxis receptors in haloarchaeal prokaryotes (sri and srii) and in eukaryotic algae (channelrhodopsins). sri and srii transmit signals by protein-protein interaction to control a phosphorylation cascade that modulates motility. channelrhodopsins are light-gated cation channels that depolarize the membrane mediating calcium ion influx into the flagellar axoneme. crystallography and molecular biophysics have begun to clarify how modifications of the same architecture enable the rhodopsins to carry out their distinctly different molecular functions. interconversions of their functions by mutation reveal the elegant simplicity by which evolution uses existing genes to create proteins with novel functions. gold colloids-fluorophore complexes for protein detection assay l. sironi 1 , s. freddi 1 , l. d'alfonso 1 , m. collini 1 , m. caccia 1 , g. tallarida 2 , s. caprioli 2 , g. chirico 1 1 dipartimento di fisica, università di milano-bicocca, italy, 2 laboratorio nazionale mdm, agrate brianza (mi), italy noble metal nanoparticles (np) are endowed with peculiar optical properties related to the surface plasmon resonances (spr). the interaction of surface plasmons of gold nanoparticles with fluorophores a few nanometers away from the surface modifies their brightness and excited-state lifetime, and this effect can be exploited to obtain nanodevices for proteinprotein recognition. we studied different types of constructs based on gold nps on which derivatives of fluorescein were bound. the interaction of this fluorophore with the gold surface plasmon resonances, mainly occurring through quenching, affects its excited-state lifetime, that is measured by fluorescence burst analysis in standard solutions. the binding of proteins to the gold nps through antigen-antibody recognition further modifies the dye excited-state lifetime. this change can therefore be used to measure the protein concentration. streptavidin-functionalized gold nps of size 5-10 nm are used to bind biotin-fluorescein and biotin-antibodies for specific proteins. we have first tested the constructs for bovine serum albumine (bsa) detection. the data reported here indicate that one can measure the concentration of bsa in solution with an apparent limit of detection of 5±2 pm. we have then extended the study to the antitumor protein p53 -p53 antibody interaction in standard solution and directly in cellular extracts. calcium transport and phototransduction in isolated rod outer segments g. rispoli dip. biologia ed evoluzione, università di ferrara, italy ca 2+ concentration in photoreceptor rod outer segment (os) strongly affects the generator potential kinetics and light adaptation. light stimuli may produce voltage changes exceeding 40 mv: since the os ca 2+ extrusion is entirely controlled by the na + :ca 2+ ,k + exchanger (nckx), it is important to assess how the nckx ion transport is affected by voltage and intracellular factors. the nkcx regulation was investigated in whole-cell recorded os, using ionic conditions that activated maximally forward and reverse exchange. in all species examined of amphibia and reptilia, the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. since hyperpolarization increases ca 2+ extrusion rate, the recovery of the dark level of ca 2+ (and of the generator potential) after light stimuli results accelerated. mg-atp doubled the size of forward and reverse exchange current without modifying their voltage dependence, indicating that mg-atp regulates the number of active exchanger sites and/or the nckx turnover number. ca 2+ jumps achieved via photolysis of caged-ca 2+ produced current transients, possibly originating from electrogenic partial reactions. no monovalent cation substituted for na + at the nckx binding sites, but rb + substituted for k + , while sr 2+ , ba 2+ , mg 2+ substituted for ca 2+ with an apparent permeability ratio of 0.78, 0.20, <0.05:1, respectively. a. pfeifer 1 , k. zikihara 2 , s. tokutomi 2 , j. heberle 1 , t. kottke 1 1 bielefeld university, bielefeld, germany, 2 osaka prefecture university, sakai, japan the blue light receptor phototropin regulates the growth of plants towards the light. it contains two light-, oxygen-, or voltage-sensitive (lov) domains and a kinase domain. lov domains bind noncovalently flavin mononucleotide (fmn) as chromophore. upon illumination, the triplet excited state of flavin reacts within few microseconds with a nearby cysteine under formation of a photoadduct, which represents the signaling state. in response to adduct formation, a jα helix adjacent to the lov2 domain dissociates and allows for autophosphorylation by the kinase domain. the mechanism of the photoreaction and the signaling pathway from fmn to the jα helix are still unclear. we have investigated the lov2 domain of arabidopsis phototropin 2 by microsecond ft-infrared spectroscopy. the difference spectrum recorded at 2 µs provides evidence that the flavin is unprotonated in the triplet excited state. therefore, a previously proposed ionic mechanism of bond formation is disfavored. changes in secondary structure were detected concomitant with adduct formation that relax with a time constant of 120 µs. this early adduct intermediate has not been previously characterized. the final adduct state is formed in milliseconds by further alterations in secondary structure. these findings raise the question of whether the early or late adduct intermediate propagate the signal to the jα helix. -photosensory biophysics the psychostimulant amphetamine increased no generation measured by epr as well as amino acid release in the rat brain nitric oxide (no) is a novel messenger that modulates many functions of the nervous system. the involvement of no in brain damage was shown mainly by indirect evidence and the data are controversial. the short half-life of no makes its direct detection difficult. we measured no generation using epr spectroscopy based on determination of the amount of paramagnetic mononitrosyl-iron complexes. the aim was to elucidate whether psychostimulant drug amphetamine (amph) modulates formation of no and lipid peroxidation (lpo) products as well as the neurotransmitter release in rat brain. the output of glutamate, aspartate, gaba and acetylcholine (ach) was monitored in striatum by microdialysis with hplc detection. amph produced 2fold elevation of no generation and lpo formation in brain areas. while amph increased the aspartate, gaba and ach release, the glutamate output was not affected. pretreatment with the neuronal nos inhibitor was highly effective in abating the rise of no and neurotransmitter levels but failed to influence the lpo intensity elicited by amph. the findings suggest that activation of no synthesis is a potent factor in the amph-induced neurotransmitter release. light scattering study of dna over a wide chain length range: comparison with wormlike model p. baeri, m. zimbone dipartimento di fisica e astronomia, università di catania, italy this work reports light scattering measurements on dna in aqueous solutions (100 mm nacl ,1mm edta and 10 mm tris-hcl buffer, ph 7.8) over a wide range of molecular weights (10 2 -10 5 base pairs) and shows that, in the above standard solvent, shorter chains ( < 10 4 base pairs) behave as a "wormlike chain" and their diffusion coefficient as obtained by dynamic light scattering measurements, confirm the prediction of standard wormlike model, whilst longer chains ( >10 4 base pairs) behave in a different manner. dynamic and static light scattering and sem analysis indicate that dna molecules 10 5 base pairs long, condense into compact structures in our solvent condition. calculations done using a wormlike model are also presented and discussed in comparison both to our experimental data and to other data reported in the literature. a. asandei, t. luchian 'alexandru i. cuza'university, faculty of physics, laboratory of biophysics and medical physics, blvd. carol i no. 11, iasi, romania amphotericin b (amb) is an antifungal antibiotic which, despite the severe side effects, is still used for the treatment of systemic fungal infections. in this study we investigated the influence of ph upon the selectivity and the transport properties of amb channels inserted in reconstituted, ergosterolcontaining zwitterionic lipid membranes. our electrophysiology experiments carried out on single and multiple amb channels prove that at ph=2.8 these channels are anion selective, whereas at neutral and alkaline ph's (ph=7 and ph=11) they become cation selective. we attribute this to the ph-dependent ionization state of the carboxyl and amino groups present at the mouth of amb molecules. surprisingly, our data reveal that the single-molecule ionic conductance of amb channels varies in a non-monotonic fashion with ph changes, which we attribute to the ph-dependent variation of the surface and dipole membrane potential. we demonstrate that when added only from one side of the membrane, in symmetrical salt solutions across the membrane and low ph values, amb channels display a strong rectifying behavior, and their insertion is strongly favored when positive potentials are present on the side of their addition. we report a detection method for the redox state of proteins which combines fret-based fluorescence/confocal microscopy on dye-labeled protein with cyclic voltammetry. by using this combined method, electron transfer properties can be revealed from protein to electrode or from redox enzyme to substrate. we applied the fluorescent detection to azurin, a blue copper protein from the bacterium ps. aeruginosa, fluorescently labeled on the n-terminus for monitoring the redox state of the protein. the dye fluorescence is quenched by energy transfer to the copper in oxidized, but not in reduced azurin. fluorescence results demonstrated that cy5-labeled wtazurin switched in fluorescence intensity by up to 85% by varying the applied potential. labeled zinc-azurin was used as a control sample and did not show any fluorescence switching. for single molecule studies wt-azurin was labeled with atto 655 dye and a mixed sam was used, with 8-hydroxy-1octanethiol as a blocking agent to prevent non-specific binding of protein on the surface, and 1,10 decanedithiol for the specific covalent binding to the protein. preliminary data show that single-molecule fluorescence switching with the potential is indeed possible. -single molecule biophysics is there a specific erythrocyte membrane receptor for fibrinogen? an atomic force microscopy approach f. a. carvalho 1 , s. connell 2 , r. a. ariëns 2 , n. c. santos 1 1 instituto de medicina molecular, univ. lisbon, portugal, 2 university of leeds, u.k. fibrinogen (fg) contributes to erythrocyte (rbc) hyperaggregation by an increase in. rbc-fb protein binding considered to be non-specific. glycoprotein α iib β 3 is a specific integrin receptor for fibrinogen on platelets. we showed that there is a single molecule interaction between fg and an unknown receptor on rbc membrane, with a lower affinity when compared with platelet binding. we evaluated if rbc-fg binding is through an integrin-like receptor or not. interactions between fg and platelet/rbc receptors were studied by force spectroscopy. force curves were performed between fg-functionalized atomic force microscope tips and rbc or platelets. to evaluate if the fg-rbc binding is calciumdependent, similar studies were performed in the presence of ca 2+ or edta. we also carried out studies in the presence of a α iib β 3 inhibitor and of methyl-ß-cyclodextrin (to disrupt lipid rafts by cholesterol depletion). rbc-fg single forces were of 300-380 pn and of 400-480 pn for platelet-fg binding in presence of calcium. a significant decrease of the platelets-fg force-rupture was obtained in the presence of edta or α iib β 3 inhibitor. significant lower fg-rbc force value was obtained in the presence of mβcd, but not in the presence of edta. conclusion: fg-rbc binding seems not to be calcium-dependent but the existence of cholesterol on rbc membrane is important. nanoscopic and spectroscopic investigation of p53-based complexes at single molecule level a. r. bizzarri biophysics and nanoscience centre, cnism, facoltà di scienze, università della tuscia, largo dell'università -01100 viterbo, italy p53 is a transcription factor that plays a widely recognized role in preventing cancer development in response to dna damage. the tumor suppressor activity of p53 involves the formation of several complexes whose detection and study, at single molecule level, could be extremely relevant to understand, in detail, the mechanisms governing the cancer defense processes, as well as to develop ultrasensitive biosensors. p53-based complexes, are investigated at molecular level, by combining atomic force microscopy (afm), atomic force spectroscopy (afs) and surface enhanced raman spectroscopy (sers) with the support of computational docking. our attention is mainly devoted to study complexes between p53 and the electron transfer azurin which has been demonstrated to interact with p53, by promoting its stabilization. a possible competition between azurin and the cellular oncogene mdm2 is also investigated. unwinding the dna helix in force clamp condition p. bianco 1 , l. bongini 2 , m. dolfi 1 , l. vincenzo 1 1 physiolab, dbe, university of florence, italy, 2 university of florence and centro interdipartimentale studio dinamiche complesse, firenze, italy we use a dual-laser optical-tweezers (dlot, smith et al., science, 1996) to define the highly cooperative conformational transition in the molecule of dna, where the natural b-dna has converted into a new overstretched conformation called s-dna (bensimon et al., phys. rev. lett., 1995; cluzel et al., science, 1996) . single molecules of double stranded λ-phage dna (in a solution with 150 mm naci, 10 mm tris-hcl, 1 mm edta, ph 8.0. and 27 • c) are stretched either in length clamp or in force clamp mode. when the dna molecule is stretched in length clamp mode with a ramp lengthening, it shows the previously described highly cooperative overstretching transition at ∼60 pn, attributed to unwinding from the b-form to the 1.7 times longer s-form. stretching the molecule in force clamp mode with a staircase of force steps at 5 s intervals (step size 1-2 pn, rise time 1-2 ms) shows, for any given clamped force f in the region of the overstretching transition, different amounts of dna elongation (∆l) with exponential time courses. the analysis of the elongation rates allows to recover all the necessary parameters for an effective two-state model able to reproduce the out-of-equilibrium properties of the system. the results imply an unwinding cooperativity of 27 bps. this value is significantly lower than that obtained assuming force independent rate constants. supported by miur, ente cassa di risparmio di firenze and itb-cnr (milano). c. m. becker 1 , a. benedix 1 , b. l. de groot 2 , a. cafisch 3 , r. a. böckmann 1 1 saarland university, saarbrücken, germany, 2 mpi for biophysical chemistry, göttingen, germany, 3 university of zürich, zürich, switzerland modifying the stability or the binding behavior of the involved proteins by mutation can influence the activity of cellular processes. for an efficient identification of possible mutation-sites a fast calculation of the free energy of proteins is crucial. here we developed a fast and reliable method (cc/pbsa) [1] for the prediction of the change in stability of proteins and binding affinity of protein-protein complexes upon mutation. the energy function of cc/pbsa is based on gas phase energies, solvation free energies and entropic contributions. the protein flexibility is taken into account by generating random conformations based on geometrical constraints only applying the concoord [2] program. we applied cc/pbsa on the tem1-blip complex, which is important in bacterial antibiotic resistance. the results of single-and double-point alanine scanning are used to detect hot spots, cooperative effects, and the corresponding energy distribution. cc/pbsa is freely accessible on our web-server: http://ccpbsa.bioinformatik.uni-saarland.de the human recombinase hrad51 is a key protein for the maintenance of genome integrity and for cancer development. this protein plays a central role is the dna strand exchange occurring during homologous recombination. here we report the polymerization and depolymerization of hrad51 on duplex dna observed with a new generation of magnetic tweezers, allowing the measurement of dna twist with a resolution of 5 • in real time. at odds with earlier claims, we show that, after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition of single proteins, involving dna twisting steps of 65±5 • . simple numerical simulations support that this mechanism is an efficient way to minimize nucleoprotein filament defects. this behavior, consisting of different stoichiometry for nucleation and growth phases, may be instrumental in vivo. fast growth would permit efficient continuation of strand exchange by rad51 alone while the limited nucleation would require additional proteins such as rad52, thus keeping this initiation step under the strict control of regulatory pathways. besides, our results combined with earlier structural information, suggest that dna is somewhat less extended (4.5 versus 5.1å per bp) and more untwisted (18.2 versus 15 • per bp) by hrad51 than by reca, and confirm a stoichiometry of 3-4 bp per protein in the hrad51-dsdna nucleoprotein filament. biofunctional micropatterned surfaces to study the spatio-temporal organisation of lfa-1 r. diez-ahedo 1 , d. normanno 1 , c. g. figdor 2 , a. cambi 2 , m. f. garcia-parajo 1 1 bionanophotonics, ciber-bbn and ibec, barcelona, spain, 2 tumor immunology, nijmegen center for molecular life sciences, the netherlands lymphocyte function associated antigen-1 (lfa-1) adhesion depends on receptor occupancy and lateral organization on the cell membrane. however, the signals and mechanisms which dynamically reorganize lfa-1 into high avidity clusters are still a subject of many studies. to obtain deeper insight on the mechanisms that control and regulate lfa-1 clustering, patterned surfaces of immobilized lfa-1 ligand areas were fabricated using microcontact printing. the diffusion of lfa-1 expressed by monocytes stretched over patterned surfaces was followed in time using single molecule tirf microscopy. single lfa-1 nanocluster trajectories on individual cells showed an increase of immobile lfa-1 fraction and a slow-down of diffusing lfa-1 on the ligand areas compared to the non-ligand areas. moreover, single-cluster intensity analysis indicated a reorganization of lfa-1 nanoclusters in microclusters upon ligand binding. finally, single particle motion analysis of lfa-1 trajectories in close neighborhood to the ligand areas showed no assisted diffusion of lfa-1 towards the adhesive regions, consistent with random ligand-encountering and binding. we are currently investigating the effect of cell membrane organizers to regulate the spatio-temporal organization of lfa-1. r. diez-ahedo et al, small, in press. optical and electrophysiological detection of single phages across a lipid membrane n. chiaruttini 1 , p. boulanger 2 , m. de frutos 3 , l. letellier 2 , u. bockelmann 1 , v. viasnoff 1 1 nanobiophysique, espci paristech, cnrs, paris, france, 2 ibbmc, université paris xi, cnrs, orsay, france, 3 lps, université paris xi, cnrs, orsay, france we present an investigation study of the ejection of single t5 bacteriophages. in vivo studies of dna ejections from the bacteriophage capsid show that the t5 genome is introduced in the bacterial host in two steps. first 8% of the genome is ejected then after a pause of a few minutes the rest is internalized. bulk in vitro studies showed that various mutants of t5 eject their genome in solution following a single or a multistep process. by immobilizing single bacteriophages on a surface and following their ejection by fluorescence microscopy we showed that in all cases the ejection occurs in one step, but some mutants seem to have a subpopulation for which the triggering signal of the ejection is transmitted more slowly to the capsid entrance. we then reconstituted the phage receptor fhua into giant liposomes and followed the ejection of the dna into the liposome by fluorescence. finally we incorporated fhua in a suspended bilayer and followed the infection of the phages through the bilayer both by fluorescence labeling and electrophysiological measurements. we will discuss the influence of the cross membrane potential on the ejection speed of the dna. helixlike pili is a prerequisite of uropathogenic e. coli to adhere to host and withstand urine flow the gram-negative uropathogenic escherichia coli (upec) bacteria, invades the urinary tract region and cause in some cases severe infections, pyelonephritis, if they can withstand the rinsing action of urine and ascend to the kidney, via the bladder and ureters. to mediate adhesion, upec express quaternary surface organelles that are assembled from ∼10 3 identical subunits into a helix-like coil, with a single adhesin located at the tip. it is believed that the single adhesin mediate attachment to host cells while the helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow. to unravel the biomechanical properties of such quaternary structures, in particular in terms of their force-elongation and kinetic behavior, force-measuring optical tweezers (fmot) have been used. a plethora of different types of pili have been identified in the literature and we show, using fmot, that those dissimilarities might reflect the host environment. for example, we have found differences among pili expressed at diverse environment inside the urinary tract, which imply that pili presumably have evolved to resist specific forces under in vivo conditions. it is thus worth striving for understanding bacterial adhesion in order to figure out alternative to the over-abundance of antibiotics worldwide. single molecules studies have probed protein conformational fluctuations and monitored the oscillating activity of enzymes that remained masked in ensemble measurements. various statistical models have attempted to connect the conformational motions to the fluctuating activity of enzymes and suggested that they adopt inter-converting conformations each one of them exhibiting different catalytic activity. the main drawback of these studies is the non specific interactions that may bias the observed catalytic behavior. we have developed new innovating tools to study the behavior of single enzymes. as a first step we utilized liposomes as scaffold to confine enzymes while keeping them under physiological conditions. keeping them in their native conformation allowed us to monitor the inherent properties of their behavior. as a second step we developed a new model that accurately connects enzyme activity behavior to conformational motions. using this approach we accurately predicted the behavior of single lipases in a highly controlled environment. we modulated the enzyme's conformational mobility and activity by systematically varying its accessibility to liposomes. as we anticipated, that resulted in altering the time the enzyme spends on active conformations.our results provide new insights on interpreting the behavior of enzymes. simulations of single-molecule fret experiments on ribozymes m. hajdziona, a. molski adam mickiewicz university laboratory for dynamics of physicochemical processes, poznan, poland rnazymes are important biological molecules and that can catalyze the cleavage of their own nucleotide chains. this precess is called self splicing [1] . there are several methods to to study kinetic properties of ribozymes at the singlemolecule level. in this work we focus on fret (förster resonance energy transfer) of single, immobilized molecules. single-molecule fluorescence spectroscopy gives an insight into the behavior of individual molecules rather than the average behavior of an ensemble of molecules, which makes it possible to observe the kinetic heterogeneity. we simulated fret trajectories for single immobilized ribozymes. in our computer simulations we consider two-and three-state kinetic models motivated by actual experiments, published in [2] and [3] . we fitted the histograms of on and off times to recover the kinetic parameters of the models. we investigated the bias and standard deviation of the recovered parameters when one or two thresholds are applied to fret trajectories between adjacent states. we found that two thresholds give better parameter estimates than one-threshold analysis. references frap probes the average dynamical properties of a population of fluorescently labeled molecules whereas spt obtains these properties from observations of the trajectories of individual molecules tagged with a colloidal particle (the "bead"). when results of frap are compared with those of spt, frap yields significantly higher diffusion constants than spt. to understand the origin of this difference, we have developed and tested a model system to evaluate the influence of the bead on the dynamics of a diffusing molecule. we use a dsdna tether to attach a bead to a supported lipid bilayer (slb). the dna tether is modified at one end by cholesterol for anchoring to and diffusion in the membrane, and at the other end by biotin for tagging with a bead. with this system, we can vary several parameters: distance between slb and bead, size and nature of the bead, lipid composition of the slb, external force. we can also model the brownian motion of the bead and the hydrodynamic flow around it. we will present results using φ = 200 nm neutravidin-coated latex particles attached to an eggpc slb via a 15 bp to 90 bp dna tether. elucidation of the mechanism of the lambda bacteriophage epigenetic switch l. finzi physics department, emory university, 400 dowman dr, atlanta, ga 30322, usa the lambda bacteriophage epigenetic switch determines the growth lifestyle of the virus after infection of its host (e. coli ). it is now clear that the switch consists of a ∼2.3 kbplong dna loop mediated by the lambda repressor protein. using tethered particle microscopy (tpm), magnetic tweezers and afm, our laboratory has novel, direct evidence of loop formation and breakdown by the repressor, the first characterization of the thermodynamics and kinetics of the looping reaction and its dependence on repressor non-specific binding and dna supercoiling. these in vitro data provide insight into the different possible nucleoprotein complexes and into the lambda repressor-mediated looping mechanism which leads to predictions for that in vivo. the significance of this work consists not only of the new insight into a paradigmatic epigenetic switch that governs lysogeny vs. lysis, but also the detailed mechanics of regulatory dna loops mediated by proteins bound to multipartite operators and capable of different levels of oligomerization. mechanical force at the molecular level is involved in the action of many enzymes. for example, the phi29 dna polymerase mechanically unwinds the dna helix as it moves processively along the dna replicating one strand of the dna molecule. using optical tweezers we have developed a single molecule mechanical assay to elucidate the physical mechanism of dna unwinding by the phi29 dna polymerase as the protein replicates the dna. a single dna hairpin is hold between an optical trap and a mobile surface. as a single polymerase works on the dna hairpin, its replication and unwinding reactions can be measured in real time by measuring the change in extension in the dna polymer, revealing the fluctuations of its rate in response to the dna sequence. moreover, by gradually pulling on the opposite strands of the dna hairpin we can promote the controlled mechanical unwinding of the dna helix and determine the effect of increasing unwinding forces on the polymerization and unwinding rates. the effect of force and dna sequence on the activities of the wild type and an unwinding-deficient polymerase mutant will allow us to determine the inner workings of this molecular motor. single centrosome manipulation reveals its electric charge and associated dynamic structure we demonstrate laser manipulation of individual early drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively-charged organelle with a very low isoelectric region (3.1 ± 0.1). from this single-organelle electrophoresis, we infer an effective charge smaller or of the order of 10 3 electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. by investigating the centrosome hydrodynamic behavior, we show that its charge has a remarkable influence over its own structure. specifically, we find that the electric field which drains to the centrosome expands its structure to a physiological size a 60% larger than previous electron microscopy determinations, a self-effect which modulates its structural behavior via environmental ph. this methodology further proves useful to study the action of different environmental conditions, such as the presence of ca 2+ , over the thermally-induced dynamic structure of the centrosome. magnetic contrast neutron reflectivity delivers significant improvements in resolution for membrane structure analysis neutron reflection (nr), with its high resolution and use of contrast variation, is a unique tool to gain information on the orientation and structure of lipid bilayers in the z-axis perpendicular to the surface. to improve the use of nr we have used a highly oriented and stable layer of membrane proteins and lipids made possible by self-assembly upon a gold surface. this also provides a model of the bacterial outer membrane. as the dimensions of the layer can be predicted with accuracy, the system provides a molecular ruler for improvements in methodology and the complexity of the layer structure adds significantly to the modelling challenge. early improvements in resolution were obtained through sample preparation and gold smoothness. further improvement however required clearer discrimination between similar models. this was achieved using the new approach of magnetic contrast variation which uses a magnetic layer to provide two different scattering length densities for oppositely polarised neutrons. during this data collection the sample is unchanged. we show that this approach delivers significant improvements in data analysis and resolution of the protein, lipid and solvent structures. the method will provide a new level of understanding of membrane structure and dynamics. single molecule force spectroscopy and recognition imaging p. hinterdorfer institute for biophysics, johannes kepler university of linz, altenbergerstr. 69, a-4040 linz, austria in single molecule force spectroscopy, interaction forces of ligand-containing tips with receptors on probe surfaces are quantified. here he attachment of human rhino virus 2 (hrv2) to the cell surface, the first step in infection, was characterized. sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra. this suggests that multiple receptors bound to the virus in a timely manner. unbinding forces required to detach the virus from the cell membrane increased within a time frame of several 100 ms. the number of receptors involved in virus binding was determined and estimates for on-rate, off-rate, and equilibrium binding constant were obtained. furthermore, we show that accurate free energy values of membrane protein unfolding can be obtained from single molecule force measurements. by applying a statistical theorem developed by jarzynski, we derived equilibrium unfolding free energies of from unfolding force data acquired at different force loading-rates and temperatures. finally, we present a method for the localization of specific binding sites and epitopes with nm positional accuracy. a magnetically driven afm tip containing a ligand covalently bound via a tether molecule is oscillated at a few nm amplitude, during scanning along the surface. in this way, topography and recognition images on membranes and cell surfaces were obtained simultaneously. mapping of the forces acting on biomolecules in cell membranes has spurred the development of effective labels, e.g. organic fluorophores and nanoparticles, to track trajectories of single biomolecules [1] . standard methods use particular statistical observables, namely the mean square displacement (msd), to extract cues on the underlying dynamics. yet, msd is not an appropriate tool to access force fields and becomes easily a biased estimator in the presence of positioning noise. here, we introduce general inference methods [2] to fully exploit information hidden in the experimental trajectories, providing sharp estimates for the forces and the diffusion coefficients within membrane microdomains. rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. the inference method is then applied to infer forces and potentials acting on the receptor of the ε-toxin labelled by lanthanide-ion nanoparticles. results show a constant diffusivity inside a complex force field confining the receptor inside a specific domain. our scheme is applicable to any labelled biomolecule, and results presented here show its general relevance to the issue of membrane compartmentation and protein motion. large scale domain motions are structural rearrangements often adopted by enzymes to achieve their full functionality. understanding the mechanism responsible for such movements by means of computational approaches is of great interest especially in rational drug design, since induced fit or population shift effects are closely related aspects of the problem. unfortunately, the simulation time required to sample these events by conventional techniques such as plain molecular dynamics, is unfeasible. here, the domain motion required by adenosine kinase (ak) to achieve its (pre-)catalytic conformation was studied using well-tempered metadynamics [barducci, a. et al. phys rev lett. (2008) , 100: 020603] with path collective variables (pcvs) [branduardi, d. et al. j chem phys. (2007) , 126: 054103]. first, a low energy path for the apo form of the enzyme was obtained, and the potential of mean force along the pcvs was reconstructed. then, the large scale movement was simulated in the presence of two inhibitors known to bind to the enzyme in different conformational states. the adopted approach was proven to be successful both to understand the mechanistic features of the ak domain motion and to provide a picture of the population shift effects upon ligand binding. force measurement study of the interaction between 23s rrna and the ribosomal protein l20 single molecule force measurements enable to probe the complex structure and folding dynamics of rna molecules and furthermore to investigate the numerous interactions with proteins that affect rna folding in vivo. we use a double optical tweezers setup to study a region of the ribosomal rna 23s from e.coli and its interaction with the essential ribosomal protein l20. the apparatus permits us to probe the dynamics of the rna structure with milisecond time-resolution. first, we pull the rna and show that it mechanically unfolds in several reproducible steps. we use these results in combination with structure prediction tools to evaluate the possible structures of the rna fragment in vitro. the most probable structure exhibits a unique binding site for l20. then, force measurements are done on the rna in presence of l20. we show that the protein specifically binds the rna and stabilizes it. the binding site is recognized with a resolution of a few bases. finally, two bases are mutated on the rna fragment. in presence of these mutations, in vivo and in vitro binding of l20 to 23s rna is abolished. the single molecule approach gives an explanation of this result: the force measurements show that the mutated rna folds differently from the natural one and that it does not bind l20. control of the translocation of single dna molecules through alpha-hemolysin nanopores g. maglia, h. bayley department of chemistry, university of oxford, ox1 3ta, oxford, uk the analysis of single nucleic acid molecules by electrophoretic threading through nanopores is under intense investigation as a rapid, low cost platform for dna sequencing. biological nanopores such as staphylococcal alpha-hemolysin (hl) have an added advantage over solid-state nanopores because they can be modified by genetic engineering with atomistic precision. although we showed that all four dna bases can be identified in an immobilized ssdna molecule (1), the translocation of free dna is too quick to observe single bases. here we show that by a small increase of the net internal charge of the hl nanopore (e.g. by introducing a ring of 7 arginine residues), we have augmented the frequency of dna translocation events through the pore and dramatically lowered the voltage threshold required for dna translocation (2) . by further increasing the net positive charge of the transmembrane barrel region of the pore (e.g. by introducing 49 extra positive charges) we have also reduced the speed at which dna translocate the pore by more than two orders of magnitude. these experiments provide a means of controlling dna translocation with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification. the atomic force microscope (afm) is a tool for imaging, measuring and manipulating matter at the nanoscale. single-molecule pulling experiments give information on the thermodynamics and kinetics of biomolecules. the purpose if this work was to develop software to simulate single-molecule pulling experiments and to analyze singlemolecule pulling data. the long term objective is to asses the accuracy and precision of the parameters recovered from single-molecule pulling data.we carried out simulations for two different models [1, 2, 3] , using a wide range of loading rates. from the simulated force-extension curves we extracted the kinetic parameters and compared them with the values used for simulations. the kinetic parameters were the intrinsic rate coefficient (k 0 kramers rate), the location of transition state (x ) and the free energy of activation (∆g). we have found that the loading rates have a small effect on the recovery of the free energy of activation, but have a significant effect on the recovery of the kramers rate. we report the tracking of single myosin v molecules in their natural environment, the cell. myosin v molecules, labeled with quantum dots, are introduced into the cytoplasm of living hela cells and their motion is recorded at the single molecule level with high spatial and temporal resolution. we perform intracellular measurements of key parameters of this molecular transporter: velocity, processivity, step size and dwell time. our experiments bridge the gap between in vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells. a mathematical model of neurotransmission at the input stage of the cerebellum t. nieus 1 , s. solinas 3 , l. mapelli 2 , e. d'angelo 3 1 dept. neuroscience and brain technologies, iit, italian institute of technology, genova, italy, 2 dept. of biomolecular sciences and biotechnology, milan, italy, 3 dept. physiological and pharmacological sciences and cnism, university of pavia, italy the granule cell (gc) of the cerebellum has some peculiar properties compared to other cells of the vertebrate brain. the gc has a small soma (diam=6 microm) and just a few (2 to 6) excitatory and inhibitory inputs (e&i). the e&i gc synaptic inputs are formed inside the cerebellar glomerulus, which favors neurotransmitter diffusion between neighboring sites (1) protracting postsynaptic receptor activation and (2) causing cross-tall between e&i synapses through presynaptic receptors. neurotransmitter release probability (p) can be regulated by long-term plasticity (ltp and ltd at e synapses) and by gaba b and mglu presynaptic receptors (both at e&i synapses). the p change in turn modifies shortterm plasticity, affecting the first response in a train much more than the followings. to gain insight into the role that p changes might have in computations performed at the cerebellum input stage, we have built detailed biophysical models of the e&i synapses. the model has allowed to investigate glomerular processing of high frequency inputs reaching the cerebellum. optimal synaptic transmission through the mfs inputs resulted when gos inhibited synchronously the gcs. the role of feed-forward inhibition onto synaptic transmission is under investigation. unzipping dna with a nanopore j. muzard, n. chiaruttini, u. bockelmann, v. viasnoff cnrs/espci nanobiophysique, 10 rue vauquelin, 75005 paris, france the use of proteinaceous or artificial nanometer size pores has become a promising approach for sensing biomolecules at the single molecule level. it was shown that alphahemolysin, a toxin from staphylococcus aureus, can be employed to sense the translocation of dna strands through a lipid bilayer. the pore dimension allows the electrophoretically driven passage of single stranded dna whereas double stranded structures need to open prior their translocation. we study the unzipping process of the double stranded part of dna both experimentally and theoretically. we show that in a first approximation the duplex opens progressively in a sequence dependent manner. the experimental results can be accounted for by modeling the unzipping process as a free diffusion of the unzipping fork in the energy landscape defined by the sequence of the basepaires. we then discuss the effect of the pore/dna interaction on the translocation process. we further characterize the effects of the pore geometry, showing that the pre-confinement of the dna in the pore vestibule is essential to the unzipping process. we eventually discuss the possibility to use this nanopore approach to sequentially probe rna secondary structures. we report on the interaction forces in the range 100 --400 pn determined between pairs of the lectin soybean agglutinin (sba) and a modified porcine submaxillary mucin (tn-psm) using a single-molecule approach. lectins are carbohydrate-binding proteins with biological activities related to i.e. cellular recognition, adhesion, growth and metastasis. here, dynamic force spectroscopy is used to investigate pairs of sba and tn-psm with the aim to understand the mechanisms of binding and cross-linking of multivalent lectins. the unbinding force increased from 103 pn to 402 pn with increasing force loading rate and the lifetime of the complex in the absence of applied force was 1.3 -1.9 s. published kinetic parameters describing the rate of dissociation of other sugar lectin interactions are in the range 3.3 x 10 −3 -2.5 x 10 −3 s. the long lifetime of the sba -tnpsm complex is compatible with a previously proposed "bind and jump" mechanism. this mechanism has also been suggested for lectins binding to multivalent carbohydrates and globular glycoprotein. the bind and jump mechanism is also similar to that observed for binding of proteins to dna, and suggest a common conserved binding mechanism of ligands to the two biopolymers and possibly between ligands and all biopolymers, as recently suggested. the rate of topoisomerase ii activity correlates with persistence length of dna q. shao 1 , l. finzi 1 , d. dunlap 2 1 dept. of physics, emory university, atlanta, georgia, u.s.a., 2 dept. of cell biology, emory univ. med. school, atlanta, georgia, u.s.a. type ii topoisomerases catalyze the transection of one double helical segment by another to modify the topological state of dna. reversible 5' linkages to the phosphate-sugar backbones are established on each strand of the "gate" segment to create an opening through which the enzyme drives the "transfer" segment. a recent crystal structure shows that the "gate" segment bends approximately 150 • upon binding to the enzyme. bending a stiff polymer like dna requires considerable energy and could represent the rate limiting step in the catalytic (topological) cycle. using modified deoxyribonucleotides in pcr reactions, more rigid dna fragments have been produced and used as substrates for topoisomerase ii-mediated relaxation of plectonemes introduced in single molecules using magnetic tweezers. before relaxation the persistence lengths were measured by fitting force extension data with the worm-like chain model. topoisomerase ii was found to release mechanically introduced supercoils more slowly in stiffer dna suggesting that dna bending might be a rate limiting step in topoisomerase ii activity. h. seidel 1 , t. klose 2 , h. lilie 2 , c. g. hübner 1 1 institute of physics, ratzeburger allee 160, 23538 lübeck, germany, 2 institute of biochemistry and biotechnology, kurt-mothes-str. 3, 06120 halle, germany one essential element of a virus is its protein shell, the viral capsid, which encloses the viral genome. the murine polyomavirus is a non-enveloped dna tumor virus with an icosahedral t=7d structure. besides the knowledge of the structure, it is of utter importance to understand the process of viral assembly [1] . the assembly reaction of polyoma vp1 does not show the typical sigmoidal kinetics in light scattering experiments. the apparent kinetics is of fourth order, which appears rather unrealistic. in order to gain knowledge on capsid composition during assembly beyond ensemble average, we apply methods of single molecule fluorescence, namely fluorescence correlation spectroscopy (fcs), fluorescence-intensity-distribution-analysis (fida), and single-particle-imaging (spi). the molecular brightness and the diffusion time reported by fcs are in agreement with the results from light scattering. fida as well as spi, moreover, point to a polymerization of subunits to complete capsids along the assembly pathway without pronounced intermediates. mixing experiments show that already early in the course of the assembly reaction no exchange of pentamers between capsids occurs, and that the effect of breathing [2] can be excluded for polyoma vp1. genetic information coded in dna provides complete instructions to cells regarding metabolism and proper functioning. a number of endogenous and exogenous sources can damage the genomic dna. unrepaired dna damage can give rise to mutations and may cause cell death. cells have evolved different mechanisms to repair damaged dna and to protect genetic integrity. uracil in dna occurs as a result of incorporation of dump in the place of dtmp during replication and deamination of cytocine, resulting in u:a and u:g base pairs, respectively. uracil-dna glycosylase (ung) is a dna repair protein that searches and removes uracil from dna. ung removes mismatched base by flipping it out from the base stack into the active site. the exact mechanism by which ung searches dna for uracil is unknown. therefore, in our study we use atomic force microscopy (afm) to investigate the interaction between single ung molecules with dna carrying the u:a or u:g mismatches. furthermore, we study the complexation of dna and ung protein. afm images of ung bound to dna reveal the structural changes at the level of single complex, e.g. dna kinking that may occur upon binding of ung to dna. imaging of dnaprotein complexes can provide a a new level of understanding of ung-dna interaction. atomic force microscopy (afm) has been a useful device to visualize cellular and molecular structures at a single-molecule resolution. especially, afm imaging under the trec t m (topography and recognition) mode (recognition imaging) can map a specific protein of interest within an afm image. in this study, we employed an antibody-coupled afm cantilever in recognition imaging, and dissected the structural/functional domains of α actinin-4, an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction. a use of monoclonal anti-α actinin-4 antibody enabled us to map its epitope in the amino-terminal actin-binding domain within a single molecule afm image of α actinin-4. counting fluorescent molecules by photonantibunching h. ta, m. schwering, d. p. herten bioquant, heidelberg university, germany information on the stoichiometry of labelled biomolecules is highly desirable. a method called photo-antibunching has successfully been used to determine the number of fluorescence emitters in multichromophoric systems. a statistical model to estimate the number of fluorescent molecules in a confocal observation volume is established based on photonantibunching in time-correlated single-photon counting (tcspc) scheme with 4 avalanche photon diodes (apds) for detection of individual photons. numerical simulations based on realistic experimental conditions show that the model is able to estimate the number of molecules with moderate errors. experiments on immobilized double-strand dna oligonucleotides with photon stabilizing agents show promising results even when the number of molecules is ∼ 20. the proposed method allows us to monitor labelled single molecules and in the near future we plan to implement it in complex biological systems (cell extracts/live cells). single molecule afm force spectroscopy and steered molecular dynamics of contactin-4 protein j. w. strzelecki, k. mikulska, a. balter, w. nowak institute of physics, nicolaus copernicus university, grudziadzka 5, 87-100 torun, poland contactin-4 (cntn4) is an axonal cell adhesion protein that contains six igc2 and four fniii domains and is responsible for creating neural outgrowths. recent research shows that mutation of cntn4 gene may be responsible for autism, while its absence in transgenic mice results in lack of smell sense. we use a homemade afm single molecule puller and steered molecular dynamics simulation to test its elastic properties through force spectroscopy. stretching experiments show unfolding of fniii domains while igc2 domains stay coiled as they are stabilized by disulfide bonds. unfolding of contactin-4 molecule appears to play role of buffer that helps to protect neural contacts from damage when neural cells are subjected to shock or tumor. single molecule studies of the thermophilic bacillus ps3 f 1 -atpase have revealed kinetic and structural information that cannot be discerned using other methods, including the presence of 40 and 80 degree physical substeps (yasuda et al. 2001 ) and the order and kinetics of chemical substeps. we are interested in using single molecule techniques to observe the effects of mgi mutations on enzyme kinetics and torque production in f 1 from the yeast saccharomyces cerevisiae. using a high speed imaging camera, we have captured the rotation of wild-type and mutant forms of yeast f 1 -atpase. rotation data for the wild-type and preliminary data for some mgi strains will be presented. we show for the first time that at saturating atp, wild-type yeast f 1 rotates approximately four times faster than the thermophilic f 1 . kinetic and substepping behaviour in yeast appears to be similar to that observed in bacterial f 1 . -single molecule biophysics biophysical characterization of a dna gquadruplex formed in the promoter of human mef2d gene w. zhou 1 , l. ying 2 1 molecular medicine, national heart and lung institute, imperial college london, london, sw7 2az, uk, 2 chemical biology center, imperial college london, sw7 2az, uk g-quadruplex is believed to be involved in many crucial biological processes, such as the gene regulation and the maintenance of human telomere. mef2d, a member of mef2 (myocyte enhancer factor-2) family of transcription factors, regulates the response of heart to cardiac stress signals. we found that a g-rich sequence starting at -145bp of mef2d promoter can form a very stable intramolecular g-quadruplex. here we present a detailed biophysical characterization of this quadruplex. we also found that this quadruplex is more stable than its duplex form under physiological conditions by cd melting and single molecular fret. we observed subpopulations in smfret measurements possibly due to different conformations of the quadruplex. we characterized its unfolding process by monitoring the change in fret when it hybridizes to its c-rich complimentary strand. finally, we proposed several possible structures of this quadruplex based on the smfret and fluorescence dms footprinting results. this research is supported by national heart and lung institute foundation. by stretching a polymer in solution using single molecule techniques it is possible to infer about its physical properties. afm stretching experiments allow for a full characterization of the elasto-mechanical properties of the sample under study. in the presented work, single molecule afm force spectroscopy experiments were used to determine mechanical properties of a peptide obtained from exon 28 (ex28) of the human elastin gene. elastin is a protein with important mechanical properties and, in particular, it shows quasi ideal elastic behavior associated to the presence of many hydrophobic unstructured domains (such as ex28) into the protein structure. ex28 coded polymer was used as a starting point to obtain bio-materials with specialized elastomechanical functions. in particular, a mutated polypeptide based on the ex28 sequence was synthesisized with the aim of obtaining a new polymer with the same mechanical and physical properties of the native molecule but with increased aggregation properties, induced by a cross-linking reaction. afm stretching experiments were used to verify the mechanical properties of the engineered proteins at a single molecule level. the obtained results allowed not only to address this question, but also to give some insight into the first aggregation steps of the polymer towards the formation of reticulated structures. luminescent lanthanide-ion doped nanoparticles (nps) are attractive single-biomolecule labels. they are synthesized directly in water, are highly photostable, and display narrow emission without intermittency. we coupled y 0.6 eu 0.4 vo 4 nps to ε-toxins produced by clostridium perfringens, which bind to a specific receptor on mdck cells. single-molecule tracking experiments using these labels produce long (5 min) uninterrupted trajectories with temporal resolution down to 20 ms or localization precision down to 15 nm. we found that the toxin receptor exhibits confined motion in cell membrane microdomains. to analyze the receptor trajectories, we used a novel approach based on an inference method [1] . this method fully exploits the information of the ensemble of the trajectory, in contrast to the usual mean square displacement analysis. applying both techniques to recorded trajectories, we highlight the difference in extracted parameters. from the shape of the confining potential, obtained by mapping the forces inside the domain, we can deduce information about the mechanism of confinement. in combination with experiments on cholesterol depletion and cytoskeleton destruction, this technique will shed light into the nature of the membrane micropatterning. background: implantable cardioverter defibrillators (icds) are save-life device for patients at risk of sudden cardiac death, and help cardiac patients to avoid slow beat-rate by means of anti-bradichardia pacing (abp) feature. however, recent literature evidenced the presence of ventricular tachyarrhythmias (vts) immediately following abp, leading to the hypothesis that icd devices might be pro-arrhythmic. aim: study differences between pacing-associated tachyarrhythmias (pat) and other spontaneous vts. signals and methods: 165 spontaneous vt episodes are retrieved from 37 patients with icd. vts are examined and pat episodes, classified. characterization of vts is based on the analysis of 20 seconds immediately preceding vt-onset quantified by: heart cycle (hc-prevt), prevalence of premature ventricular contraction (pvcprev) and prevalence of paced beats (pprev). significant differences are evaluated by student-t or chi-square tests with p<0.05. results: 20 vt episodes from 8 patients are pat episodes (12%). cardiac activity preceding pats vs. non-pats differs: pat episodes have higher hc-prevt (p<0.01), greater pprev (p<0.001), and greater pvcprev (p<0.001). analysis also shows that pat episode often occur when the paced beat immediately follows a premature contraction. conclusion: the study indicates that new icd generations should avoid abp after premature ventricular contraction. towards mechanistical understanding of adsorption: combining technologies in situ and in real time p. h. bjöörn q-sense ab, västra frolunda, sweden a growing number of researchers in different surface related fields present evidence from more than one analytical technique when detailing their findings. thus a logic and useful development is to combine technologies for simultaneous measurements on a single sensor surface. to develop a biosensor platform such as an assay fast and accurately, mechanistical understanding of why the platform works is essential. quartz crystal microbalance-with dissipation (qcm-d) technology and instrumentation provides an open platform and enable easy and precise quantification of mass, thickness and viscoelastic properties of surface bound molecules. these parameters provide both a reference tool in assay development, but also detecting and identifying your target molecules as the combination of mass and material property info in many cases provide a unique response for a specific molecule. by combining qcm-d with other technologies, a range of useful info is put within easy reach of the researcher. recent advances will be presented where simultaneous real time and in situ measurements using qcm-d together with electrochemistry, ellipsometry and fluorescence microscopy enables manipulation of films as well as quantification of the variation of water content as a result of conformational changes in immobilized molecules. examples will include new data from the formation of protein films, polyelectrolyte multilayers and polymer brushes. intrinsic gravity versus metabolically inert infrastructure and basal metabolic rate in living mass i. r. bhattacharjee 1 , r. kashyap 2 , b. shaptadvipa 2 1 assam agricultural university, jorhat-785013, india, 2 international institute of intrinsic gravitation biology (i3gb), jorhat-785013, india 'self gravitation bio' is an emerging concept in biophysics. intrinsic or 'self' gravitational force might exert when mass increases beyond critical level in biological particle pyramid. (http://en.wikipedia.org/wiki/biomechanics of intrinsic gravity) 'metabolically inert infrastructure' (mii) consists of total body mass (body water, dissolved substances, mineral and organic deposits) and serves as storage of nutrients, transport and distribution of these materials. to act independently as living body, mii is suggested also to provide structural support to the organism with density-gradient buoyant force against intrinsic and extrinsic gravitational attraction for the biological mass. it is shown that 'amniotic fluid', 'isotonic saline to ailing patient', 'growth factors', 'cultural medium' and other 'medium matrices' act as counter-gravity mechanism for micro to macro living organisms under center-of-biomass reference frame. controversy over relationship between bmr (basal metabolic rate), rmr (resting metabolic rate), pal (physical activity level), on one hand, and mass of the living organism, on the other, (as described in rubner's 2/3 law, kleiber's 3/4 law that continued to be contested by many) can be favorably resolved substituting the concept of self gravitation bio, considering 'mass' as synonym of gravitational force under center-of-biomass reference frame. bioenergetics would be an unequal but opposite entity of self-gravity reinforced by extrinsic gravity. platelet arrest on von willebrand factor (vwf) occurs transiently via the platelet receptor gpib. depending on the combination of shear stress and surface density of vwf binding sites the platelets either only adhere, pull tethers or generate microparticles. these processes are influenced by the properties of the platelet membrane and the cytoskeleton. under shear flow conditions these platelet characteristics were examined with reflection interference contrast microscopy and a viscosimeter. in addition we quantified platelet adhesion energy and tether pulling forces. disrupting platelet f-actin had several effects. the tethers were shorter, the membrane contact area was larger, the receptor αiibβ3 density of the microparticles was depleted and no platelet spreading occurred. breaking up the microtubular system had different implications. the number of severed tethers tripled, the contact area was smaller, microparticle formation was increased and the area of spread platelets was reduced. however changes in the membrane elasticity had no effect on the platelets. our results suggest that platelet attachment and adhesion is not only determined by the platelet adhesion receptors and their cytoplasmic linker proteins, but that cytoskeletal structures have also a crucial influence on how platelets interact with thrombogenic surfaces. gene expression is orchestrated by a host of regulatory proteins that coordinate the transcription of dna to rna. regulatory proteins function by locating specific binding sequences of dna and binding to these sequences to form the transcription initiation complex. in many instances, these regulatory proteins only have several hundred copies that must efficiently locate target sequences on the genome-length dna strand. the non-specific binding of regulatory proteins to random sequences of dna is believed to permit the protein to slide along the dna in a stochastic manner. periodically, a thermal kick or an interaction with another bound protein will disengage the regulatory protein from the dna surface, leading to three-dimensional diffusion. eventually, the protein will reattach to the dna at some new location that is dictated by both the diffusivity of the protein and the dna configuration. cycling through these random events constitutes a search strategy for the target site. we build a reaction-diffusion theory of this search process in order to predict the optimal strategy for target site localization. the statistical behavior of the dna strand acts as a necessary input into the theory, and we consider several governing behaviors for the dna strand. we explore the impact of dna configuration and protein occlusion on target site localization in order to predict how protein expression will vary under different experimental conditions. a. di garbo 1 , s. alloisio 2 , s. chillemi 1 , m. nobile 2 1 istituto di biofisica cnr, via g. moruzzi 1, 56124 pisa, italy, 2 istituto di biofisica cnr, via de marini 6, 16149 genova, italy in the nervous system of vertebrates there are more glial cells than neurons: from 10 to 50 times, depending on the animal type. glial cells are not able to generate action potentials but, nevertheless they play an important role for the functioning of the different brain's area. the astrocytes are the more abundant cells of the macroglia and through their processes they modulate synaptic activity. in this contribution a biophysical neural network model consisting of a pyramidal neuron, an interneuron and the astrocyte is studied. the corresponding dynamical properties are mainly investigated by using numerical simulations. the results show that the presence of the atp and of the interneuron strongly impacts the neural activity. moreover, it is shown that the fluxes of calcium through the cellular membrane strongly affect the modulation of the neural activity arising from the astrocyte. microscopic origin of the very-low energy vibrational dynamics in proteins g. d'angelo 1 , v. conti nibali 1 , c. crupi 1 , a. paciaroni 2 , u. wanderlingh 1 1 department of physics, faculty of science, messina university, italy, 2 department of physics, faculty of science, perugia university, italy important functions of biological processes require large atomic rearrangements and conformational fluctuations. for proteins, atoms are mostly displaced along the soft directions identified by the delocalized, low frequency vibrations. the study of very low energy vibrational spectrum of proteins is therefore of particular interest. as a step toward understanding their functional role, we have investigated the low frequency vibrational motions (0.03÷10 mev) in different proteins by performing inelastic neutron scattering and low temperature (1.5÷30 k) specific heat measurements. for the first time for biological systems, the well-known boson peak found in neutron scattering spectra at ∼ 3 mev is put in relationship with a clear anomaly of the measured specific heat located at around 7 k. this departure from the debye-like behavior further expands the analogy of proteins with glassy systems. quite interestingly, in the very low sub-mev energy range and below ∼3k, we observe an additional anomalous behaviour, which could be ascribed to the existence of twolevel-systems states. increasing the hydration degree, the low energy vibrational region is drastically altered, revealing that the addition or removal of the hydrogen bond network around the protein deeply modifies the interatomic forces, affecting the character of the vibrational modes. a. cupane 1 , m. levantino 1 , m. cammarata 2 , g. schirò 1 1 department of physical and astronomical sciences, university of palermo, via archirafi 36, i 90123 palermo, italy, 2 european synchrotron radiation facility, grenoble, france our efforts in recent years have been to study in great detail the way hemoglobin works in confined geometries [1] [2] [3] . to this aim we have contributed to the development of a new experimental technique, time-resolved wide-angle x-ray scattering (tr-waxs), that enables one to watch proteins at work in solution [4, 5] . a very recent and challenging application of this technique is the study of hemoglobin functioning inside intact red blood cells (rbc). our preliminary results show that, by using laser pulses of about 150 ns, it is possible to photolyse hemoglobin inside rbcs obtaining about 40% -60% photolysis. good structural signals from hemoglobin are obtained, with limited radiation damage to the cells: this opens the possibility of studying the conformational transitions of hemoglobin in its "natural" environment. preliminary results concerning the timing of the r->t quaternary transition inside the rbc and comparison with the behaviour in dilute solution will be discussed. physical review letters and physical review e invite submission of your best work in biological physics. submissions must present significant new results in physics; topics may range from the microscopic to the macroscopic. we will provide information on the different types of articles published in the journals, on authors and referees, and on the review process. for publication in physical review letters, the work should be important and of broad interest. for rapid communications in physical review e, the work should be important for the field. biological physics papers published in physical review are indexed in medline. in an effort to bring important research to the attention of a wider community, the physical review journals have recently begun highlighting important work with viewpoints in the online publication physics. all physical review journals may be accessed through the website http://publish.aps.org/. modeling of fibrin gels using confocal microscopy, light scattering and turbidimetry f. ferri 1 , d. magatti 1 , b. cardinali 2 , a. profumo 2 , m. rocco 2 1 dipartimento di fisica e matematica, università dell'insubria, como, italy, 2 istituto nazionale per la ricerca sul cancro (ist), genova, italy fibrin gels are biological networks playing a fundamental role in blood coagulation. confocal microscopy of fibrin gels shows a collection of straight fibers, not uniformly distributed in space, connected together at low-order (3) (4) branching points. although each fiber is quite straight (mass fractal dimension d m =1), for the overall system d m >1. based on the confocal images, we generated threedimensional (3d) synthetic gels made of cylindrical sticks of diameter d, joined together at randomly distributed nodal points. the resulting 3d network is no more random but ordered on length scales of the order the average fiber length, and exhibits a fractal morphology with d m ∼1. 2-1.7 . the gel structure is analyzed by means of its 3d correlation function g 3d (r)=<n(x) n(x+r)> x , where n(x) is the gel local density. since the gel is isotropic, g 3d depends on the modulus r=|r| and can be obtained by averaging 2d correlation functions evaluated at different heights of the gel volume. from this analysis we recover the fiber diameter d (fwhm of g 3d ), the fractal dimension d m (power-law decay of g 3d ) and the gel mesh-size ξ (minimum in g 3d ). furthermore, the 3d-fourier transform of g 3d (r) gives the gel power spectrum i(q), which compares quite well with elastic light and multi-wavelength turbidimetry data taken on real gels. a model coupling vibrational and rotational motion for the dna molecule e. drigo filho, j. r. ruggiero, r. a. d. s. silva unesp -sao paulo state university, brazil a largely used mechanical model for vibrational motion of dna has been proposed by peyrard and bishop (pb). in this model, dna is represented by a pair of harmonic chains coupled by a nonlinear potential. the most frequently used nonlinear potential for this purpose is the morse potential. some extensions of the original model are proposed considering, for example, the helicoidal structure of dna. an important objective for this kind of model is to understand the phenomenon of thermal denaturation and, through this, get some knowledge about other processes such as the genetic transcription and drug intercalation. it is possible to obtain from this type of model interesting properties, such as the average stretching between base pairs as a function of the temperature using the transfer integral operator. dynamical properties of this model were also explored and several phenomenological quantities are studied, such as energy localization. in this work, we extend the original pb model by introducing rotational motions for the nucleotides. in this way, both the vibrational and rotational motion for each nucleotide are considered. the stretch of the base pairs is given by the morse potential in the same way as in the original pb model. however, the coupling between the two kinds of motion, rotation and vibration, is obtained through a nonlinear combination of them in the morse potential. a. dobovišek 1 , m. brumen 2 , p.županović 3 , d. juretić 3 1 university of maribor, faculty of natural sciences and mathematics, maribor, slovenia, 2 jožef stefan institute, ljubljana, slovenia, 3 faculty of science, university of split, split, croatia mepp is a physical principle, widely used for quantitative explanation of non equilibrium phenomena in physics, chemistry and biology [1] [2] [3] . here, we applied mepp to study two and three state reversible michaelis-menten kinetic schemes of enzymatic reactions. by applying constraints as a diffusional limit for kinetic constants, constant free energy differences between enzymatic states and constant thermodynamic force, we calculated shannon information entropy and entropy production of the entire reaction system as a function of forward rate constants. we found maxima in the net steady-state metabolic flux, total entropy production and shannon entropy for equal values of forward rate constants. moreover, for these values an analytical expression derived gives a relation between substrate and product concentrations at which enzymes operate in the optimal way. in conclusion, we demonstrated that mepp is an appropriate selection principle for evolutionary optimization of enzymes. attractive interaction between like-charged lipid surfaces mediated by spherical nanoparticles with spatially distributed charge is theoretically described by using functional density theory and mc simulations. the spatial distribution of charge within a single nanoparticle is considered by two effective charges at a finite distance. the minimization of the free energy is performed to obtain the equilibrium configuration of the system. both, the rigorous solution of the variational problem and the mc simulations show that orientational ordering of nanoparticles subject to the gradient of the electric field gives rise to an attractive interaction between charged lipid surfaces for high enough charge densities of the interacting surfaces and large enough separations between charges within the nanoparticle. the attraction is explained by orientational ordering of dimeric charges in the electric field which lowers free energy. viral genomes encode for a series of membrane proteins which are embedded or attached to the lipid membrane of the host, or penetrate them during the very first step of viral invasion. we are focussing especially to those of the former which are known to assemble and from channels or pores for small ions or substrates. with these channel forming proteins the virus manipulates the host cell interior for the benefit of its replication. strategies are developed to answer the question about the assembly process of these proteins based on the 'two stage model' and the substrate flux. only few experimental data are available to answer these questions, consequently we stress computational methods to derive the adequate answers. the methods applied are ab inito molecular dynamics (md) simulations based on density functional theory (dft) and conventional md simulations. potential routes for assembly of the three transmembrane domains of 3a protein from sars-cov will be outlined and compared with computational data from other viral membrane proteins. with a novel pore lining motif suggested for 3a, the dynamics of ions at selected positions within the putative pore will be assessed. probing the molecular mechanism of antibiotics diffusion through the ompf channel e. hajjar 1 , a. kumar 1 , m. winterhalter 2 , p. ruggerone 1 , m. ceccarelli 1 1 department of physics, university of cagliari, italy, 2 jacobs university, bremen, germany in gram-negative bacteria, the outer membrane porin-f (ompf) is the preferred entry point of antibiotics. bacteria can resist to antibiotics by altering the expression and the structures of ompf. a key feature in the structure of ompf is the presence of a constriction zone, characterized by both spatial and electrostatics restrictions. to study the process of antibiotics translocation at a molecular scale, we performed molecular dynamic simulations accelerated with the metadynamics algorithm. we studied the diffusion of antibiotics with different structural and chemical properties through ompf wild-type and variants. the free energy surface suggests faster translocation for the cephalosporins compared to the penicillins antibiotics, and also for ompf mutants compared to the wild type. further, the conservation of favored orientation and affinity sites of antibiotics inside the ompf channel reveal which specific interactions govern translocation. the calculated energy barriers and rate determining interactions for translocations compared well with the electrophysiology measurements and liposome swelling assays from our collaborators. this study demonstrates how theory and experiments can be combined to reveal the structural determinants and mechanism of ompf permeation. this will benefit to the design of antibiotics with improved transport properties. m. g. gauthier, j. bechhoefer dept. of physics, simon fraser univ., burnaby, bc, canada dna replication requires two distinct processes: the initiation of pre-licensed replication origins and the propagation of replication forks away from the fired origins. experiments indicate that these origins are triggered over the whole genome at a rate i(t) (the number of initiations per unreplicated length per time) that increases throughout most of the synthesis (s) phase, before rapidly decreasing to zero at the end of the replication process. we propose a simple model for the control of dna replication in which the rate of initiation of replication origins is controlled by the interaction with a population of ratelimiting proteins. we find the time set by reaction-diffusion kinetics for such proteins to find, bind to, and trigger a potential origin. the replication itself is modeled using a formalism resembling that used to study the kinetics of first-order phase transitions. analyzing data from xenopus frog embryos, we find that the initiation rate is reaction limited until nearly the end of replication, when it becomes diffusion limited. initiation of origins and hence i(t) is suppressed when the diffusionlimited search time dominates. we find that, in order to fit the experimental data, the interaction between dna and the rate-limiting protein must be subdiffusive. we also find that using a constant nuclear import of the limiting proteins leads to a more accurate description of the experimental data. the microsoft research -university of trento centre for computational and sisytems biology, trento, italy we propose a new method for inferring the structure of a biochemical network from the time-series of the reactant species. it consists of two parts: the first is the quantification of the correlation between the time-series profiles. correlation in time series data can be used to reveal dependencies between variables and to infer the graph of connectivity among species. the second part consists in the elimination from the connectivity graph of the relationships that have a non-null correlation coefficient, but that are not biochemically plausible. the cutting of false correlations from the graph is performed through the estimation of the parameters ("calibration") of the network. to calibrate the network for detecting null dynamics correlations, we developed the software tool kinfer (knowledge inference). based on a new probabilistic model of the variations in reaction concentrations, kinfer infers the values of the kinetic rate constants, their confidence regions and the level of noise in the input data by maximizing the likelihood to obtain the observed variations given the network model. the a priori knowledge required as input is minimal, as it consists only in the time-series of reactant concentrations. the minimal a priori knowledge and the probabilistic formulation of the calibration method make the accuracy of the predictions strong against experimental, biological and stochastic noise, and allows to use it to cut the null-dynamics edges of the connectivity graph. a. kuzmanic, b. zagrovic mediterranean institute for life sciences, split, croatia root-mean-square deviation (rmsd) is a measure used to give information on the global structure of macromolecules. for example, pairwise rmsd (prmsd) is used to assess similarity of the lowest energy nmr structures or for clustering large ensembles of structures. on the other hand, to obtain information on the local structure of a macromolecule and its dynamics, root-mean-square fluctuation (rmsf) is often used. rmsf can be calculated from md simulations, but also from experimental x-ray b-factors. since prmsd and rmsf report on different features, it is interesting to ask what the relationship between them is. first, we provide a mathematical derivation showing that, given a set of conservative assumptions, the <prmsd> rms is directly related to the <rmsf> rms and, consequently, experimental b-factors. second, we demonstrate this on structures taken from distributed-computing md simulations of the native and unfolded state of villin headpiece. both our analytical and computational results suggest a strong correlation: <rmsf> rms = [(s-1)/2s] 1/2 <prmsd> rms , where s is the number of compared structures. furthermore, if <prmsd> rms is defined as a generalized radius of gyration in the space of 3d structures and using rmsd as a measure of distance, the following identity holds: <rmsf> rms = <prmsd> rms. our results provide a basis for determining the level of structural diversity of molecular ensembles, as captured by <prmsd> rms , directly from experiment. charge migration along dna helices may be biologically important because extended electronic states could play a role in the processes of sensing of dna damage and/or dna repair via long-range charge transfer. we measured conductivity and other physical characteristics of several models of natural and diversely damaged molecules of dna. dna polymers were mimicked by various sequences of 32nucleotide-long double helices with fully watson-crick (wc) paired bases, with several central bases mismatched, and also with chemical modifications that included removal of bases from a few central nucleotides (abasic dna), and neutralization of phosphate charges by their derivatization. the model dnas were investigated by scanning tunneling microscopy, time-resolved thz spectroscopy, raman spectroscopy, circular dichroism, and modeled by molecular dynamics simulations. dna has the highest conductivity in its biologically most relevant double helical form with wc base pairs and negatively charged phosphates equilibrated with counterions. mismatches and all chemical modifications always lower the conductivity. the mechanism of charge transfer is consistent with electron or hole hopping between parallel stacked bases. these observations and data showing that the natural dna has also the most regular double helical form suggest that the continuous base stacking is critical for charge transfer. to control the passage across the bacterial cell wall nature created a large number of "nano"-channels which may act as selective gates for water soluble molecules. here we focus on porins from e. coli which control permeation through interactions with the channel surface. comparing single channel temperature dependent conductance measurements with all atom modeling allow conclusions on the mode of permeation. for example, surprisingly modeling ompf-conductance revealed not only a good agreement with the experiment over a broad range of temperature but also the selectivity for ions. the primary task of porins is to provide facilitated diffusion. we investigated facilitated diffusion of maltooligosaccharide or antibiotics. time resolved conductance measurements allows conclusion on the flux and molecular modeling identifies the limiting interactions with the surface, reveal potential barriers and pathways. exploiting the selectivity of natural or bioengineered channels has promising applications for detecting molecules, characterizing molecular interaction, sequencing dna, protein folding etc. traditionally, studies of diffusion-controlled reaction of biological macromolecules have been made in diluted solutions. however, the high concentration of macromolecules in intracellular environments results into non-specific interactions (macromolecular crowding), which have a great importance on the kinetics and thermodynamics of possible reactions that occur in these systems. in the literature there are studies concerning monte carlo (mc) simulations, giving results that are satisfactory agreement with experimental data, showing, for example, that the protein diffusion in cell cytoplasm is reduced considerably. in addition, there are mc studies about enzymatic reaction, which predict a temporal dependency of the velocity constant in macromolecular crowding. in this work, we try to compare the predicted behavior by mc simulation with the results obtained from the study of the diffusion and reaction of a model protein (alpha-chymotrypsin) using spectroscopic techniques in highly confined media in order to study experimentally the temporal dependence of its diffusion and reaction coefficients. a. paciaroni 1 , a. orecchini 1 , c. petrillo 1 , a. de francesco 2 , f. sacchetti 1 1 university of perugia, italy, 2 cnr-infm, genova, italy the single-particle and collective dynamical properties of protein hydration water have been studied by neutron scattering experiments in a wide temperature and hydration range. an unprecedented accuracy has been achieved thanks to the availability of a large amount of fully deuterated protein powder and the use of the high-flux spectrometers in5 and brisp. the protein under investigation was the maltose binding protein (mbp), which is a well-known and widely studied model of biosensor systems. we found that the low-temperature single particle dynamics of mbp hydration water shows clear features that can be traced back to amorphous systems. more in detail, its vibrational density of states is simply described as the superposition of the contributions of low-density and high-density amorphous ice. the quasielastic signal, which appears at the higher temperatures, can be excellently described with a fractional power law which may put in relationship with the peculiarities of fractal systems. quite strikingly, there is a strong similarity, on both the qualitative and quantitative point of view, with the behaviour of hydrated proteins. the collective dynamics of protein hydration water is characterised by the presence of two modes, whose dispersion curves are reminiscent of those of bulk water. however, the relevant damping factors suggest a strong similarity with glassy systems. m. malferrari 1 , f. francia 1 , s. sacquin-mora 2 , g. venturoli 1 1 università di bologna, bologna, italy, 2 cnrs upr 9080, paris, france the coupling between electron transfer and protein dynamics has been compared in reaction centers (rc) from the wild type (wt) and the carotenoid-less mutant r26, by combining brownian dynamics simulations and the kinetic analysis of charge recombination. upon incorporation of the rc into a progressively dehydrated trehalose matrix the electron transfer between the primary photoreduced quinone and the photoxidized donor accelerates progressively and becomes broadly distributed. this behaviour reflects the hindrance of protein relaxation following charge separation and the inhibition of interconversion between conformational substates. in extensively dehydrated matrices the recombination kinetics is two-times faster and three-times more distributed in the wt rc, indicating a larger inhibition of the internal protein dynamics. in line with this findings brownian dynamics simulations reveal a larger rigidity of the carotenoid-containing structure, in which a cluster of residues close to the quinone acceptors is stiffened as compared to the r26 rc. the in silico and experimental results indicate that the introduction of an internal void in the rc structure has long-range effects on the protein dynamics and that the coupling between the glassy matrix and the rc interior depends markedly on the local mechanical properties of the protein. n. maghelli 1 , v. krstic 2 , n. pavin 2 , f. julicher 2 , i. tolic-norrelykke 1 1 mpi-cbg, dresden, germany, 2 mpi-pks, dresden, germany in the fission yeast schizosaccharomyces pombe, the nucleus is positioned at the cell center. since the nucleus determines the cell division site, keeping the nucleus at the center is crucial for ensuring symmetrical cell division (1 ) . microtubules push against the cell ends and exert force on the nucleus (2 ), but how the cell regulates these forces in order to center the nucleus remains unknown. here we tackle this problem by using a combination of live cell imaging, cell manipulations by optical tweezers, and a theoretical model. we show that microtubule pushing forces can center the nucleus because of a larger number of contacts between the microtubules and the proximal cell end than the distal one. moreover, kinesin-8 motors (klp5/6) increase the rate of microtubule catastrophe (transition from growth to shrinkage) in a microtubule length-and contact-dependent manner. thus, the motor behavior results in a longer contact between a microtubule and the proximal than the distal cell end. taken together, our experimental and theoretical results provide a novel centering mechanism, where kinesin-8 motors increase the efficiency of nuclear centering. electropermeabilization is a commonly used physical method which can induce a transient permeabilization of the cell membrane allowing the entry of therapeutic molecules into the cell and is thus of great interest in the fields of cancer treatment and gene therapy [1] . however, very little is known about the mechanisms occurring at molecular level. there is clearly some microscopic reorganization of the membrane which is responsible for this change in its transverse transport properties. rather than studying the change of these transport properties, we adopt a simple strategy based on the use of giant unilamellar vesicles and videomicroscopy, as described below. we apply a series of permeabilizing electric pulses to the liposomes, and we observe a size decrease down to a critical radius beyond which their size no longer changes. this decrease in size points to the fact that during the physical processes leading to electropermeabilization, lipids are lost from the vesicles. our results published in [2] suggest different possible modes for lipid loss, which can be small vesicles, pores, or tubules formation. imaging of brain activation using core techniques as fmri, pet and synchrotron radiation in parallel c. poitry-yamate, g. margaritondo, r. gruetter ecole polytechnique fédérale de lausanne, switzerland functional magnetic resonance imaging (fmri), magnetic resonance spectroscopy (mrs), positron emission tomography (pet) and synchrotron x-ray emission imaging form a highly complementary set of imaging core technologies for studying brain function and energy metabolism. owing to differences in the information each conveys and the temporal and spatial scales on which they measure labeled substances, a single working hypothesis can be tested from different angles to provide a cross-validated, consistent and coherent explanation. the cibm is a unique research facility in europe for advancing our understanding of biomedical processes in health and disease. the housing of a 7-tesla human magnet, 9.4 and 14.1-tesla animal magnets, an animal pet imaging facility and fully-equipped neurochemistry and rf laboratories has enabled us to develop and perform well-targeted experiments around one research theme. in parallel, a longterm collaborative project using synchrotron x-ray transmission and emission imaging at elettra enables us to combine these core technologies towards understanding brain function in vitro and in vivo, from tissue to cells. a brief presentation of these methods will be followed by their application in studying the visual system from man to mouse. p. picone 1 , r. carrotta 2 , d. giacomazza 2 , m. di carlo 3 1 dip. chimica e tecnologie farmaceutiche, università di palermo, italia, 2 ibf -cnr, palermo, italia, 3 ibim -cnr, palermo, italia diabetes and alzheimer's disease are connected in a way that still is not completely known. diabetes has been implicated as a risk factor for developing alzheimer's disease. some diabetes drugs appear to decrease the cognitive decline associated with alzheimer's disease. it has been recently demonstrated that extracellular injection of insulin is able to protect neurons against a-beta amyloid cell death. one of the proposed theories to explain such an effect is that the hematic glucose levels affect the metabolism of the hippocampus, a part of the brain (associated with memory, emotion and motor skills), which is strongly damaged in alzheimer's patients. the aim of this study is to investigate the effect of insulin on the a-beta induced degeneration and oxidative stress on the neuroblastoma lan5 cell line. in particular, the present study looks into the role of insulin in the inhibition of abeta specific degenerative apoptotic pathways. preliminary results indicate that insulin dissolved in culture medium in its hexameric form (as tested by absolute scale light scattering) is able to reduce neurodegeneration induced by a-beta amyloid in a dose dependent manner. the link between diabetes and alzheimer's disease may provide new targets for future alzheimer's treatments. moreover, due to the increased incidence of diabetes in western countries, a deeper understanding of such a link is relevant in order to control the escalation in the number of people dealing with dementia. a. pelizzola dipartimento di fisica, politecnico di torino, torino, italy many features of protein folding have been shown to be described by an ising-like model (one-dimensional, with longrange, multispin interactions) whose equilibrium thermodynamics is exactly solvable [1] [2] [3] . we have generalized such a model to the problem of mechanical unfolding. the equilibrium thermodynamics is still exactly solvable, and the characteristic kinetic responses found in force ramp and force clamp experiments are well reproduced [4, 5] . unfolding and refolding pathways and intermediates can also be studied, again with good agreement with experiments [6]. applications to various proteins and rna fragments will be discussed. [ the key role of water in living systems has been widely studied in literature, along with its anomalies, consequence of the extensive three-dimensional hydrogen bonding of water molecules. moreover protein-water interactions take place at protein surface where cell water has been recognized to behave differently from bulky water. the two-states theory of water assumes that water is a mixture of microdomains of different structure and density, the low-density water (ldw) and the high-density water (hdw) domains, and ions partition selectively into ldw or hdw domains. the idea developed in this work was to explore the ordered water structure by measuring delayed luminescence (dl) from salt aqueous solutions in which water structuring is anticipated. it appeared that dl signal from salt solutions is significantly relevant when prevalence of ldw domains is foreseen, with a decay time probability distribution function characterized by a broad maximum in the microsecond range. the obtained results support the ability of dl to reveal the different properties of ldw and hdw domains induced by salt molecules. moreover, the results reveal the existence of clusters, whose characteristics strongly depend on the specific ion effects, of surprisingly long lifetimes not observed till now. this could give new insight into biological water properties. self assembly of patchy particles and dnafunctionalized dendrimers f. sciortino dipartimento di fisica e infm-cnr-soft università 'la sapienza', roma, italy i will report numerical results on the phase behavior of very simple models of patchy particles with the aim of understanding the interplay between phase separation and selfassembly and how the fraction of surface allowing for attractive interactions controls the collective behavior of the system. the case of janus particles, particles characterized by a surface divided evenly into two areas of different chemical composition, will be discussed. i will also discuss the self-assembly of a simple model for four single strands of dna tethered to a central core, and show that the model exhibits a rich phase diagram that includes at least four thermodynamically distinct amorphous phases (polyamorphism) in a one-component system. the dense phases consist of a hierarchy of interpenetrating networks, reminiscent of a woven cloth. peptide dimer motifs in the phospholipid environment -structure, interaction and molecular design p. e. schneggenburger 1 , a. beerlink 2 , t. salditt 2 , u. diederichsen 1 1 iobc, universität göttingen, germany., 2 irp, universität göttingen, germany. based on recently reported homodimeric peptide pores with a d,l-alternating configuration a novel double helical hairpin-motif of a membrane active gramicidin a analog was designed. [1, 2] the cd spectroscopic analyses of the peptide-lipid complexes revealed the structural preservation and elucidated the importance of a zwitterionic interaction of the peptide termini. [3] the peptide design was enhanced regarding the versatile functionalization with analytical probes as well as molecular recognition moieties like peptide nucleic acids (pnas) to observe the effects of aggregation and specific organization within model lipid membranes even at high peptide-to-lipid ratios. [3] x-ray reflectivity on lipid bilayer stacks in combination with heavy atom labeling and spectroscopic studies of vesicle systems provides information about the peptide structure and interaction in the native fluid state of the membrane system. [2, 3] for this, the fmoc-diiodo-allylglycine building block was created to serve as a novel iodine label pinpointing at a certain position with respect to the membrane normal. we study thermal undulations of giant unilamellar vesicles (guvs) of lipids by flickering spectroscopy. getting values for the mechanical parameters of lipid bilayers requires the experimental fluctuation spectra to be scrutinized in view of the classical helfrich's theory. pure bending modes are revealed unable in predicting the large fluctuations systematically found at high wavevectors. hybrid curvature-dilational modes have been invoked as a more efficient mode of motion in producing high curvatures. a bimodal spectrum of the thermal undulations has been theoretically developed for the shell-like topology. from this new description, two important consequences emerge a priori, the dependence of the fluctuation dynamics on either vesicle size and on bending/compression parameters. for popc and dopc vesicles containing cholesterol the experimental fluctuation spectra are well described by the new spectrum. reconciliation between experiments and theory is achieved when this bimodal spectrum is considered. the new theory opens enormous possibilities for better exploring membrane mechanics in guv models. under normal conditions, platelets circulate in the vascular system having very low interaction with each other and with other cells. the platelets become activated when the biological system is disturbed, for instance by vascular damage in which blood gets in contact with collagen. upon activation, different types of receptors/molecules are exposed on the cell membrane to support adhesion, spreading and aggregation of the platelets onto the damaged vessel. the measurement of altered platelet function is particularly important in cardiovascular diseases such as thrombosis. we are investigating biosensor technologies for the detection of functional properties of platelets. an important study is the specific and non-specific stimulation of platelets in a biosensor cartridge. we will present experimental results on biosensor platelet activation using the platelet-specific membrane markers p-selectin and gp1b. multi-joint analysis of locomotion in the first neonatal rats flown in space d. sulica, j. vinersan "carol davila" university of medicine and pharmacy, bucharest, romania the first mammalian neonatal animals in space were the rats flown on the space shuttle endeavor during a 9-day mission, sts-72. the development of locomotion in weightlessness was evaluated using two litters of neonatal rats, launched at postnatal days 7 and 15. age-and cage-matched animals were used as ground controls. free walking was videotaped from the landing day. although preliminary analysis of walking showed differences in both hindlimb and forelimb joint angles and a hyperextension of the hindlimbs was apparent, the numerical values reached the significance level only for the ankle angle measured at specific moments of the step cycle: foot contact, maximum loading with weight, foot lift and maximum flexion during swing. we report here on the behavior of all the joints during the whole step cycle, by computing the integral of these angles over the step cycle. the results were affected by the differences in the walking speed (the young animals walked faster than the controls), so we scaled the integrals by the step cycle duration. we found that, besides the ankle, the knee was also more extended throughout the whole step cycle in both groups of animals. moreover, all the joints (including the toe and the hip) were affected in the same way (hyperextended), since the differences were still significant when we added together these angles. the animals recovered slowly, with significant differences remaining after 14 days of readaptation. the effect of dextran 70 concentration on red blood cell deposit formation j. strzelecka, b. grzegorzewski department of biophysics, collegium medicum in bydgoszcz, nicolaus copernicus university 85-067 bydgoszcz, poland red blood cell (rbc) deposit formation was examined by means of an optical method. blood was obtained from healthy donors and measurements were performed at initial hematocrit 40%. the intensity of scattered light was measured during sedimentation of rbcs suspended in saline -dextran 70 solutions at different polymer concentrations (2 -6 g/dl). the changes in the intensity of the scattered light manifest rbc aggregate formation, their sedimentation and the process of deposit formation. the deposit formation curve was determined. it is shown that the concentration of dextran affects the deposit formation. an empirical model has been used to describe the experimental data. the parameters of the deposit formation curve as a function of dextran concentration are analyzed. water is essential to life and a major scientific interest lies in a detailed understanding of how it interacts with biological macromolecules in cells. we studied water dynamics in whole cells with neutron scattering [1, 2, 3] . the cellular environment is extremely crowded with macromolecules and water molecules are permanently in close contact to biological interfaces. we measured water dynamics in e. coli and human red blood cells with neutron scattering [2, 3] . the data revealed two populations of water in the cells: a major fraction which has dynamical properties similar to those of bulk water (relaxation times ∼ps) and a minor fraction in the order of ∼10% which is interpreted as bound hydration water with significantly slower dynamics (relaxation times >49 ps). in this contribution we report on investigation of model membrane dynamics by means of quasi elastic and inelastic incoherent neutron scattering and on the effect of membrane inserted pore forming peptide gramicidin. model membrane are realized by highly oriented, hydrated phospholipid bilayer stacks of dmpc (1,2-dimyristoyl-snglycero-3-phoshatidylcholine) hydrated with d2o in excess of solvent condition. the bilayer were supported on mica substrates and prepared at different concentrations of gramicidin, a 15-residue oligopeptide showing antimicrobial activity by forming pores on the membrane surfaces which allow water and small ions to permeate across the membrane. incoherent qens and ins spectra, measured on in5 and in13 spectrometer at ill, allows to obtaining information on the mean dynamics of the hydrogen atoms in the system. moreover, by proper orientations of the membrane plane respect to the scattering wave vector q, we were able to derive information on in plane and out of plane motions of the phospholipids. the using of media products for the creating attracted bioenergetic brain rhythmus advertisement v. i. vlastopulo, v. g. nikolajev str. gen. petrova 92 , app. 44, 65065 odessa, ukraine the technical efficiency of bioenergetical influence of advertisement is present with assistance of consciousness of memory at revision and hearing of advertisement on tv, radio stations, mobile phones, at supermarkets and other places. in a basis of useful model it is put a task to improve the method of creating the attracted advertisement, in which the creation of bioenergetical influence by the oscillation of not less 2 electromagnetic fields or video-images is introduced with their creating as the base on the spatial or flat structure formative macro matrix or matrixes with repeatable structure with the brain α-rhythm frequency of extreme attention and δ-rhythm frequency of meditation. the point of the patent on the device is in the bioenergetical influence increases in addition to the information influence by advertisement of pictures and audio oscillations the bioenergetical influence increases at the consciousness contribution of human memory at the moment of watching or hearing of television, radio stations, working in the internet, music in the supermarkets, banks, clubs, metro and other places. cell adhesion and motility are processes involved in fundamental biological phenomena. they imply multimolecular scaffolds as anchorage points and actin cytoskeleton filaments to build internal stress and eventually crawl onto the substrate. these processes, very dynamic by nature are out of equilibrium. we study cell adhesion on micro-patterned substrates where the introduction of a finite distance between the possible anchorage points of the cell modifies drastically the organization of the cytoskeleton and the anchorage point's distribution. because statistical quantification shows that some shapes are more likely than other, we believe they represent particular organizations of the system which should minimize the energy dissipation. we checked this hypothesis by using the cellular potts model. shapes obtained by simulation are in excellent qualitative agreement with experimental shapes. they depend on phenomenological parameters such as interaction between cells and the extra cellular matrix, a line tension and an elastic modulus. the aim of this work is to link model parameters to physico-chemical properties of cells and to establish phenomenological relations between relevant biochemical regulators controlling the cytoskeleton organization. c. canale, d. ferrera, f. benfenati, l. gasparini the italian institute of technology, 16163 genova, italy alzheimer disease (ad) is characterized by cerebral extracellular deposits of β-amyloid (aβ) fibrils. aβ aggregation is a multi-step process involving the formation of various conformational species including soluble intermediate species (i.e. aβ oligomers), protofibrils and fibrils. such aggregates may have various effects on neuronal and glial function and differentially contribute to ad neurodegeneration. aim of this study was to investigate the structural properties of distinct aβ aggregated species and dissect out their effects on neuronal viability. recombinant aβ42 and aβ40 peptides were aggregated in vitro in conditions differing by ionic strength, temperature and ph and were analyzed by gel electrophoresis, thioflavin t binding assay and atomic force microscopy (afm). afm analysis was performed using both hydrophilic and hydrophobic substrates, to analyze the full spectrum of structural species. stable low molecular weight oligomers were obtained when aβ42 was incubated at 37 • c for 5 days in low salt concentration buffer. doughnut-shaped conformational species were detected by afm alongside globular aggregates (1.6-5.2 nm height range). acidic ph promoted aggregation of aβ42 into thioflavin-positive fibrils and protofibrils. protofibrils appeared as beaded chains having a mean height of 3.65±1.49 nm. effects of aβ on viability of mouse hypppocampal neurons were assessed and correlated with their conformational features. a. bellova 1 , e. bystrenova 2 , m. koneracka 1 , p. kopcansky 1 , f. valle 2 , j. bagelova 1 , f. biscarini 2 , z. gazova 1 1 1 institute of experimental physics, slovak academy of sciences, kosice, slovakia, 2 2 ismn cnr, bologna, italy peptide amyloid aggregation is a hallmark of amyloid diseases including azheimer's disease or type ii diabetes. recent works have addressed the potential of nanoparticles to affect amyloid aggregation. the experimental data are very controversial suggesting that particle characteristics markedly influence the final effect of nanoparticles on the amyloid aggregation (initiation, acceleration or inhibition of amyloid aggregation). we investigate the ability of electrostatically stabilized magnetic nanoparticles of fe 3 o 4 to affect the amyloid aggregation of lysozyme, as a prototype amyloidogenic protein. we have used a combination of spectroscopic (tht fluorescence) and local microscopic techniques (afm). we found, that the ability of magnetic nanoparticles to inhibit formation of amyloid aggregates or destroy pre-formed amyloids exhibit concentrationdependence. the values of inhibition ic50 and depolymerization dc50 were determined suggesting that nanoparticles interfere with lysozyme aggregation at stoichiometric concentrations. the observed features make magnetic nanoparticles of potential interest as a therapeutical agent against amyloid diseases. (this work was supported by project of esf 26220120021 and by slovak academy of sciences in frame of cex nanofluid, vega grants 7055, 0056 and 0038 and eu-strp 0032652 biodot.). a. bellova 1 , l. balogova 2 , b. chelli 3 , e. bystrenova 3 , f. valle 3 , j. imrich 2 , p. kristian 2 , l. drajna 2 , j. bagelova 1 , f. biscarini 3 , z. gazova 1 1 institute of experimental physics, sas, kosice, slovakia, 2 faculty of sciences, p. j. safarik university, kosice, slovakia, 3 ismn cnr, bologna, italy numerous diseases have been linked to a common pathogenic process called amyloidosis, whereby proteins or peptide clump together to form amyloid aggregates in the body. an attractive strategy to develop therapies for these diseases seems to be reduction of polypeptide aggregation. we have tested several acridine derivatives characterized by various glycosyl groups for their potential to affect the lysozyme amyloid aggregation in vitro. the ability of glycosyl acridines to interfere with lysozyme aggregation was investigated by tht assay. we found that structure of acridine side chain is factor affecting their anti-aggregation activity significantly. for the most effective compounds the values of ic50 and dc50 were obtained. the reduction of protein aggregation was confirmed by afm. to investigate influence of the glycosyl acridines on the cell processes we examined effect of compounds on cell viability. we performed glycosyl acridines characterization by high anti-aggregation activity and low toxicity suggesting their possible application for therapeutical purpose. (this work was supported by project of esf 26220120021 and by slovak academy of sciences in frame of cex nanofluid and vega grants 7055, 0056 and 0038 and eu-strp 0032652 biodot.). a. j. beevers, a. m. dixon department of chemistry, university of warwick, uk due to the immense medical importance of proteins which span the membrane of cells, detailed molecular structural information of these systems is essential. practical difficulties in employing high-resolution structural elucidation techniques have resulted in a relative paucity of fully resolved membrane protein structures. therefore a variety of lower-resolution techniques are used to determine structural information of the transmembrane (tm) domains of proteins. one example of such a membrane protein is erbb-2, a receptor tyrosine kinase responsible for triggering cell division and which is prone to a mutation in its transmembrane domain resulting in permanent activation and oncogenic effects. we have predicted an interface for the mutated tm domain dimer using site-specific infrared spectroscopy containing a repeating sequence of ile, val and leu 1 . applying the in vivo toxcat assay to the tm domain sequence and to specific mutants of it, confirms this proposed interface whilst another proposed interface is discounted. current studies are focussing on the effect of the tm mutation to the activation of the erbb-2 receptor and to any possible change in this interface. bacteriophages are complex molecular assemblies which multiplication relies on bacteria infection. the process starts with the binding of the phage on its specific host receptor and the injection of its genome into the host cytoplasm. our work aims to determine the physical mechanisms and forces driving the dna transfer from the phage capsid. the in vitro dna ejection has been analyzed by using light scattering and gel electrophoresis measurements for three phages (t5, spp1 and lambda) belonging to the same family (syphoviridae). our results reveal two forces contributing to drive the dna transfer: the first one is originated from the pressurization due to the strong confinement of dna into the capsid; the second one comes from a pulling mechanism originated by the presence of condensed dna outside the capsid. these two contributions were characterized in in vitro conditions but they likely play a role in the in vivo transfer. the ejection kinetics was also analysed and the characteristic time of the mechanism was studied as a function of the temperature. it appears to follow an arrhenius law, allowing the determination of the activation energy that governs the transfer. the energy values are close for the different phages, suggesting that the mechanism regulating the ejection is common for a given phage family. below these general features, our studies also reveal differences between the three phages. the effect of &beta-amyloid peptide on polymer cushioned membranes s. dante 1 , r. steitz 2 , t. hauss 2 , c. canale 1 , n. a. dencher 3 1 istituto italiano di tecnologia, genova, italy, 2 bensc, helmholtz center berlin, germany, 3 tu-darmstadt germany beta-amyloid (aβ) is a peptide implicated in the neurodegenerative process characteristic of the alzheimer's disease (ad). to clarify its mechanism of action it is crucial to elucidate the interaction of aβ with the neural membrane. in previous work we demonstrated the capability of aβ to penetrate and perturb stacked lipid bilayers. in this study we considered polymer cushioned lipid bilayers as a model for neural membranes. the polymer cushion is aimed to preserve the membrane natural fluidity; it is obtained depositing charged polyelectrolites layer-by-layer; the lipid membrane is built on the top of the polymer film by fusion of unilamellar vesicles. the floating membranes were kept always in contact to the subphase. the kinetics of adsorption of the lipid double layer at the polymer/water interface was monitored by neutron reflectivity; different experimental conditions to obtain the best surface coverage were exploited. after administration of aβ to the subphase the lipid membrane still adhered to the polymer cushion, but its structure was modified by the interaction with aβ. neutron reflectivity showed a change of the scattering density profile in the direction perpendicular to the membrane plane, suggesting a penetration of aβ inside the double layer. a change in the surface morphology was detected by afm imaging; afm film-rupture experiments showed that aβ weakens the lipid packing. x. cheng 1 , r. pacheco-gomez 2 , a. rodger 1 , h. matthew 1 , d. i. roper 1 1 university of warwick, u.k., 2 university of birmingham, u.k. ftsz, the ancestral homolog of eukaryotic tubulins, is a gt-pase that assembles into a cytokinetic ring structure (z ring) essential for cell division in prokaryotic cells. the z ring also recruits other proteins (e.g. zapa, ygfe, zipa) to the division site, where they participate in formation of the septum that separates the two daughter cells. we have studied ftsz polymerization and its dynamic behaviour in real time by right angle light scattering. similar to tubulin, ftsz polymerizes into dynamic protofilaments in the presence of gtp; polymer assembly is accompanied by gtp hydrolysis. the kinetics of inorganic phosphate (p i ) released from the gtp hydrolysis have been studied as well, employing a fast and sensitive colourimetric assay. at ph 6.5, approximate 90% of the p i was released into the media within 20 minutes of gtp addition. the effects of gtp, ph, k + , and mg 2+ were studied in both cases, and the results were used to build up a model for the mechanism of fibre assembly and disassembly. ygfe, a ftsz accessory protein, is identified as a functional zapa orthologue. finally, we have studied the ygfe bundling to ftsz polymers. it strongly promotes ftsz bundling and is an inhibitor of the gtpase activity. many genomes of viruses encode small membrane spanning proteins which are proposed to modify membrane permeability for ions and small molecules. these channel or pore forming proteins are getting into the focus for antiviral therapy since they are essential for some of the viruses. one of the general themes of the mechanism of function of the proteins is to self-assemble to form the functional form. we present a study on the open reading frame (orf) 8a membrane protein encoded in structural region of human severe acute respiratory syndrome coronavirus (sars-covs). the full length orf8a protein is 39 residues long and contains a single transmembrane (tm) domain. full length protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers forms cation-selective ion channels. the bilayer recordings show cation selection channel activity with a major conductance level of around 8.5 ps also at elevated temperatures (38.5 • c). in silico studies with a 22 amino acid tm domain are done to assess conformational space of the monomeric orf8a helix. with this monomeric helix homooligomeric helical bundle models are built and embedded in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycerol-3phosphatidylcholine (popc) bilayer. results of both experimental channel recordings and computational modeling show sars orf8a to act as a channel forming protein. -biomolecular self-assembly microtubules are involved in many vital processes. their rigid structure can resist high forces while their intrinsic ability to switch stochastically between growth and shrinkage phases allows them dynamically to reorganise. in cells, a sizeable network of microtubule binding proteins control and regulate microtubule dynamics. alp14 and dis1 are members of the dis1/ xmap215 family that are major players in s.pombe. the deletion phenotypes of alp14 and dis1 are similar, but nonetheless distinct. both are involved in the formation of spindles but alp14 is also involved in the maintenance of cytosolic microtubules in interphase. the restrictive temperatures of alp14-deletion and dis1-deletion mutants are different. alp14 interacts with alp7, a potential member of the tacc protein family. i am working to reconstitute alp14/dis1-dependent microtubule dynamics in vitro, using purified s. pombe tubulin. both alp14 and dis1 express well in insect cells and can be readily purified. preliminary data show that both proteins bind tubulin at low salt concentrations and that both influence the dynamics of pig brain microtubules. my goal is a complete functional analysis of alp14 and dis1, individually and in combination, to test candidate molecular mechanisms for alp14/dis1-catalysis of s. pombe microtubule dynamics. the syphoviridae coliphage t5 is a well-suited model to study the assembly of large viral capsids. biochemical and biophysical approaches were used to reconstitute in vitro the assembly pathway of its capsid. the t5 structure was recently solved from cryo-em and image reconstruction. its icosahedral capsid (t = 13) is built from the major head protein (pb8, 775 copies) forming both the pentons and hexons and from the portal protein (pb7, 12 copies) located at one vertex. its assembly proceeds by steps. pb8 and pb7 first assemble into a precursor structure called prohead i, which is converted to prohead ii by proteolysis of pb8 and pb7 by a head maturation protease. packaging of the 121 kbp dsdna is then driven through the portal pore by a molecular motor, the terminase. this promotes expansion of prohead ii leading to the mature capsid. the different assembly steps and the conformational changes accompanying capsid maturation were characterized using proheads i either self-assembled from the overproduced and purified capsid proteins or isolated from a phage mutant. these precursor capsid structures were analysed by small angle x-ray scattering. the 3d structure of prohead ii and of the expanded capsid were solved from cryo-em. our data show that the assembly process of a large icosahedral capsid can be efficiently reconstituted in vitro. amyloid beta peptide fibril formation modulated by phospholipid membranes e. hellstrand 1 , e. sparr 2 , s. linse 1 1 lund univ., department of biophysical chemistry, sweden, 2 lund univ., department of physical chemistry, sweden disease-causing amyloid fibril formation can be modulated by many factors including interactions with biological lipid membranes. an increasing amount of evidence suggests that the process of fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. alzheimer's is probably the most well-known amyloid disease and the associated amyloid beta peptide originates from the membrane incorporated amyloid precursor protein (app). we use recombinant abeta m1-40 and abeta m1-42 produced in escherichia coli, which allows us to perform large scale kinetics assays with good statistics where the amyloid formation process is followed in means of thioflavin t fluorescence. the lipid membranes are introduced in the system as large unilamellar vesicles composed of dopc, dppc and sphingomyelin, with and without incorporation of cholesterol. we find that the phase behanviour of the membrane in the vesicles has a large effect on the lag time of the amyloid formation process for both abeta m1-40 and m1-42. all membranes increase the lagtime to some degree but dppc has the largest effect. by comparing different phases we can conclude that the translational diffusion in the membrane seems to be more important than the acyl chain ordering. furthermore, electrostatics, concentration dependence and membrane addition at different time points in the amyloid formation process have been investigated. equilibrium/non-equilibrium transitions in macromolecule interactions p. dumas 1 , g. gibrat 2 , s. bernacchi 1 , e. ennifar 1 1 ibmc-cnrs, strasbourg, france, 2 llb (cea/cnrs), saclay, france usually, so called 'relaxation phenomena' occur on a fast time-scale and 'p-jump' or 't-jump' techniques are required to follow such events lasting (much) less than 100 ms. we report that, during stability studies of proteins or nucleic acids, such relaxation events can be observed on astonishing long time-scales. we first performed 'melting studies' with nucleic-acid duplexes by using linear variations of temperature (t) with time (t). we observed that even very low rates dt/dt could lead to a frozen state for temperature values below a sharp temperature range, and relaxation to equilibrium beyond that range. this allows defining a 'relaxation temperature' t r separating the two regimes. numerical simulations very accurately described the related hysteresis phenomenon observed upon a heating-cooling cycle, which is the hallmark of departure from equilibrium. analogous observations were made with protein oligomers submitted to either a variable pressure, or variable concentration in denaturant. importantly, a single theoretical frame predicts that the critical relaxation value x r (x standing indifferently for temperature, pressure or denaturant concentration) depends on ln(dx/dt). one may ask whether some thermosensor rnas known for switching on or off genetic expression by 'feeling' a temperature variation, might also 'feel' dt/dt. if true, the exact switching temperature would depend on dt/dt and faster temperature changes would increase t r . -biomolecular self-assembly the major component of amyloid plaques in the gerstmann-sträussler-scheinker disease is a prion peptide fragment from 81-82 to 144-153 residues. here, we present a structural study of prp82-146 in form of oligomers and fibrils by fourier transform infrared spectroscopy (ftir) and atomic force microscopy (afm). after incubation at 37 • c, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands and by a wide distribution of oligomer volumes. after a lag phase, a conformational rearrangement of oligomers into fibrils, with a parallel orientation of the cross β-sheet structures, was observed. by afm, different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly. in addition, we also studied thermal and random aggregation. the prp82-146 peptide was found to undergo several aggregation pathways, whose end products display different structural properties and intermolecular interactions. these findings underline the high plasticity of the prion peptide, a peculiar feature of prion proteins to overcome species barriers (natalello et al. j.mol.biol. 2008;381:1349-1361). the role of proline isomerisation in the aggregation process and fibril formation of alpha-synuclein j. meuvis 1 , m. gerard 2 , v. baekelandt 3 , y. engelborghs 1 1 lab.of biomolecular dynamics, ku leuven, belgium, 2 lab.of biochemistry, campus kortrijk, belgium, 3 lab.for neurobiology & gene therapy, ku leuven, belgium alpha-synuclein (α-syn) plays a central role in parkinson's disease. the aggregation of this protein, which contains five proline residues (p108,p117,p120,p128,p138), is accelerated in vitro by fk506 binding proteins (fkbps), a family of enzymes with a peptidyl-prolyl cis-trans isomerase activity (ppiase). fkbps catalyze the cis-trans conformational change of proline, often a rate limiting step in protein folding. to elucidate the role of the proline residues in aggregation, we constructed a mutant p(108,117,120,128,138)a α-syn . the kinetics of the aggregation of the mutant were studied with turbidity and thioflavin t fluorescence (tht). turbidity measurements show the formation of early, tht negative aggregates which is as fast for both wt and mutant. fibril formation however is faster for the proline-deficient mutant. we also studied the effect of fkbp12 on the aggregation of the mutant. although wt α-syn early aggregate formation is accelerated by the addition of 1 µm fkbp12, this effect disappears in the mutant. addition of (1pm-1µm) fkbp12 accelerates the fiber formation of wt α-syn, which is abolished in the mutant. we can conclude that α-syn fiber formation is accelerated for the proline-deficient mutant, which suggests a role for the proline residues in fiber formation. furthermore all accelerating effects of fkbp12 are abolished in the mutant which suggests that the ppiase activity of fkbp12 is responsible for the accelerating effect on the aggregation of wt α-syn. materials used as gene delivery vehicles must be able to condense dna into small sizes to facilitate transport and crossing various barriers. one of the polycations investigated for dna compaction is chitosan, which has the advantage of being safe and biodegradable. as a step towards reducing the aggregation behaviour of dna-chitosan complexes, chitosans were modified by grafting peg-chains on the backbone. it is known that the transfection efficacy depends on the chitosan chain length. additionally, the degree of pegylation might influence the condensation process. here a systematic biophysical study of pegylated chitosans and how the interplay between chitosan chain length and degree of pegylation affect the compaction of dna in terms of particle size and structure, stability in pbs and when exposed to serum, and transfection efficacy is presented. three different chain lengths of chitosans are employed, and for each sample three pegylation degrees are investigated and the properties of the dna-pegchitosan complexes compared to complexes formed when employing the original, chitosan for dna compaction. it is found pegylation of chitosans can be used to increase both the stability of the dna-chitosan complexes when exposed to serum as well as increase their transfection efficacy in hek293 cells. max-planck institute for polymer research, mainz, germany model membranes mimic the essential function of a natural membrane. however, the complexity is reduced in order to allow the study of fundamental processes. tethered membranes consist in principle of a lipid bilayer that is covalently linked to a solid support through a spacer group. this architecture allows the characterization of the membrane itself as well as of incorporated membrane proteins using surface analytical techniques. we have established a versatile system of various anchor lipids, which allow membrane formation on different surfaces. the architectures have been characterized by surface plasmon techniques, neutron reflectivity and electrochemical methods. the membranes are electrically insulating and allow for the functional incorporation of ion channel proteins. polymerizable lipids allow to pattern the membrane and to study lateral diffusion processes. furthermore, the membranes can be used as a sensing platform, where embedded membrane proteins act as actual sensing units. a. perico, s. pietronave, l. arcesi, c. d'arrigo consiglio nazionale delle ricerche (cnr), institute for macromolecular studies (ismac) the electrostatic free energy (fe) of two parallel rigid likecharged polyelectrolytes (pes) is given as a function of the separation distance. 1 for high linear charge density, z , the fe shows a minimum due to the increasing of the counterion condensation and condensation volume as the two pes approach. the interaction fe is governed by a critical linear charge density, z c , inversely proportional to the counterion valence. for highly charged pes (z > z c , like dna), the pes attract the stronger the smaller is the counterion valence, because the fe is dominated by the entropic term due to condensation of counterions in a volume displaying a maximum at short distances. for weakly charged pes (z < z c /2 ) the pes remain undercritical in the whole separation range and therefore repel. for moderately charged pes (z c /2 < z < z c ), the infinitely separated pes are undercritical but become supercritical as they approach a critical distance and charge condensation and condensation volume expansion start: in these circumstances the pes may attract if the counterion valence is sufficiently large. in the case of many dna rods, hexagonal clusters may be formed. upon interaction with hydrophobic surfaces, proteins show a tendency to expose regions that are normally buried in the hydrophobic core. unfolding is generally perceived as an undesired process in studies aimed to anchor functional proteins at surfaces. upon an upset of perspective the fine control of the unfolding/re-assembly process could be regarded as a strategy to build up molecular nanostructures for the development of organic-inorganic assemblies. we show that molecular layers patterned at the nanoscale, with longrange order properties extending over the microscopic scale, can be obtained upon adsorption of proteins onto the hydrophobic and ordered surface of pyrolytic graphite. upon adsorption, proteins lose their native folding and polypeptide chains re-assemble on the surface in a layered fashion, forming a molecular bilayer. the first layer, in contact with the substrate, and the second molecular layer show corduroy-like nanopatterns of different periodicity, with a relative orientation between the first and second layer patterns of 30 • . surface-induced protein unfolding and polypeptide chain reassembly according to a layered ordered structure is a rather general phenomenon since it is observed for different proteins irrespectively of their specific structural properties. the possibility of using these ordered molecular structures as templates for the subsequent patterned deposition of supramolecular aggregates will be discussed. understanding protein-protein interactions and assemblies to control the hierarchical building of well-ordered supramolecular structures is highly relevant to new tailormade biomaterials. we previously evidenced that contrary to native calcium-loaded α-lactalbumin (holo α-la), calcium-depleted form (apo α-la) has the ability to selfassemble with lysozyme (lys) to form different supramolecular structures in temperature-dependent manner. in the present work, the events occurring at molecular scale were explored using fluorescence techniques. fluorescence anisotropy and fluorescence lifetime measurements provide a powerful and sensitive mean to measure intermolecular interactions. we showed that lys interacts with both apo α-la and holo α-la to form oligomers, assumed to be heterodimers, at 10 • c and 45 • c. the dissociation constants for dimerization, found to be in the µm range, were sensitive to the ionic strength. correlation time calculations suggest that formed heterodimers holo α-la/lys and apo α−la/lys differed in their shape and/or conformation. such conformation differences could explain why holo α-la/lys complexes are trapped as heterodimers while the apo α-la/lys complexes have the ability to further self-assemble into previously reported various supramolecular structures. polyglutamine aggregation and neurodegeneration g. nicastro, l. masino, a. pastore national institute for medical research, the ridgeway, nw71aa london, u.k. polyglutamine (polyq) diseases are rare but dominant misfolding diseases linked to neurodegeneration. they are caused by the expansion of cag codon repeats, which encode polyq tracts in the corresponding gene products. aggregation of polyq proteins is thought to be triggered by polyq expansion but be strongly modulated by the protein context. in the attempt of understanding the molecular bases of polyq diseases, we are studying the structures, interactomes and aggregation properties of selected polyq proteins. here, we present recent work on ataxin-3, taken as a representative example of the whole family. ataxin-3 is a ubiquitin specific cysteine protease, involved in the ubiquitinproteasome pathway and known to bind poly-ubiquitin chains of four or more subunits. the enzymatic site resides in the n-terminal josephin domain of ataxin-3. we have characterized, using different biophysical techniques, the structure in solution and the aggregation properties of josephin both in isolation and in a ubiquitin complex. we demonstrate that interaction with ubiquitin strongly modulates the aggregation properties of ataxin-3 and suggest the importance of protein-protein interactions in preventing aggregation. our study also provides new insights into the molecular mechanisms which determine ataxin-3 specificity for poly-ubiquitin chains of the correct length and cross-linking. förster resonant energy transfer (fret) from an optically excited to a non-excited molecule has been widely used to probe molecular interactions in living cells. changes in the molecular makeup of a cellular region occurring during the acquisition of fluorescence images place tight constraints on the fret technology and data analysis, which could not be addressed satisfactorily until recently. we will describe a method for imaging protein complex distributions in living cells with sub-cellular spatial resolution, which relies on a spectrally resolved two-photon microscope (raicu et al, 2009, nature photon. 3: 107-113) and a simple theory of fret in oligomeric complexes (v. raicu, 2007, j. biol. phys. 33:109-127) . then, we will overview recent results on the determination of the supra-molecular structure and distributions in living cells of oligomeric complexes of some g protein-coupled receptors. observing protein aggregates on surfaces m. rabe, d. verdes, s. seeger institute of physical chemistry, university of zurich, switzerland protein aggregation is an important topic of current protein research as it is associated with several human diseases including alzheimer's disease, parkinson's disease, and type ii diabetes. although protein aggregation mechanisms and conditions have been comprehensively investigated, studies on the formation and the fate of protein aggregates in contact with solid interfaces are scarce. we have comprehensively investigated the structure of protein assemblies that form spontaneously upon protein adsorption on solid interfaces using a surface sensitive fluorescence imaging technique based on super critical angle fluorescence (saf) detection. combining this technique with fret we not only succeeded to detect protein aggregates deposited on surfaces but also to characterize their behavior in real time, i.e., their emergence, growth, or spreading. the model protein bsa, for instance, was found to exhibit a certain tendency for aggregation in the buffer solution. these protein clusters can deposit onto solid surfaces and spread resulting in a large, flat structure after some time. 1 a different possibility how protein aggregates emerge on surfaces consists of a direct deposition of protein monomers to pre existing aggregates. such a growth of protein aggregates on the surface has been observed in a model system using the protein α-synuclein, which is tightly associated with the parkinson's syndrome. we present the results of a spectroscopic ellipsometry (se) study of the adsorption process of yeast cytochrome c (ycc) on gold and graphite substrates, according to methods already applied to study the growth dynamics of organosulphur sams on gold [1] . se investigation was carried out both in situ, at room temperature during protein deposition, and ex situ. on gold, se data demonstrate the formation of an about 3-4 nm thick layer, consistent with the formation of a dense monolayer of ycc molecules, confirmed by afm inspection. both in situ and ex situ measurements were characterized by well defined spectral features related to the soret band. analysis of the fine position of this feature allowed to obtain information on the oxidation state of the iron ion of the heme group. se data suggest that proteins have preserved their native structure. a completely different adsorption mechanism was observed on highly oriented pyrolytic graphite (hopg) [2] . ex-situ se data on ycc/hopg, supported by afm observations, indicate the formation of an ultrathin molecular layer (∼0.7nm) related to complete protein unfolding at the hydrophobic surface. the role of phospholipid anisotropy in the stability of inverted hexagonal phase was considered. the equilibrium configuration of the system was determined by the minimum of the free energy involving the contribution of the isotropic and deviatoric bending and the interstitial energy of phospholipid monolayers. the shape and local interactions of a single lipid molecule were taken into account. the minimization with respect to the configuration of the lipid layers was performed by the monte carlo simulated annealing method. at high enough temperature the lipid molecules attain a shape exhibiting higher intrinsic mean and deviatoric curvatures which fits better into the inverted hexagonal phase than into the lamellar phase. for the mathematical model the advanced geometry with non-spherical cross-section of inverted hexagonal phase was calculated, resulting in lower energy in non-spherical cross-section. theoretical results are in a good agreement with the small angle x-ray scattering experimental data. for a long while the conventional view has been that alzheimer disease is brought about by the beta-amyloid fibrils found in the senile plaques, but more recently it has been suggested that the main neurotoxic species would be the soluble oligomeric species, apparently prone to interact with cell structures and macromolecules potentially inducing neuronal dysfunction. peptide-peptide interactions resulting in self-assembly phenomena of beta-amyloid yielding fibrils can be modulated and influenced by small organic molecules that might also be effective therapeutic tools to ideally target both oligomeric and fibrillar species. in this perspective, polycyclic aromatic molecules are of special interest because they might disrupt the molecular architectures precursors of beta-amyloid fibrils by means of weak, non-covalent aromatic interactions, like stacking interactions. we have performed an in vitro spectroscopic study (light scattering, circular dichroism, ftir and fluorescence) of the effects on beta-amyloid fibrillogenesis of the natural pigment hypericin extracted from hypericum perforatum. our results show that, thanks to its structural characteristics and peculiar spectroscopic features, hypericin can be easily used to in vitro monitor the appearance of initial aggregation states of beta-amyloid peptides and, more importantly, that hypericin can interfere with the early stages of polymerization process, playing the role of an aggregation inhibitor. peptaibols are peculiar peptides produced by fungi associated to plants. they are composed by 4 to 20 amminoacidic residues and exhibit antibiotic and antifungal properties. due to their amphypatic nature, they can form ion channels in biological membranes. by making use of experimental models of biological membranes (biomimetic membranes) currently employed in the laboratory of bioelectrochemistry, and models of plant membranes (corn seed root), that are used in the international laboratory of plant neurobiology (linv), we characterized synthetic peptides such as trichogin gaiv and its shorter homologues (4 and 8 residues). we studied these peptaibols in a dioleoylphosphatidylcholine monolayer supported by hg using different electrochemical techniques (ac,vc,eis). the experimental technique employed in the linv (clark microelectrode coupled to mife system) allows to measure oxygen flux in the solution contacting plant cell membranes, after treatment with different peptide concentrations. preliminary results might indicate that short peptides can influence the whole metabolism of the plant and can therefore be used as "elicitors" in order to induce an acquired systemic resistance. supported biomimetic membranes (sbm) were developed for protein-membrane interactions studies. phospholipid vesicles were chemically linked onto amine grafted gold or glass surfaces; after an osmotic choc and liposomes fusion a continuous membrane bilayer was formed. the anchoring phospholipid molecule (dspe-peg-nhs) incorporated into the vesicles allowed the formation of a water-filled compartment between the surface and the bilayer. this first sbm model was used to monitor the membranes binding properties (dependent of calcium) of the adenylate cyclase toxin (cyaa) from bordetella pertussis. the sbm model was improved in order to study the translocation of the catalytic domain of cyaa across the bilayer. naturally, the cyaa catalytic domain, when it reaches the target cell cytosol, associates with intracellular calmodulin (cam) an activator of the adenylate cyclase activity of cyaa. to mimic this biological phenomenon, cam was first immobilized on the surface (gold or glass) and in a second step membrane construction was performed over the cam layer. the formation of the biomimetic membrane onto the cam layer was monitored by spr while membrane fluidity and continuity were analysed by fluorescence. our results demonstrated the potentialities of sbm for the study of protein insertion into and translocation across biological membranes. a multi-resolution approach to the structure and function of integrin αiibβ3 m. rocco 1 , c. rosano 1 , j. w. weisel 2 , d. horita 3 , r. r. hantgan 3 1 istituto nazionale per la ricerca sul cancro (ist), genova, italy, 2 university of pennsylvania, philadelphia, pa, usa, 3 wake forest university, winston-salem, nc, usa. integrins are heterodimeric transmembrane receptors involved in mechanical anchoring and two-way signaling. each α and β subunit has a modular structure with a large extracellular portion, a single transmembrane region, and a cytoplasmic domain. integrins activation mechanism is regulated by controversial conformational changes: while crystallography revealed similar bent shapes for resting and primed extracellular region constructs, ligand binding-induced large structural rearrangements in smaller constructs suggested extension, "opening" and tails separation. in a multiresolution approach, we used experimental and computed hydrodynamics to discriminate among αiibβ3 integrin models built on x-ray, nmr, and em data. in contrast with xray data and 3d em maps, an extension is needed to match the hydrodynamics of full-length, solubilized αiibβ3; an electron tomography-based model fares better. consistent with that, and with our averaged 2d em images, a conformational change in the head region (β3 hybrid domain swingout) coupled to a simple transmembrane helices shift matches priming agents-induced frictional changes in full-length αiibβ3. our multi-resolution study thus suggests that in integrins extension and immediate tail separation are uncoupled from head domain rearrangements following activation. -biomolecular self-assembly stabilizing effects of α s1 -casein, a natively unfolded protein, on the aggregation of biomolecules a. trapani, r. carrotta, p. l. san biagio, d. bulone ibf-cnr palermo, italy α s1 -casein is one of the four types of caseins, a group of calcium phosphate-binding proteins that, in the form of micellar aggregates, makes up the largest protein component of bovine milk. the structure of α s1 -casein is that of a triblock polymer with a hydrophilic tract interposed between two hydrophobic blocks. due to the lack of a compact folded conformation, this protein can be classified as one of the intrinsically disordered (or natively unfolded) proteins. this class of proteins is known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces that partially unfolded molecules may expose to the solvent. here we present results on α s1 -casein effects on the thermally induced aggregation of gluthathione stransferase, a ligand-binding anzyme, and 1-40 β-amyloid peptide involved in alzheimer's disease. by means of light scattering and circular dichroism experiments, we attempt to reveal the molecular details of α s1 -biomolecules interaction. bacterial protein self-assembly on surfaces of well-defined chemistry s-layers are one of the most common cell envelope components of prokaryotic organisms and represent the simplest biological membrane developed during evolution. these (glyco)proteins, which can self-assemble into 2-d crystalline nanostructures on lipid films, liposomes, and polymers, play already an important role in nanobiotechnology. in this work, we present new findings concerning the recrystallization of bacterial proteins on substrates with defined chemistry. manipulation of the protein-sample interaction was carried out by changing the relative height of oh and ch 3 terminated moieties in self-assembled monolayers (sams). we have found that differences in chain length lead to: i) protein bilayer-protein monolayer transition, ii) preferential protein side adsorption, and iii) increase of the crystal lattice parameters. further manipulation of the protein-sample interaction was achieved by using silane chemistry. we will show that sample hidrophobicity speeds up recrystallization kinetics and reduces the crystalline domain size (and layer compliance). the protein-adsorbed mass per unit area on these substrates is reported for the first time. self-assembly of phenylalanine oligopeptides: insights from experimental and computational studies p. tamamis 1 , l. adler-abramovich 2 , m. reches 2 , k. marshall 3 , p. sikorski 3 , l. serpell 3 , e. gazit 2 , g. archontis 1 1 dpt. of physics, univ. of cyprus, cyprus, 2 dpt. of molecular microbiology and biotechnology, tel aviv univ., israel, 3 dpt. of biochemistry, univ. of sussex, u.k. the diphenylalanine peptide (ff) forms well ordered nanotubes and its derivatives form nano-assemblies of various morphologies with promising material applications. we demonstrate for the first time by electron and laser microscopy and ftir spectroscopy that the related, triphenylalanine peptide (fff) assembles into rather planar nanostructures, rich in β-sheet. in addition, we conduct 0.4-µs replica exchange m.d. simulations of aqueous ff and fff solutions in implicit solvent. the peptides coalesce into aggregates and participate frequently in open or ring-like linear networks, as well as elementary and network-containing structures with β-sheet characteristics. polar and nonpolar interactions, as well as the surrounding aggregate medium contribute to the network stabilities. within a network, consecutive peptides are linked by head-to-tail interactions; the aromatic sidechains of neighbor peptides assume approximately "t-shaped" orientations. these features are observed in ff crystals and could characterize early formations, or stabilize the mature nanostructures. the fff aggregates acquire higher stability and peptide-network propensity compared to the ff aggregates due to energetic contributions 1,2 . chlorophyll biosynthesis is light-dependent in angiosperms because the reduction of protochlorophyllide (pchlide) into chlorophyllide is driven by a photoenzyme, nadph:pchlide oxidoreductase (por). the unique properties of por are due to its ability to assemble into dimers and oligomers within the prolamellar body (plb) membranes of etioplasts studied mainly in leaves of dark-grown seedlings under laboratory conditions. we extended these studies to plant organs developed in the nature: cabbage heads, leaf primordia inside buds, pericarp-covered regions of sunflower cotyledons, potato tubers and seedlings germinating under the soil. in electron microscopic and fluorescence spectroscopic studies we found in many of these organs poorly developed plbs in which por was mainly in monomer state. as a consequence, the chlorophyll accumulation was slow and photo-oxidation processes occurred at illumination. in vitro we artificially induced the aggregation of por monomers into oligomers in glycerol and sucrose containing buffers. this resulted in the increase of the photoreduction rate at the expense of photo-oxidation. these results underline the importance of the self-assembly of por and the plbs in chloroplast development and chlorophyll synthesis in nature. v. vetri 1 , g. ossato 2 , v. militello 1 , m. a. digman 2 , m. leone 1 , e. gratton 2 1 dsfa, university of palermo, palermo, italy, 2 lfd, university of california, irvine, ca, usa we report an experimental study on concanavalina (cona) aggregation in live cells. in vitro, close to physiological temperature, cona readily forms fibrils involving secondary structure changes leading to β-aggregate structures. the effect of cona on cell cultures and formation of protein aggregates were measured by confocal fluorescence microscopy. in particular, we monitored protein aggregation in live cells by means n&b analysis, cross-n&b and rics. n&b showed the aggregation kinetic and the progressive formation of cona oligomers at cell surface. this suggests that, at cell membrane where local concentration is higher, nucleation sites for aggregation are provided. in parallel, the morphology of the cells changes indicating the progressive cell compaction and death. aggregation and binding of small aggregates to the cell surface were assessed by rics: it is possible to distinguish regions where small aggregates are diffusing and regions where they are bound to the cell. oligomers formation may stimulate non-specific cellular responses due to the exposure of reactive regions of protein structure and of progressive formation of cross−β structures. moreover, aggregates stoichiometry was measured during the kinetic by cross-variance n&b. the two conductive pathways of p2x7 purinergic receptor: different modulation and selectivity r. barbieri 1 , s. alloisio 1 , a. di garbo 2 , m. nobile 1 1 institute of biophysics, cnr, via de marini 6, 16149 genoa, italy, 2 institute of biophysics, cnr, via g. moruzzi 1, 56124 pisa, italy the p2x 7 purinoceptor (p2x 7 r) is an atp-gated cation channel that is able to activate a cell permeabilizing pore. p2x 7 r cytosolic c-terminal tail is thought to modulate this function. this study was aimed to characterise the biophysical properties of p2x 7 r compared to those of the variant lacking the entire c-terminus tail (trp2x 7 r) by measuring whole-cell currents and intracellular ca 2+ variations. a mathematical model is used to describe the experimental results. in p2x 7 r expressing hek-293 cells, the potent agonist 3'-o-(4-benzoyl)benzoyl adenosine 5'-triphosphate (bzatp) -evoked ionic currents depending on concentration and frequency of agonist applications. the currents were strongly inhibited by extracellular mg 2+ in a noncompetitive way. by contrast, in trp2x 7 r cells, only high bzatp concentrations elicited small currents not affected by mg 2+ . interestingly, bzatp-induced ca 2+ influx was present both in p2x 7 r and in trp2x 7 r cells, albeit in the latter the intracellular ca 2+ elevation was smaller. importantly, in trp2x 7 r the intracellular ca 2+ rise maintained a competitive mechanism of mg 2+ inhibition similar to that observed in p2x 7 r. the experimental data and the modelling findings support the tenet of a functional structure of p2x 7 r possessing two distinct conductive pathways. the review of our data on the effects of physical and chemical weak signals on physicochemical properties of water, cell volume, activity and the number of membrane proteins (receptors, ionic channels and enzymes, na + /k + pump and na + /ca 2+ exchanger), intracellular signal systems in norm and pathology (cancer and nerve disorders) would be presented. light microscopic, cell voltage-and patch-clamp, isotope, standard biochemical and genetic engineering methods were used. weak signal-induced effects on cell functional activity (intracellular enzymes activity, the number of functionally active membrane proteins) are realized by changing the physicochemical properties of extra-and intracellular aqua medium. the latter induces the modulation of na + /k + pump-induced cell hydration, which serves as a primary mechanism through which the autoregulation of pump and regulation of membrane excitability and chemosensitivity are realized. by genetic engineering method in oocytes it was shown that the correlation between na + /k + pump and na + /ca 2+ exchanger, which is realized through intracellular messenger systems, plays a crucial role in weak signals transduction in cells and determination of aging-induced increase of cell pathology. listeriolysin pore forming ability in planar lipid membranes at different ph listeriolysin o (llo) is a cholesterol-dependent cytolysin secreted by the intracellular pathogen listeria monocytogenes. its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. llo exhibits optimal cytolytic activity at low ph but it is still able to bind membranes at physiological or even slightly basic ph values in a cholesterol-dependent fashion. high cholesterol concentrations in the membrane restore the low activity of llo at high ph values. based on this broad ph activity we investigated the electrophysiological properties of pores formed by llo at room temperature and at different phs using planar lipid bilayer technique. llo is able to form pores both at ph 5.5 and 7.5 with a similar permeabilizing ability and similar heterogeneous conductances in the range of picosiemes to nanosiemens. cholesterol content directly correlates with llo activity but it does not change the pore characteristics. collectively, our results demonstrate that llo activity at physiological ph cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for listeria pathogenesis. s. aimon, g. toombes, p. bassereau institut curie, paris, france the physics of membrane/channel and channel/channel interactions is difficult to investigate in cells where it is nearly impossible to modify relevant parameters to deduce physics laws. to overcome these difficulties we built a model system in which voltage gated ion channels were reconstituted in giant unilamellar vesicles (guvs) for the first time. as a first step, we successfully expressed kvap (a voltage gated potassium channel) in e-coli. the channel was purified, fluorescently labelled and reconstituted in small liposomes. its functionality was checked with electrophysiology via fusion of these liposomes into black lipid membranes (blm). as a second step, guvs were formed from these small proteoliposomes using electroformation in a buffer containing 100 mm kcl salt. the proper incorporation of proteins into guvs was controlled using confocal microscopy. functional proteins were detected using the patch clamp technique. with our protocol, we are thus able to prepare guvs containing functional voltage-gated ion channels. one goal is now to study the effect of channel activity on its spatial distribution in these guvs. ryr activation in cultured shr cardiomyocytes at the end of the prehypertensive period the rate of [ca 2+ ] i elevation after the ryanodine receptor (ryr) activation by 4-chloro-m-cresol (4-cmc) and l-type ca 2+ channels (dhpr) activation by bay k8644 was studied in cultured (5 days) cardiomyocytes of spontaneously hypertensive (shr) and normotensive rats (wky, wistar) during 6 weeks of postnatal development. the differences in dh-prs and ryrs activities began to be evident after 3 weeks age when cicr formation has finished and became more expressed at the end of prehypertensive period (6 weeks). in response to 4-cmc (2 mm), a drastic increase in the rate of [ca 2+ ] i accumulation (2.85±0.8 times) in shr myocytes was registered after 20 days age versus a decrease in the rates of ca 2+ efflux from the sarcoplasmic reticulum of wistar and wky rat cardiomyocytes. bayk (80 µm) also induced more sharp [ca 2+ ] i elevation in shr myocytes (3.35±0.25 times) as compared with wistar (2.32±0.16 times) and wky (1.22±0.09 times) ones of the same age. our results argue that in shr and wky cardiomyocytes, as opposed to normotensive wistar rats, gradual growth of dhpr activity is observed, which follows in parallel with cicr formation in the excitation-contraction coupling during early postnatal ontogenesis, and drastic activation of ryr2 in shr myocytes after the process termination. cytochrome ba 3 from thermus thermophilus belongs to the large family of structurally related heme-copper oxidases. it accepts electrons from cytochrome c 552 at the p-side of the membrane and uses them to reduce oxygen to water. the energy released in this reaction is used for proton pumping across the membrane to form of an electrochemical proton gradient, used by the cell for formation of atp. in this work we followed the kinetics of single-electron injection into the oxidized nonrelaxed state (o h →e h ) of cytochrome ba 3 by time-resolved optical spectroscopy. two main phases of electron transfer were resolved. the first (τ∼17 µs) includes oxidation of cu a and simultaneous reduction of both low and high spin hemes. the second (τ∼420 µs) reflects reoxidation of both hemes by cu b . this is in significant contrast to the o h →e h transition of aa 3 -type oxidases, where the fastest phase is due to transient reduction of the low-spin heme a only. on the other hand, the single-electron reduction of the relaxed o state in ba 3 oxidase consisted of only rapid electron transfer from cu a to heme b, which is similar to that in aa 3 oxidase. this indicates a functional difference between the relaxed o and the pulsed o h states of cytochrome ba 3 . as opposed to the phospholamban pentamer, sarcolipin forms anion-selective channels in biomembranes l. becucci 1 , c. b. karim 3 , d. d. thomas 3 , g. veglia 2 , r. guidelli 1 1 chemistry department, florence university, 50019 florence,italy, 2 chemistry department, university of minnesota, minneapolis, mn 55455, 3 3department of biochemistry, molecular biology, and biophysics, university of minnesota, minneapolis, mn 55455 sarcolipin (sln) and the phospholamban pentamer (pln) are two membrane proteins that inhibit ca-atpase of the sarcoplasmic reticulum at low concentrations. in contrast to pln, sln stimulates maximal ca 2+ uptake rates. sln and pln were incorporated in a bilayer lipid membrane (tblm) tethered to a mercury electrode through a hydrophilic spacer. electrochemical impedance spectroscopy measurements show that sln forms channels permeable to chloride ion, weakly permeable to phosphate ion and impermeable to inorganic cations such as na + and k + . a relationship between this property of sln and its regulatory function on ca-atpase of sarcoplasmic reticulum is proposed. atp increases the permeability of a tblm incorporating sln to phosphate ion by associating to sln with an association constant of 0.1 µm. an explanation for this behavior is provided. sln can be identified with the " p i transporter" described by a.g. lee et al. conversely, both electrochemical impedance spectroscopy measurements and molecular dynamics simulations provide strong evidence that the pore of the pentameric form of pln does not act as a chloride channel. "social" domain organization and dynamics of nicotinic acetylcholine receptor at the cell membrane f. j. barrantes unesco chair biophys. & mol. neurobiology. univ. nac. sur, 8000 b. blanca, argentina a combination of experimental techniques (patch-clamp, confocal frap and fcs, single-particle tracking, highresolution fluorescence microscopy) has been used to analyze the supramolecular organization of the acetylcholine receptor (achr), the dynamics of the receptor at the cell surface, and the kinetics of receptor internalization. changes in cholesterol (chol) content affected muscle and neuronal-type achr organization and dynamics at the cell surface. chol depletion produced gain-of-function of single-channel dwell time. submicron-sized (∼250 nm) domains, stable over a period of hours at the cell membrane, could be resolved into achr "nano-clusters" with a peak size distribution of ∼55 nm by sted microscopy. chol depletion reduced the number of nanoclusters, increasing their size, and changed their supramolecular "social" organization on larger scales (0.5-3.5 microns). frap, fcs and spt experiments provided information on the dynamics of achr nanoclusters, disclosing the dependence of their mobility on chol content and cortical cytoskeleton. chol content at the plasmalemma may thus modulate cell-surface organization and dynamics of receptor domains, and fine-tune receptor channel function to temporarily compensate for acute achr losses from the cell surface. m. czaplinska, k. gwozdzinski, a. koceva-chyla division of research of structure of biopolymers university of lodz, lodz, poland doxorubicin (dox) and paclitaxel (ptx) are anticancer drugs commonly used in chemotherapy of breast cancer therapy, however, the use of these drugs is limited by the risk of developing heart failure. generation of reactive oxygen species contributes to the cardiotoxicity of doxorubicin. nitroxides are low molecular weight, stable free radicals reacting with ros and they present antioxidant properties. the aim of this study was to analyze the effects of pirolid (pd) on the oxidative stress induced by dox and taxane in mcf-7 breast cancer cell line. results from mtt test revealed that ptx is more cytotoxic towards mcf-7 cells than dox. the ic 50 was 0.3µm and 3µm, respectively. pd alone does not influence cell viability. pd in combination with both drugs did not change viability of cells. both drugs increased the level of carbonyl groups in cells. the highest level of peroxide was observed in cells incubated with dox (approx. 3-fold). nitroxide alone did not influence the level of peroxide in the whole range of concentrations. combination of pd with dox and ptx reduced the level of carbonyls depending on its concentration. pd did not affected on the level of peroxide in cells suspension. dox and ptx increased (2-fold) the level of carbonyls. pd decreased the level of peroxides in cells treated with dox and ptx. the lowest concentration of peroxide was observed at 50µm of pd. these results show that pd protect mcf-7 cells against oxidative stress induced by drugs. open channel structure of mscl from fret microscopy and simulation mechanosensitive channels open in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing and osmoregulation. here, we have determined the likely structure of the open state of the mechanosensitive channel of large conductance from e. coli (mscl) in a natural environment using a combination of patch-clamp studies, fret spectroscopy, epr data, molecular and brownian dynamics simulation. structural rearrangements of the protein are measured while controlling the state of the pore by modifying lipid bilayer morphology. fret efficiency changes can be related to distance changes using a monte carlo analysis program in conjunction with detailed orientational analysis. these measurements are used as restraints in all atom molecular simulations in order to determine the likely structure of the open state, whose probable conductance is derived from brownian dynamics simulations. transition to the open state occurs via large rearrangements throughout the protein that create a wide pore nearly 30 a in diameter. both transmembrane helices are found to line part of the pore. the n terminal helix is found to lie along the face of the membrane where it can act to sense membrane tension and directly transfer this to the pore lining helices. the method of coupling spectroscopic data with simulations is likely to be of great value for studying conformational changes in a range of membrane proteins. putative potassium channels in synechocystis sp. pcc 6803 v. checchetto, m. zanetti, g. m. giacometti, i. szabò, e. bergantino department of biology, university of padova, italy we are interested in the identification and characterization of potassium channels in the cyanobacterium synechocystis sp. pcc 6803, an organism which is considered the ancestor of plant chloroplasts. a bioinformatic screening of synechocystis proteome identified, among others, two proteins on which we focused our attention. the first one (syncak) displays sequence homology to mthk, a ca 2+ -dependent potassium channel from m . thermoautotrophicum. the second one (synk) is predicted to contain six transmembrane regions and the typical selectivity filter of all potassium channels. our goal is to understand their roles in the physiology of cyanobacteria. we cloned their coding sequences in fusion with gfp and the hybrid proteins were expressed in chinese hamster ovary cells. we evaluated the presence of both proteins in plasma membrane by fluorescence microscopy and then we proceeded to their functional characterization using patch clamp technique.this analysis will allow us to gain information about channel activity, regulation and pharmacology. we also plan to evaluate the importance of syncak and synk channels in photosynthesis. to test the hypothesis that they could be involved in regulating this process, we will produce deletion and site-specific mutants in synechocystis. finally we would also identify the homologous of these channels in the higher plant a. thaliana and obtain some information about their localization and function. j. braunagel max-planck institute for polymer research, ackermannweg 10, 55128 mainz, germany the cyclododecadepsipeptide valinomycin is composed of two amino acids (l-valine and dvaline) and two hydroxyl acids (d-α-hydroxy-isovaleric acid and l-lactic acid). they form a 36membered ring of alternating amino and hydroxyl acids. in the cyclic structure, the polar groups are oriented towards the central cavity, whereas the rest of the molecule is relatively nonpolar. this enables the complexation of ions and valinomycin acts as a selective ion transporter (k + ) in lipid membranes. when one of the amino acids is exchanged, e.g one l-valine by an l-lysine, selected functionality can be engineered into the depsipeptide while maintaining its ion conducting properties. we induced several modifications into valinomycin, i.e. a biotin binding unit or a ferrocene group to induce an electrochemical active center. the ion conducting properties of the modified ion carriers have been probed in planar lipid bilayers as well as in solid supported membranes. the role of the membrane dipole potential (ϕ d ) is of a particular interest due to a powerful impact of this potential on the membrane permeability and lipid-protein interactions. channel forming activity of gramicidin a, alamethicin, syringomycin e, hpa3 peptide, and ompf porin are influenced by ϕ d . we have studied the effect of the membrane dipole modifier, phloretin, on the properties of single channels formed by a wild-type alpha-hemolysin in planar lipid bilayers. the single channel of a ∼1000 ps conductance exhibits transitions into a number of low-conductance states as the transmembrane voltage exceeds ∼130 mv (regardless of the voltage polarity). the phloretin addition to the bathing solutions (20 µm) (after the hemolysin channel was formed in the membrane) shifts dramatically the channel voltagedependence. transitions to the low-conductance states are observed at ∼30 mv. the effect of the phloretin addition was not observed in the case when it was introduced into the bathing solution before alpha-toxin. since phloretin reduces ϕ d , the data may report on the influence of the electric potential profile on the energy of the low-conductance state of the alpha-hemolysin channel. the alternative explanation of this effect consists in a specific interaction between the phloretin and toxin channel. the work is supported in part by rfbr (# 09-04-00883), ss (# 1135.2008.4) , and the program of the ras «molecular and cell biology». atypical mechanism of conduction in potassium channels c. domene 1 , s. furini 1 1 physical and theoretical chemistry laboratory, department of chemistry, university of oxford, oxford, u.k., 2 department of electronics, computer science and systems, university of bologna, bologna, italy potassium channels can conduct k + ions with rates of up to ∼ 10 8 ions per second at physiological conditions, and they are selective to these species by a factor of 10 4 over na + ions. ion conduction has been proposed to involve transitions between two main states, with two or three k + ions occupying the selectivity filter separated by an intervening water molecule. the largest free energy barrier of such a process was reported to be of the order of 2-3kcal mol −1 . here, we present an alternative mechanism for conduction of k + in k + channels where site vacancies are involved, and we propose that coexistence of several ion permeation mechanisms is energetically possible. conduction can be described as a more anarchic phenomenon than previously characterized by the concerted translocations of k + -water-k + . we exploited the au-deposited self-assembled monolayers of the type: [−s−(ch 2 ) n −ch 3 ] (where n = 4, 9 and 15) with hydrophobically adsorbed redox protein -azurin to verify intrinsic electron transfer mechanisms according to the charge-transfer theory. the enthalpies and volumes of activation were determined through the variation of temperature (2-50 o c) and pressure (5-150 mpa) and experimental values were compared with those expected on the theoretical grounds. for the case of n = 4 the activation enthalpy definitely contains a large contribution originated from frictional-like dynamics of protein, and the activation volume has a small positive value. for n = 15 the value of activation enthalpy directly matches 1/4 of that for the reorganization energy, and the activation volume attains substantially negative value. for n = 9 we observed the intermediary performance. the whole kinetic pattern is consistent with the smooth changeover between adiabatic and nonadiabatic mechanisms of electron transfer. two gating modalities in the pore of the miniature k + channel kcv kcv is a viral protein that forms functional k + channel in heterologous systems. because of its miniature size (94 amino acids) we use kcv as a model system to study and manipulate basic properties of the k + channel pore. by analysing single-channel recordings we highlighted two voltage-dependent modalities of gating in kcv: a slow and a fast gating. the presence of a slow gating is revealed by the very low (in the order of 1-3%) mean open probability. slow gating is not related to the presence of a bundle crossing, as shown by accessibility of the cavity to mts reagents. channel opening might involve the transient formation of salt bridges between residues at the n and c termini of the channel, as suggested by mutational experiments inspired by molecular dynamics simulations of kcv. fast gating, analyzed by beta distributions, is responsible for the negative slope conductance in the single-channel i/v curve at extreme potentials and can be explained by depletion-aggravated instability of the filter region. aca8 is a type 2b caatpase with a regulatory n-terminus whose autoinhibitory action can be suppressed by binding of calmodulin (cam). aca8 n-terminus is able to bind a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. to define the role of this interaction in autoinhibition we have analysed a number of single point mutants produced by mutagenesis of aca8 e252-n345 sequence. mutation to ala of any of 6 acidic residues (e252, d273, d291, d303, e302, d332) originates an enzyme with normal activity in the presence of cam, but less camstimulated. these results highlight the relevance of a negative charge of the surface area of the small cytoplasmic loop in aca8 autoinhibition. the most deregulated mutant is d291a aca8, which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the phenotype of the d291a mutant is stronger than that of d291n aca8 suggesting a more direct involvement of this residue in autoinhibition. of the other mutants (i284a, n286a, p289a, p322a, v344a, n345a), only p322a aca8 has a basal activity higher than that of the wt. these results provide the first evidence that the small cytoplasmic loop of a type 2b caatpase plays a role in the attainment of the autoinhibited state. complex i, the first member of the respiratory chain, serves as a proton pump catalyzing transfer of two electrons from nadh to ubiquinone coupled with the translocation of four protons across the membrane. so far the mechanism of energy transduction by complex i is unknown. the nadh-binding cavity of complex i has a very prominent feature -the presence of two invariant amino acid residues, glutamate and tyrosine, that are exposed to the solvent and located in the vicinity of the fmn, the primary electron acceptor in the enzyme. it was suggested that they might be involved in the binding of nadh through interaction with its nicotinamide moiety. in this work we assessed the function of corresponding glu95 from the nuof subunit of e. coli complex i by mutation for glutamine. we showed that the negative charge of glutamate in the catalytic site is needed for the electrostatic repulsion of negatively charged phosphates of nucleotides. this process facilitates release of the product nad + and, as a result, accelerates turnover of complex i. we also found that glutamate, as one of the four negatively charged amino acid residues surrounding the isoalloxazine ring of the fmn at a distance of 4-6å, has a share of 40 mv of the overall 130 mv depression of the midpoint potential of this redox cofactor. l. erokhova 1 , p. kügler 2 , p. pohl 1 1 institute of biophysics, johannes kepler university, linz, austria, 2 ricam, austrian academy of sciences, linz, austria the mechanism of water transport through epithelia is still under debate. in the present work we tested the hypothesis of isosmolal water transport using mdck cells stably expressing the human sodium-glucose cotransporter (hsglt1). tagging hsglt1 with egfp enabled determination of its abundance in the plasma membrane by fluorescence correlation spectroscopy (fcs). by monitoring tiny shifts in the concentration of water-soluble dyes in the vicinity of epithelia, fcs also allowed assessment of the water fluxes through confluent cell monolayers grown on permeable supports. fitting the set of differential equations for the osmotic drift and for the back diffusion to the experimentally determined dye distribution permitted calculation of water flow both in the presence and in the absence of an osmotic gradient. from the calorimetric measurements of glucose transported across the cell monolayer, the water:glucose stoichiometry was derived. dividing the increment in osmotic water flux due to hsglt1 expression by the number of hsglt1 copies in the plasma membrane resulted in a single transporter water permeability p f of 4.6 x 10 −14 cm 3 /sec. thus, p f is close to the single channel water permeability of aquaporin-1. consequently, even small osmolyte concentration differences between the cytoplasm and the basolateral buffer solution are sufficient to drive a substantial water flux. m. emre, s. kavak cukurova university school of medicine, department of biophysics, balcali-adana, turkey experimental studies have shown that the at1-receptor antagonists telmisartan (tel) has a ppar-activating property, but there does not appear to be a class effect. to test telmisartan's importance, we investigated its effect on electrical activities (ea) in diabetic (d) rats. the purpose of this study was to investigate the effects of the tel on ea of diabetic papillary muscle (dpm) with stz-induced. in this study, we used four groups: (1) nondiabetic control (ndc) group (c), (2) tel-treated ndc group (c+tel), (3) diabetic group (d), and (4) tel-treated d group (d+tel). diabetes was induced by a single i.v injection of stz. in the study, membrane potential (mp) and action potential (ap) recorded after the establishment of diabetes. 1) mp was decreased significantly in both tel-treated c and d rats (from -71,4±0,7 to -64,7±0,8 mv and from -69,9±0,6 to -63,8±0,7 mv). 2) ap unchanged in d group, whereas c+tel and d+tel groups showed increase in ap compared with c and d groups. 3) repolarization time was prolonged in diabetic rats. 4) in c+tel and d+tel groups depolarization rate values increased significantly. on the other hand, in d group repolarization rate values descreased increased significantly compared to baseline values in tel solution. as a result, our data suggest that the beneficial effects of tel-treatment on the ea of the dpm appear to be due to the diminished k + currents. cytochrome c oxidase is a terminal complex (cco, complex iv) of a respiratory chain that is located in an internal membrane of mitochondria or plasma membrane of bacteria. cco is an electron transfer enzyme that reduces o 2 and uses the redox energy of the o 2 reduction for the proton translocation across the membrane. the electron-and proton-transfer generate a transmembrane electrochemical gradient (∆µh + ) that is used for atp synthesis and all other kinds of work for the cell needs. the proton translocation mechanism of cco requires 'channels' for the h + uptake and expulsion within the enzyme. the proton transfer occurs on a time-scale of micro-to-milliseconds. in order to study the proton transfer in cco, a flow-flash approach based on a time-resolved ftir spectroscopy was developed and applied. our ftir flow-flash approach (the measurement of the reaction of cco with o 2 ) allows to reach a time resolution up to tens milliseconds. with this approach and site-specific mutants of cco where the catalysis is slowed down, separate steps of the proton transfer were studied. the results showed that a unique cross-linked tyr-280 (in a combination with a time-resolved visible spectroscopy and electrometry) serves as a proton donor for the dioxygen bond cleavage during the o 2 reduction by cco. furthermore, the protolytic transitions of glu-278 -a key amino acid in the proton transfer mechanism in cco -were shown for the first time. role of calcium ions in nickel potentiation of nmda currents p. gavazzo, i. zanardi, p. guida, c. marchetti biophysics institute ,cnr, genova, italy nmda receptors are glutamate-gated channels distributed throughout the brain in the excitatory synapses and are critical for the nervous system function. they are assembled from two types of subunits, the essential nr1 and at least one nr2(a,b,c,d). nickel (ni) modulates the current flowing through nmda receptors in a different way depending on the nr2 subunit present. we have recently identified several domains of the channel involved in ni interaction, but many aspects of this modulation remain elusive. in this work we intended to determine the role of calcium (ca) ions in the potentiation induced by ni on the current through nr1/nr2b recombinant nmda receptors. when ni was applied in the presence of the physiological concentration of ca (1.8 mm) , a voltage-independent potentiation of the current was observed with a kp of 2.5 µm. this effect was progressively reduced by decreasing ca concentrations and it was no more detectable with 0.18 mm ca or in the presence of barium (ba, 0.3 mm). in this last case the effect of ni on nr1/nr2b receptors was mainly inhibitory (ki(-60mv)=156µm). therefore a physiological concentration of ca is necessary to induce ni amplification of the current. many data in the literature indicate a correlation between ca ion entrance through the channel, nmda current facilitation and cytoskeleton; however, in our experiments, the application of the actin perturbing agent cytochalasin-d did not produce major modifications in ni effect. heteromerization properties of voltage dependent potassium channels f. gambale 1 , l. pedemonte 1 , a. naso 1 , i. testa 2 , c. usai 1 , a. diaspro 2 , c. picco 1 1 istituto di biofisica, cnr, genova, italy, 2 lambs-microscobio, department of physics, genova, italy voltage-gated potassium channels are either homomeric or heteromeric tetramers composed of four α-subunits. in order to bring a contribution to the comprehension of channel heteromerization we have been investigating the properties of two plant voltage-gated k + -channels by using electrophysiological and fluorescence techniques. experiments were focussed on kdc1 and kdc2, coexpressed in xenopus laevis oocytes. kdc1, the first potassium channel cloned from daucus carota, belongs to the subfamily of α-modulatory silent channels as it doesn't form functional homomeric channels by itself. on the contrary kdc1 forms functional heteromeric channels when coexpressed with homologous subunits. kdc2, the last k + channel cloned from d. carota, belongs to the kat1 family and shares an overall identity of 63% with kat1. to correlate kdc1 functional properties with its localization in oocytes, kdc1 and/or kdc2 subunits were labelled with gfp and their properties investigated by confocal microscopy and voltage-clamp. we found that the kdc1-egfp fusion protein is not targeted to the plasma membrane unless it is coexpressed with kdc2. moreover electrophysiological experiments demonstrated that the heteromeric kdc1-kdc2 channel has altered selectivity and activation properties with respect to homomeric kdc2 channel. circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome (ards) [1] . we present a microfluidic investigation of the roles of actin organization and myosin ii activity during the different stages of leukocyte trafficking through narrow capillaries using specific drugs. the deformation rate during entry reveals that cell stiffness depends strongly on f-actin organization and hardly on myosin ii activity, supporting microfilament role in leukocyte sequestration. in the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. conversely, membrane unfolding was independent of leukocyte stiffness. the surface area of sequestered leukocytes increased by up to 160% in absence of myosin ii activity, showing the major role of molecular motors on microvilli wrinkling and zipping. finally, cell shape relaxation was largely independent of both actin organization and myosin ii activity, whereas a deformed state was required for normal trafficking through capillary segments [2] . [1] g.s. worthen et al., science, 245, 183-186 (1989) excitation-contraction coupling in skeletal and cardiac muscle is tightly regulated by the calcium release channel of the sarcoplasmic reticulum, the ryanodine receptor (ryr). we could previously show that suramin is a potent activator of the ryr via the calmodulin binding site. calmodulin shows dualistic action, i.e. activation or inhibition of the ryanodine receptor, depending on the absence or presence of ca 2+ . screening of suramin analogues identified nf676 as a use-dependent inhibitor of the skeletal muscle ryr (ryr1). here we show that nf676 inhibits high affinity [ 3 h]ryanodine binding and single channel recordings of the purified ryr1. nf676 induced a reduction of open probabilities in a concentration dependent manner, with no effect on current amplitude and unitary conductance. importantly, nf676 triggers flickering episodes of channel openings and closings before the ryr1 is frozen in a complete non-conducting state, which is fully reactivated by the ryr agonist atp. moreover, zwitterionic behaviour of nf676 facilitates plasma membrane permeation, which prevented caffeine induced ca 2+ transients in skeletal muscle cells and cardiomyocytes. conversely, ip 3 mediated ca 2+ signals were not altered by nf676. this work was supported by herzfelder'sche familienstiftung and fwf . beyond steady-state protein dynamics t. hauß 1 , j. pieper 3 , a. buchsteiner 1 , r. e. lechner 2 , n. a. dencher 2 1 helmholtz-zentrum berlin für materialien und energie, berlin, germany, 2 technische universität darmstadt, germany, 3 technische universität berlin, germany to study protein dynamics beyond steady-state experiments we have developed a novel laser-pump:neutron-probe experiment which allows us to monitor temporal changes in protein dynamics during a working cycle of a protein. protein dynamics has been extensively studied, but so far, the correlation of internal protein dynamics with the function of proteins was investigated only indirectly in steady-state experiments by variation of external parameters by variation of external parameters like temperature or hydration. the method comprises of an in-situ optical activation of a protein and a time-dependent sampling of the dymamic response using quasi-elastic neutron scattering. with the membrane protein bacteriorhodopsin, a light driven proton pump, we can demonstrate for the first time temporary alterations in the protein dynamics after triggering the working cycle. this observation is a direct proof for the functional significance of protein structural flexibility, in connection with the largescale conformational changes in the protein structure occurring during the operation of a "molecular machine". the slow vacuolar (sv) channels are ubiquitous in all tissues of higher plants. the sv channel is a non-selective cation channel permeable to both monovalent and divalent cations. sv currents recorded in a typical patch-clamp experiment require unphysiologically high cytosolic and low vacuolar calcium concentrations for full activation. we aim at looking for endogenous plant substances which might be able to modify or shift the voltage activation threshold of this channel towards more physiological conditions. flavonoid naringenin [nar] is present in all plant species where it plays a central role in the flavonoid biosynthetic pathway. nar is stored in the vacuoles in glycosylated form called naringin. when nar was added to cytosolic bath solution, we recorded a dose-dependent reversible decrease in sv channel activity. when we investigated the effect of nar on the voltage dependence of the channel, we observed that the activation threshold of the sv channel is shifted towards more positive voltages. our group has evidences that approximately 10% of the total sv current at high (e.g.> 50 mv) positive voltages is mediated by calcium. therefore, in order to verify whether nar affects both potassium and calcium conductance, we performed experiments by combining the patch clamp technique with fluorescence measurements using the fluorophore fura-2: both sv currents and calcium signals were abolished by 1 mm [nar]. determination of calcium currents in cation channels using a novel fluorescence/patch-clamp approach p. v. k. gutla, a. gradogna, a. carpaneto istituto di biofisica, consiglio nazionale delle ricerche, via de marini 6, 16149 genova, italy the patch-clamp technique combined with fura-2 fluorescence detection is suitable to investigate calcium fluxes. we used the excised patch configuration and focused the photomultiplier to the tip of the recording pipette where the fluorescent dye was present (fluoresence combined with excised patch = flep). this configuration has several advantages, i.e. absence of delay in loading the fluorophore, of interference by endogenous calcium buffers and of photobleaching. here we present an application for the determination of fractional calcium currents (pf) in a plant non-selective cation channel, showing that pf can be modulated by cytosolic calcium and potassium. flep is very efficient for measuring small calcium currents (<1 pa) of sufficiently long duration; fluorescence signals are amplified by integration in time, as the calcium/fura-2 complex accumulates at the tip of the recording pipette and diffuses slowly. we propose this technique not only for the study of calcium transport pathways, but also for other transporters of divalent cations as nickel and manganese known to quench fluorescence thus reducing both 340 and 380 nm components. moreover, using the appropriate fluorophore the technique may be extended to further ion species, e.g. bcecf for investigating proton transport pathways. ref: we introduced an original method for the monitoring of the changes in the electrostatic surface potential, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. this approach opens new way to understanding the dynamic processes within the proteins. our experiments revealed that the conformation of the na + /k + -atpase large cytoplasmic loop (c45) in the presence of the atp (without magnesium) substantially differed from the conformation in the presence of mg 2+ or mgatp or in the absence of any ligand not only in the sense of geometry but also in the sense of the electrostatic surface potential. moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. this property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain. influx of antibiotics into the periplasm of gram-negative bacteria is facilitated by porins that form channels in the outer membrane. we propose that certain natural antibiotics have been optimized by co-evolution to take advantage of the charge distribution in non-specific porins to achieve binding and thereby facilitating their uptake in bacteria. we investigate the permeation pathways of antibiotics into bacteria by reconstitution of a single porin into an artificial lipid bilayer and measuring the binding of antibiotic molecules through the time-resolved modulation of a small ion current. we have been able to characterize facilitated translocation of several antibiotics through escherichia coli and enterobacter aerogenes porins. noise analysis of ion currents through a porin in the presence of effective antibiotics revealed binding kinetics at a single molecule level. we report for the first time temperature dependent antibiotic translocation that revealed complete energy profile. combining these results with microbiological assays and molecular dynamics simulations, we conclude the molecular mechanism of antibiotic permeation. our approach may contribute to the rational design of new antibiotics against clinical bacterial strains for the most efficient delivery to target sites. mitochondria regulate ca 2+ influx and determine patterns of er ca 2+ refilling in acinar cel o. kopach, i. kruglikov, t. pivneva, n. voitenko, n. fedirko bogomoletz institute of physiology, kiev, ukraine store-operated ca 2+ entry (soce) is mediated by activation of soc-channels of plasma membrane following the emptying of endoplasmic reticulum (er) ca 2+ stores. the soce is required for calcium signaling, secretion of neurotransmitters and proteins, but the mechanisms of natural soce regulation are not well understood. we utilized several imaging methods to measure ca 2+ signals in cytoplasm ( ] mit transients, observed both in egta-and bapta-buffering inside solutions. these data suggest that ca 2+ sequestration by mit is associated with the formation of microdomains and prevents ca 2+ -dependent socc inactivation. we also found that inhibition of mit under prolonged cell stimulation resulted in complete inhibition of soce as well as decrease and deceleration of er refilling. thus, mit regulate calcium recycling and maintain the soce controlling dynamic interplay between soce and sustained er refilling under prolonged stimulation. bioelectrochemical devices composed of au electrodes coated by self-assembled monolayers (sams) of different composition and thickness are ideal systems to probe et patterns and mechanisms for redox proteins. representative proteins, cytochrome c and azurin were studied by using the combinations of four different strategies including the variation of sam thickness ([−s−(ch 2 ) n −ω], with n running over the range 3 to 20, throughout), solution viscosity (varied by adding of the viscose additive -glucose), temperature (0 to 50 o c) and hydrostatic pressure (up to 150 mpa), aiming the identification of different intrinsic et patterns and interplay between them in the framework of generalized charge-transfer theory. we demonstrated the full adiabatic (frictional) control for the case of thinner sams, the intermediate (mixed) regime, and the complete changeover to the nonadiabatic mechanism (long-range tunneling) for the case of thick sams owing to the variation of electronic coupling, in a nice agreement with theoretical predictions. h. nury 1 , f. manon 1 , b. arnou 2 , m. le maire 2 , e. pebay-peyroula 1 , c. ebel 1 1 institut de biologie structurale (ibs) cea cnrs ujf grenoble france, 2 cea ibitec cnrs ura2096 univ. paris-sud gif-sur-yvette france adp/atp carriers (aacs) are major and essential constituents of the inner mitochondrial membrane. they drive the import of adp and the export of newly synthesized atp. they were described as functional dimers from the 1980s until the structures of the aac shed doubt on this consensus. we aimed to ascertain the published biophysical data claiming that aacs are dimers and to characterize the oligomeric state of the protein before crystallization. analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine aac is a monomer in 3-laurylamido-n,n 2 dimethylpropylaminoxide (lapao), whereas in triton x-100 and reduced triton x-100, higher molecular mass species can also be identified. neutron scattering data for monomeric bovine aac in lapao does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. we discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of aacs within the mitochondrial membrane. local anaesthetic binding to shaker channels: role of aromatic residues j. nilsson, h. ullman, k. sahlholm, p. arhem the nobel institute for neurophysiology, department of neuroscience, karolinska institutet, se-171 77 stockholm, sweden local anaesthetic, antiepileptic and antiarrhythmic drugs acting on nav and herg channels have been assumed to bind to aromatic residues in the internal vestibule; to f1764 and y1771 in nav (ragsdale et al., 1996 and to y652 and f656 in herg (mitcheson et al., 2000) . despite a lack of such residues in kv channels, local anaesthetics, antiepileptic and antiarrhythmics bind to kv channels with a considerable affinity. to explore the role of aromatic residues for the binding we investigated the effect of bupivacaine, benzocaine, phenytoin and quinidine on shaker channels mutated to residues corresponding to the most c-terminal of the two aromatic residues in the s6 segment of the nav and the herg channels, (v473y and p474f respectively). the channels were expressed in xenopus oocytes and the currents measured with the two-electrode voltage-clamp technique. the results suggest that aromatic residues do not increase the binding affinity of the studied compounds to kv channels. rather, the affinity decreases (as reflected in typical k d values for bupivacaine on v473f, p474f and wildtype channels, being 550, 740 and 300 µm, respectively). thus, aromatic residues seem not to be necessary for high-affinity binding of the studied compounds to kv channels. how this relates to their suggested roles in the nav and herg channels, remains to be evaluated. (molina et al, 2006) . the occurrence of a similar behavior in other channels points out to clustering and coupled gating as a potentially important drug target to modulate channel activity. we have identified molecular determinants involved in single and coupled channel gating in kcsa. first, we detected that clustering and coupled gating of kcsa is modulated by anionic lipid. also, a model for the interaction between two kcsa open channels was built. the docking predicts intermolecular sites which includes the non-annular lipid binding site. this explains how an excess of anionic lipid disrupts interactions between channels, destabilizing clustering and coupled gating in kcsa. in addition, the docking model reveals molecular determinants involved in single and coupled channel gating. this interaction involves w87, which affects the neighbouring channel through specific interactions in the extracellular mouth stabilizing the selectivity filter in an open conformation. the coupled gating is also explained since this mechanism affects the opposite channel in a mutual manner. finally, mutants kcsa e71a and kcsa w87a disrupt the coupled gating of kcsa, thus, supporting the model. we think that this coupled gating phenomenon could correspond to the second gate previously detected by fluorescence methods (blunck, et al, 2006) . supported by grants from the spanish bfu2008-00602/bmc and csd2008-00005. a. kumar, e. hajjar, p. ruggerone, m. ceccarelli university of cagliari, monserrato, italy the striking presence of outer membrane (om) in gramnegative bacteria of e.coli represents a strong barrier for any molecule to penetrate inside bacteria. in particular for β-lactam antibiotics, which have their target located inside the bacteria; the first step towards reaching inner part of bacterium is the cellular uptake. ubiquitous presence of porins (such as for instance ompf, ompc) in the om, function as a channel facilitating the transport of molecules (such as, for instance antibiotics) across the om. bacteria can exhibit resistant towards antibiotics by: (i) decreasing their uptake by under-expressing the porins or/and (ii) production of inactivating enzymes such as ß-lactamases. to combat the latter mechanism ß-lactamase inhibitors (such as, sulbactam for instance) are prescribed together with the antibiotics. like the antibiotics, the inhibitors must penetrate the om, the main path being through porins. it is thus evident the biological relevance of investigating the mechanisms by which porins can regulate entry/exit across the om. to achieve this goal, molecular dynamics simulations were performed to explore the structure and dynamics of pores formed by ompf and ompc porin. from the analysis of data obtained from our simulations, we identified the key residues buried behind the l3 loop, which may play be crucial for porins to exert their biological role. as a case study, we report results about the diffusion of sulbactam through the two porins. the pentadecapeptide gramicidin forms a cation-specific ion channel in membrane environment. the two main conformations are the head-to-head helical dimer (hd) known as the channel conformation and the intertwined double helical form (dh) often referred to as non-channel conformation. in this comparative study [1] , the energetics of single potassium ion permeation by means of the potential of mean force (pmf) for both gramicidin conformations embedded in a dmpc bilayer has been addressed by molecular dynamics simulations. a significantly decreased free energy barrier by ∼25 kj/mol for potassium ion passage through dh as compared to hd is reported. favorable electrostatic side chain-cation interactions in hd are overcompensated by phospholipid-cation interactions in dh. the latter are coupled to an increased accessibility of the channel entrance in dh due to distributed tryptophans along the channel axis. this result underscores the importance of the lipid environment of this channel not only for the equilibrium between the different conformations but also for their function as cation channels. y. sawada 1 , m. murase 2 , m. sokabe 1 1 dept. physiol. nagoya univ. grad. sch. med., nagoya, japan, 2 icorp/sorst cell mechanosensing, jst, nagoya, japan the bacterial mechanosensitive channel of large conductance mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer α-helices, and its 3d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. although several models for the opening process have been proposed with molecular dynamics (md) simulations, as they do not include mscllipid interactions, it remains unclear which amino acids sense membrane tension and how the sensed force induces channel opening. we performed md simulations for the mechano-gating of mscl embedded in the lipid bilayer. upon tension in the bilayer, phe78 in the outer helix was dragged by lipids, leading to a tilting of the helices. among amino acids in the outer helix facing the bilayer, phe78 at the water-lipid interface showed the strongest interaction with lipids, thus may work as a major tension sensor. neighboring inner helices cross each other in the inner leaflet, forming the most constricted part of the pore. as tension increases, the crossings move toward the cytoplasm associated with an expansion of the constricted part. during the movement, a hydrophobic water block environment around the constricted part was broken followed by water penetration and permeation. the k + channel kcsa is an integral membrane protein from s.lividans, used as a model system for studies on ion channels and oligomeric membrane proteins. its atomic structure has been solved by x-ray diffraction, which shows an assembly of four identical subunits around a central aqueous pore, including the so-called selectivity filter. this channel is able to permeate k + at high flux rate and it is blocked by na + (physiological blocker). fluorescence, circular dichroism and fourier transform infrared experiments carried out in our laboratory demonstrated that k + and na + are able to bind to kcsa in a competitive manner. this binding is indeed associated with channel conformational changes, which seem to be related to the permeation and blockade processes. to further investigate this phenomenon we carried out a detailed study of chemical and thermal denaturation of wild-type and mutant kcsa channels. these two types of experiments were in agreement and indicate that both cations are able to stabilize the channel through conformational changes, being k + the more efficient one, even more when lipids are present. particularly, mutant channels with a structurally altered selectivity filter show that ion and lipid-induced global conformational changes are intimately associated to the conformation of this selectivity filter (conductive and non-conductive forms). modulation of the voltage-gated sodium channel nav 1.5 by rcssii, a toxin from the scorpion centruroides suffusus c. picco 1 , g. corzo 2 , l. d. possani 2 , g. prestipino 1 1 institute of biophysics, cnr, genova, italy, 2 instituto de biotecnologia, unam, cuernavaca, mexico the main cardiac voltage-gating sodium channel, na v 1.5, generates the fast depolarization of the cardiac action potential and plays a key role in cardiac conduction. its importance for normal cardiac function has been exemplified by the description of numerous naturally occurring genetic variants of the gene scn5a, which encodes na v 1.5, that are linked to various cardiac deseases. subsequently, studies of this channel localization have led to its identification in immature and denervated skeletal muscle and in the brain neurons. in our effort to identify high affinity ligands for this channel, we have investigated the effects of the recombinant cssii (rc-ssii), a four disulfide-bridged scorpion toxin isolated from the venom of the scorpion centruroides suffusus. human cardiac sodium channel α subunit scn5a was expressed in cho cells and macroscopic na + currents were recorded with patchclamp technique in whole cell configuration. the electrophysiological experiments have highlighted a strong affinity for the channel at low nanomolar concentration. compared with control conditions, rcssii toxin affects in reversible way the kinetics of activation and inactivation and marked decrease the peak na + influx. the extrusion mechanism of substrates in rnd family efflux pumps: a molecular dynamics study a. v the rnd transporters of the acrab-tolc (e.coli) and mexab-oprm (p.aeruginosa) systems are able to export structurally and chemically different substrates outside bacteria through the membrane, being responsible of multidrug resistance. on the basis of crystallographic information, an extrusion process conceived as a three-cyclic peristaltic pumping has been proposed, but further microscopically well-funded investigations are needed to understand the mechanism. using different computational methods like adaptive bias force (abf) and targeted molecular dynamics (tmd), we have investigated the mechanism of substrate uptake and pumping at a molecular level. with the first method we have investigated the passage of antibiotics from the periplasm into the internal pore of the pump, while tmd has been used to assess the effect of conformational changes on the extrusion of drugs (which have been located into one of the proposed binding pockets). comparison between the active pumps acrb and mexb (which show different resistance patterns despite their homology) provide insights into the microscopic details of their functioning. in arabidopsis thaliana there are twenty genes, grouped into three subfamilies, encoding for homologues of animal ionotropic glutamate receptors (iglrs). each protein displays a pore-forming loop, flanked by two conserved helices (plus a third c-terminal helix), a glutamate-binding domain and an n-terminal region. through pharmacological and/or genetic approaches, many physiological functions have been attributed to plant glurs, such as the regulation of cytosolic calcium, photomorphogenesis, water balance and carbon/nitrogen sensing and assimilation. according to the endosymbiotic theory, the cyanobacteria are considered to represent the precursors of the present chloroplasts. the first prokaryotic glutamate receptor (glur0) was identified in the cyanobacterium synechocystis. the putative products of the atglr3.4 and atglr3.5 genes display a possible targeting sequence for chloroplast location and show a high degree of homology with glur0. using specific antibodies and confocal microscopy, we have localized the two members of the atglr subgroup 3, glr3. 4 and glr3.5 (splicing variant) , to the chloroplast in arabidopsis and to the inner envelope membrane in spinach. electrophysiological experiments indicate the presence of an activity which is compatible with that of glutamate receptors. furthermore, oxygen evolution measurements suggest that chloroplast-located glutamate receptors may play a role in the regulation of photosynthesis. under extreme conditions many cells control their volume responding to osmotic challenges by unloading or loading solutes to recover their original volume. a faster volume regulatory role triggered by membrane tension has been disclosed for aquaporins in kidney proximal tubule cells, where aqp1 is the main water channel, using isolated brush border membranes. in conventional osmotic studies in animal cells it is common to disregard internal hydrostatic pressures because they are insignificant compared to osmotic forces. however, by using low osmolarity buffers in small radii vesicles, we detected a rise on the internal pressure that creates surface tension and causes membrane stress, with a negative outcome on aquaporin water permeability. these findings suggested a mechanism for volume regulation in kidney proximal tubule epithelia where massive solute and fluid transport occurs. to further explore aquaporin regulation by membrane tension, yeast cells were used as a model that could bare surface tension some orders of magnitude higher than animal cells due to the existence of a cell wall. the effect of increasing levels of membrane tension on yeast water channel activity was evaluated. an impairment of aquaporin activity correlated with the increase of membrane tension corroborates the volume regulatory role of aquaporin in different cells. deuterium isotope effects on fast gating of the chloride channel clc-0 g. zifarelli, a. r. murgia, p. soliani, p. michael istituto di biofisica, cnr, via de marini, 6, i-16149 genova, italy gating of the torpedo cl − channel clc-0 is modulated by intracellular and extracellular ph, but the mechanism responsible for this regulation has remained so far elusive. using inside-out patch clamp measurements we studied the dependence of the fast gate on ph int and [cl − ] int . only the closing rate, but not the opening rate showed a strong dependence on these intracellular factors. using mutagenesis we excluded several candidate residues as mediators of the ph int dependence. we propose a model in which a proton generated by the dissociation of an intrapore water molecule protonates e166 leading to channel opening. deuterium isotope effects confirm that proton transfer is rate limiting for gate opening and that channel closure depends mostly on [oh − ]. the model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on ph int and [cl − ] int . deletion of the c-terminus destabilizes phosphorylated na/k pump state containing 3na ions n. vedovato, d. c. gadsby the rockefeller university, new york, u.s.a. the na/k pump's extended c-terminus (compared to the serca ca pump's) links 3 transmembrane helices, and its truncation lowers cytoplasmic na affinity for forming the occluded e1p(na 3 ) state. here we test the effects of c-terminal truncations on interactions with external na. we deleted the last 2 (yy) or 5 (kesyy) residues in xenopus α1 β3 pumps made ouabain resistant by mutations q120r-n131d (rd) or c113y (c-y), and then used two-microelectrode voltageclamp recording in xenopus oocytes to measure pump currents as 10 mm ouabain-sensitive currents while endogenous na/k pumps were silenced with 1 µm ouabain. inhibition by external na of steady outward pump current ([k] o =15 mm) at large negative voltages was somewhat weaker in both rd and c-y pumps than in wt pumps, but was severely impaired in all c-terminal truncated pumps. consistent with this, the voltage dependence of transient charge movements under na/na exchange conditions ([k] o =0 mm) was strongly shifted to more negative potentials in the truncated pumps relative to the parent rd or c-y pumps, shifts comparable to those seen in wt pumps on decreasing [na] o several-fold. together, the results suggest that these c-terminal deletions lower the apparent affinity for external na ions to bind and become occluded in the na/k pump. the c-terminus therefore provides contacts important for stabilizing the occluded e1p(na 3 ) conformation, regardless of the route of na ion entry into the binding pocket. muscle contraction is driven by molecular motors that adapt their energy utilization according to the demands made on them. we test the hypothesis that rate constants controlling the biochemical steps involved in atp hydrolysis by myosin atpase are affected by the force of the muscle. here we use fluorescence lifetime imaging microscopy (flim) of a fluorescently labelled atp analogue to investigate changes in the environment of the myosin atpase, caused by different loads applied to skeletal muscle. single muscle fibres were subjected to cycles of stretches and releases in the presence of rigor solution and 10 µm of coumarin-labelled atp. flim acquisition was synchronised with stretch/release cycles and force measurements, which allow us to investigate the effect of strain on the lifetime of the labelled atp bound to the actomyosin complex. characterization of the fluorescence decay by a bi-exponential function resolved the time constant of two populations, namely, free fluorophore (τ 1 = 0.47 ± 0.03 ns; mean ± s.d.) and fluorescent nucleotide bound to the actomyosin complex (τ 2 = 2.21 ± 0.06 ns at low strain). these experiments showed that while the time constant of the free fluorophore did not change with force, the time constant of the fluorescent nucleotide bound to actomyosin showed a linear dependence with the force applied to the muscle of 0.43 ± 0.05 ps/kpa. neck linker docking coordinates the kinetics of kinesin´s heads i. derenyi, a. czovek, g. j. szollosi department of biological physics, eotvos university, pazmany p. stny. 1a, h-1117 budapest, hungary conventional kinesin is a two-headed motor protein, which is able to walk along microtubules processively by hydrolyzing atp. its neck linkers, which connect the two motor domains and can undergo a docking/undocking transition, are widely believed to play the key role in the coordination of the chemical cycles of the two motor domains and, consequently, in force production and directional stepping. although many experiments, often complemented with partial kinetic modeling of specific pathways, support this idea, the ultimate test of the viability of this hypothesis requires the construction of a complete kinetic model. considering the two neck linkers as entropic springs that are allowed to dock to their head domains and incorporating only the few most relevant kinetic and structural properties of the individual heads, we have developed the first detailed, thermodynamically consistent model of kinesin that can (i) explain the cooperation of the heads during walking and (ii) reproduce much of the available experimental data (speed, dwell time distribution, randomness, processivity, hydrolysis rate, etc.) under a wide range of conditions (nucleotide concentrations, loading force, neck linker length and composition, etc.). besides revealing the mechanism by which kinesin operates, our model also makes it possible to look into the experimentally inaccessible details of the mechanochemical cycle and predict how certain changes in the protein affect its motion. the positive role of noise on the transport efficiency of na, k atpase c.-h. chang 1 , t. y. tsong 2 1 institute of physics, national chiao tung university & physics division, national center for theoretical sciences, hsinchu, taiwan, 2 institute of physics, academy of sciences, taipei 115, taiwan na, k atpase is a molecular motor which is able to transport ions through cell membranes, even against the transmembrane ion concentration gradient. while in vivo this nanoscale soft machine consumes atp, it may be driven by external fluctuating electric fields, no matter they are periodic or random. theoretically, the motor conformations can be described by a conformation vector v(t) governed by a multi-dimensional kinetic equation. given an oscillating electric field with a slight fluctuation, the boltzmann distributions of these conformations will change with time. the instantaneous transported ion flux is a functional of the quasi-cyclic trajectory v(t) of this non-autonomous dynamical system. various interesting dynamical properties of this ion pump, including stochastic resonance, can be studied theoretically, some of which have good agreement with recent experimental findings. in situ measurements of the molecular motor of muscle with nanometer-microsecond resolution in a contracting muscle, arrays of the dimeric motor protein myosin ii pull the actin filament towards the centre of the sarcomere during cyclical atp driven interactions. when the external load is smaller than the array force, the sarcomere works as a motor, converting metabolic energy into mechanical work; when the external load is larger than the array force, the sarcomere acts as a brake resisting the load with reduced metabolic cost. to investigate the molecular basis of the work production and the braking action of muscle, we use sarcomere-level mechanics and x-ray interferometry in intact single cells from frog skeletal muscle. during isometric contraction, each motor bears a force of about 6 pn. during shortening against high and moderate loads, the number of myosin motors attached to actin reduces in proportion to the external load while the force per attached motor is maintained similar to the isometric value (piazzesi et al., cell 131, 784-95, 2007) . rapid stretches of 1-5 nm between each overlapping set of myosin and actin filaments in a muscle sarcomere cause the stiffness of the array of myosin motors to increase up to twice the isometric value within 2 ms (brunello et al., pnas usa 104, 20114-19, 2007) , indicating that the high resistance of active muscle to stretch is due to recruitment of the second motor domain of the myosin molecules with the first domain already attached to actin. supported by miur and ente crf (italy), nih (usa), mrc (uk), embl, esrf. kinesin-1 is a molecular motor that moves cellular cargo along microtubules. its functional mechanism is well understood for individual motors. however, the way that many kinesin-1 motor proteins bound to the same cargo move together is not. we addressed the structural basis for this phenomenon using video microscopy of single microtubulebound full-length motors and various spectroscopy methods were employed to study synthetic peptides derived from hinge-1 region. these peptides show an unexpected profile of secondary structure forming propensities. video microscopy of single microtubule-bound full-length motors reveal the sporadic occurrence of high compliance states alternating with longer-lived, low compliance states. the deletion of hinge-1abolishes transitions to the high compliance state. from the results we hypothesize that strain accumulated during multiple kinesin motility populates the high compliance state by unfolding helical secondary structure in the central hinge 1 domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple motor situations. titin is a giant protein of vertebrate skeletal and cardiac muscles. cardiac titin is expressed in two main isoforms: short n2b (∼3000 kda) and long n2ba (∼3400 kda). we have studied changes of titin isoform composition in myocardium of hibernating ground squirrels and spontaneously hypertensive rats (shr). using electrophoresis we have revealed considerable decrease (by 2-3 times) in the content of titin relative to myosin heavy chains in shr heart as compared with that for normotensive rats. surprisingly that the data of qrt-pcr showed the increase in mrna content of n2ba and n2b-isoforms in hypertrophic heart more than 2 times in comparison with norm. we suppose that such a result is an effect of depressed translation of mrnatitin in pathology. we have observed the decrease (by 1,5 times) of total titin amount in heart of hibernating animals in comparison with that for summer active animals. however n2ba/n2b ratio in the heart upon hibernation was increased by 2 times. similar trend was not revealed for the mrna level of corresponding isoforms, although we have showed the decrease of mrna of both titin isoforms in heart of hibernating ground squirrels as compared to their content of summer animals. the decrease in total mrna level may be explained by repressed transcription or mrna degradation in the cell during hibernation. these discrepancies in protein and mrna levels may be considered as the posttranscriptional regulation of titin isoforms expression. actomyosin cross-bridges formed when the globular heads of myosins bind to actin filaments are the molecular engines that drive muscle contraction, fuelled by atp hydrolysis. critical to this process is the change in shape of the cross-bridge and the change in the interactions with actin, in response to force applied to the muscle, and to the status of the nucleotide in the binding pocket. although molecular detail is known from x-ray crystallography and biochemistry, understanding of the interplay between cross-bridge shape and chemical state requires studies in muscle fibres generating force. we use fluorescence life time imaging microscopy (flim) as a probe of the cross-bridge environment.with a fluorescent analogue of atp 1 , fluorescence life-time (flt) changes when the crossbridge binds to actin. now, we show preliminary experiments on the effect of force on flt. the essential light chain of myosin (elc) is a ∼20 kda peptide that wraps around a 9nmlong α-helix of the myosin cross-bridge known as the lever arm which tilts during force generation. using a recombinant elc, labelled with a fluorophore at a strategic cys, we replace the native elc and introduce the fluorescent elc in muscle fibres. preliminary experiments demonstrate that the elc fluorophore also is sensitive to force applied to the muscle fibre. in addition, förster resonance energy transfer occurs between the nucleotide and elc fluorophores, opening the way for studying structural changes in cross-bridges during force generation by fret. muscle contraction: pitfalls in the determination of the contractile response e. grazi dipartimento di biochimica e biologia molecolare, università di ferrara, ferrara, italy the contractile response of an active muscle depends on the load. the load is a force /cross-section. there are three fundamental dimensions: the mass, m; the space, l; and the time, t. from these three dimensions are built up all the physical dimensions. as an example the acceleration, a, is given by, a=l.t −2 . once the direction and versus are settled the modulus fully defines the physical effect of the acceleration. what about the force? the force, f, is given by, f=m.a. at variance with the acceleration, once the direction and versus are settled, the modulus does not define the physical and the biological effects of the force: the same force is generated by an infinite number of mass-acceleration couples that display different physical and biological effects. the same occurs with the load. thus defining the load that opposes the contractile force does not define the contractile system. in the studies on muscle contraction the acceleration of the load is not considered nor it is provided a way to extract this information. thus these systems are poorly defined from the physical as well as from the biological point of view. models of muscle contraction that consider explicitly both the mass and the acceleration of the load show that, at the same load, the decrease of the acceleration of the load significantly delays the pre-steady state of the contraction and decreases the stiffness of the active fibre. r. shahapure 1 , f. difato 1 , a. laio 1 , d. cojoc 3 , e. ferrari 3 , j. laishram 1 , g. bisson 1 , v. torre 2 1 int. school for advanced studies, trieste, italy, 2 iit-sissa unit, trieste, italy, 3 lab. nazionale tasc, trieste, italy polymerization of actin filaments is the main source of motility in lamellipodia and is controlled by many regulatory proteins. the underlying molecular mechanisms are only partially understood and now a determination of the dynamical properties of force generation is needed. using optical tweezers we measured with millisecond temporal resolution and pn sensitivity the force-velocity (fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. when force and velocity are averaged over 3-5 s, fv relationships can be flat. on a finer time scale, random occurrence of fast growths and sub-second retractions become predominant. maximal power dissipated by lamellipodia over a silica bead with a diameter of 1 µm is 10 −16 w. due to the presence of adhesion forces, beads in close contact with a lamellipodium can seal on its membrane reducing the amplitude of brownian fluctuations often by more than 10 times. under these conditions, when lamellipodia grow and push the beads, discrete jumps varying from about 5 to 50 nm are detected. when lamellipodia retract, pulling the beads, no discrete events are observed. our results on the dynamical properties of force generation are: a) force generation is a probabilistic process; b) underlying biological events have a bandwidth up to at least 10 hz; c) fast growths of lamellipodia leading edge alternate with local retractions; d) force generation is produced in discrete steps with varying amplitude up to 0.2 pn. pushing on microtubules: dominant spindle centering mechanism in c. elegans embryo? j. pecreaux, s. redemann, a. a. hyman, j. howard mpi-cbg, pfotenhauerstr 108, 01307 dresden, germany asymmetric cell division, where the content of the two daughter cells -as well as their sizes -differ, is found in many organisms. strikingly, the spindle, first centered, starts to be displaced out of the center only in late metaphase. in c elegans embryo, the spindle rocks and is posteriorly displaced during anaphase by force generators asymmetrically localized on cell cortex. prior to anaphase onset, the spindle is usually assumed to be centered by the same pulling force. it thus requires the force generators to be carefully repressed to distribute forces symmetrically. on live embryos, we measured positional fluctuations of centrosomes during metaphase with 20 nm accuracy. fourier analysis shows an extremely accurate centering respect to the number of force generators and microtubules. furthermore, spectrum is close to a lorentzian, modeled by a spring and a dashpot, suggesting a spindle centering more likely by pushing on microtubules than pulling. deviation at high frequencies indicates a subdominant pulling force. rnai of gpr-1/2, known to control force generation, increases slightly centering accuracy; this result supports the hypothesis of an independent centering mechanism. conversely, zyg-9(rnai), a microtubule growing factor, decreases centering accuracy, modeled spring stiffness and damping modulus. conclusion: first, the spindle centering mechanism is independent of cortical pulling force generator. second, microtubules pushing is likely to center the spindle. mechanical forces are important in the regulation of cellular adhesion and migration. the focal adhesion kinase (fak) has been suggested to transduce cellular forces and govern cell migration. to obtain more insight in the functioning of fak, a fret-based optical biosensor for fak was designed to relate integrin-mediated conformational changes in its ferm domain to focal adhesion behavior during cell spreading and migration in living cells. imaging of the kinetics of ferm-based fak conformational changes in spreading cells revealed two consecutive stages of focal adhesion activation. heterogeneous ferm conformational responses were observed in individual focal adhesions of adherent motile cells, with the active ferm conformation being enriched in growing and sliding fas, but not in stable and shrinking focal adhesions. inhibition of the cellular actomyosin system revealed the involvement of rho-rock rather than mlckinduced tension signaling in the modulation of the ferm response. our results place the ferm conformational change of fak at the interface between integrin and force sensing. the time course of inorganic phosphate release in permeabilized cardiac trabeculae of the rat c. mansfield, t. west, m. a. ferenczi imperial college london, u.k. the rate of p i release was determined in permeabilized rat trabeculae. contraction was elicited at 20 • c by laser-flash photolysis of npe-caged atp, and time-resolved p i release was monitored using mdcc-pbp, a coumarin-labelled phosphate binding protein, which increases its fluorescence intensity five-fold upon p i binding. the atpase rate during the first turnover of the total crossbridges (assuming 100 µm myosin heads) was 23s −1 . the rate decreased to a steady state of 4s −1 after the eighth turnover (0.5-0.6s after activation). this steady state rate is comparable to published values of 3 -10s −1 , made ∼15s after activation using an nadh-linked enzyme assay of adp release. the advantage of using mdcc-pbp is that the control of mechanochemical coupling can be examined from the onset of force production and as it progresses toward the steady state. force production and p i release were simulated using a seven step scheme. force was attributed to the states in the sequence a.m.adp.p i ↔ a.m.adp ↔ a.m.adp, with strain sensitivity incorporated into the isomerisation of a.m.adp. the a.m.adp.p i and a.m.adp states populated rapidly as force was increasing. in contrast, the a.m.adp state accumulated slowly after the force plateau was reached and became the dominant force bearing state at the time of the eighth crossbridge turnover. experiments are on-going to examine how the distribution of a.m states changes in response to rapid length-changes. pulling as a factor in forming the heterophasic structure of immunoglobulin proteins structure of proteins of immunoglobulin superfamily: human igg3 kuc and muscle protein titin, has been investigated by methods of electron microscopy and diffraction with the use synchrotron radiation. super elasticity of titin, the protein of immunoglobulin superfamily, is a key parameter that determines the mechanical properties of muscle. however, the structural-physical mechanism of titin elasticity under tension remains poorly understood. here both tension transduction and high elasticity of titin are explained in terms of crystalline polymer physics. x-ray data suggest a model of titin as a nanoscale, morphological, aperiodical array of rigid ig-and fn3-type domains covalently-connected by conformationally variable short loops. the line group symmetry of the model can be defined as s m with axial translation τ ∞ . homologous domains would have similar stability, but the structure of different domains on stretching is subject to different forces because they have different orientations relative to the axis of the molecule. under the force influence the structure of any domain can become either rigid or flexible depending on its orientation in the titin strand. pulling geometry forms an active axial structure from latent isotropic random coil structure of titin strand. we are suddenly faced with nanophase-separated morphology of igg3 kuc. study was supported by rfbr grant 07-02-01281. strain response of myosin essential light chain in permeabilized skeletal muscle fibres d. s. ushakov, d. ibanez-garcia, t. g. west, p. m. w. french, m. a. ferenczi imperial college london, uk we applied fluorescence lifetime imaging microscopy (flim) to investigate the relation between conformation of myosin head and mechanical force in skeletal muscle fibres. recombinant myosin essential light chain (elc) was expressed in e.coli and labelled at cys-178 with coumarin. the labelled elc was exchanged with native elc in single permeabilized rabbit m.psoas fibres. fluorescence lifetime was measured using leica sp5 upright confocal microscope equipped with becker & hickl time-correlated single photon counting module and 63x 1.2 na leica planapo dipping objective, with the two-photon fluorescence excitation at 856 nm by sapphire pulsed laser. after acquiring flim images of muscle fibres in relaxed state, the solution was changed to ca-free rigor. further images were acquired in rigor with or without 0.5-1% stretch applied by a motor. both single and double exponential fluorescence decay analysis showed that the lifetime in rigor was lower compared to relaxed (about 80 ps difference for single exponential fit) and to rigor fibres under strain (about 40 ps). these data suggest a change in the microenvironment of coumarin induced by nucleotide binding and strain. this change is likely to be due to interaction between c-terminal domain of elc and the n-terminal domain of myosin heavy chain related to the lever arm re-orientation process. supported by bbsrc. alpha-synuclein and its a30p mutant affects the actin cytoskeleton structure and dynamics v. sousa 1 , s. bellani 1 , g. ronzitti 2 , f. valtorta 1 , j. meldolesi 1 , e. chieregatti 2 1 department of neuroscience, hsr, milano, italy, 2 department of neuroscience, iit, genova, italy alpha-synuclein (syn) is a soluble protein abundant in the brain, primarily enriched at pre-synapses. syn overexpression and the expression of its a30p mutant participate in the pathogenesis of parkinson's disease. many roles have been proposed for syn, including the regulation of synaptic vesicle pools and of neurotransmitter release. the actin cytoskeleton regulates many aspects of synaptic function and its dysregulation may be a cause of neurodegeneration. working both in cell-free and in vivo conditions we demonstrate that syn and the a30p mutant have different effects on the actin cytoskeleton dynamics. our results show that syn binds actin, and decreases actin polymerization rate probably by monomer sequestration. on the contrary, a30p accelerates actin polymerization in vitro and disrupts the cytoskeleton of intact cells. in particular, during dynamic cytoskeleton remodeling, a30p induces the assembly of discrete actin-rich foci. actin trapping and the impairment of filaments reassembly lead to inhibition of cell movement and of the re-establishment of cell-cell contacts. in a30p expressing cells cytoskeleton-based processes, such as cell migration and the exo/endocytic traffic are inhibited. elucidating the dynamics of syn interaction with actin may contribute to the understanding of its role in neuronal physiology as well as in neurodegeneration. on the physics of muscle contraction m. l. shur ural state university, yekaterinburg, russian federation whichever energy source is chosen as an engine, its force will decrease with increasing velocity. this is connected with a limited power of any engine. thus, we state that hill's formula is a mere sequence of the law of energy conservation. to derive a mathematical dependence "force-velocity", all the means of consumption of fuel energy should be determined -in our case, the energy of the atp hydrolyze. moreover, the conformation energy of the crossbridges attached serves as the force source as well. we state that part of the energy release transforms into the energy of oscillations of myosin proteins; the other part goes into thermal energy of the sarcoplasmic solution. interaction of the oscillating myosin system with the sarcoplasmic solution controls the process of force generation by a muscle. it is just this interaction that leads to the temperature dependence of force. the presentation is devoted to constructing a theory based on these simple considerations. a discussion and constructive critic is especially wanted. -biological motility and molecular motors meiotic nuclear oscillations in the fission yeast schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. we report a mechanism of these oscillations based on collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. by combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. the redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, due to the intrinsic motor properties, generate regular large-scale movements in the cell (1 ) m. versaevel, s. gabriele, p. damman university mons-hainaut, 7000 mons, belgium the remodeling of blood vessels in response to changes in blood flow is mainly realized by endothelial cells (ecs) that convert mechanical stimuli from flowing blood into changes in cell signaling through a process called mechanotransduction. many of the biological responses to external forces originate at two types of microscale structures: focal adhesions linking cells to their extracellular matrix and adherens junctions that link adjacent cells. this study aims to elucidate the role of the cytoskeleton, cell-matrix and cell-cell junctions in transducing fluid shear stress into intracellular signals in ecs. by using microcontact printing of proteins, we design substrates with defined adhesive islands in order to control shapes of living cells. this confinement of ecs allows to study the organization and the contractile activity of the cytoskeleton in order to redistribute their intracellular forces in response to externally applied forces. we design microfluidic channels with sizes and geometries close to small blood vessels to apply a physiological range of shear stresses on ecs. our results indicate that cells deposited on a precisely defined adhesive area inside microchannels and subjected to shear stress reorganize their cytoskeleton, their focal adhesions and adherens junctions in response to blood flow. drugs interfering with the cytoskeleton are used to underline the role of its different components in the cellular adaptation to the mechanical environment. s. mahdavi 1 , b. ranjbar 1 , s. gharibzadeh 2 , m. toosi 2 , m. javan 1 1 tarbiat modares university, tehran, iran, 2 amirkabir university of technology, tehran, iran multiple sclerosis (ms) is the main known pathology of myelinating cells. an autoimmune reaction occurs against myelin sheets of neurons, so action potential (ap) propagation along the affected nerve fibers has been destroyed and it causes various disorders. here, we propose a novel strategy for ms symptoms treatment. we modeled neuron by orcad software and simulated the action potential propagation along the axon in normal condition. our model simulated normal neuronal behavior. then we destroyed the myelin sheet as it occurs in ms and observed destroyed ap propagation as it was reported in ms disease. we investigated the effect of changes in the voltage-gated sodium channel (vgsc) threshold on the efficiency of ap propagation. the results demonstrated that reduction of vgsc threshold improves the propagation of ap by increasing the amount of sodium flux during ap propagation. although, some researches have proposed vgsc blocker as ms symptom treatment, our result suggests that the increase of sodium current produced by reduction of vgsc threshold, improves ap propagation and probably cure some ms symptoms. so, we suggest that vgsc gating modifiers can be considered as novel strategy for ms treatment. surely, this results needs to be confirmed by experimental studies. a. gradogna, e. babini, a. picollo, m. pusch istituto di biofisica, consiglio nazionale delle ricerche, via de marini 6, 16149 genova, italy clc-ka and clc-kb are highly homologous cl − channels expressed in the kidney and the inner ear where they mediate transepithelial chloride transport. both channels heteromerize with the beta subunit barttin. mutations in clc-kb and barttin genes lead to bartter's syndrome. we analyzed the modulatory effect of extracellular ca 2+ and h + on clc-k channels using the xenopus oocyte expression system. clc-ka currents increased with increasing [ca 2+ ] ext without full saturation for [ca 2+ ] ext up to 50 mm. however, in the virtual absence of ca 2+ , clc-ka currents are about 18 % of currents measured in 10 mm [ca 2+ ] ext , demonstrating that ca 2+ is not strictly essential for opening. vice versa, clc-ka was blocked by increasing the [h] + ext with an almost complete block at ph 6. among various reaction models tested, the model that best fitted all state-steady data predicts an allosteric regulation of channel opening by separate binding sites for ca 2+ and h + . moreover, the best fit suggests that one ca 2+ and two h + bind to the channel. kinetic analysis of current responses upon [ca 2+ ] ext and ph jumps confirmed the allosteric character of modulation. in support of the presence of two separate binding sites we identified several mutations that selectively altered ca 2+ or h + sensitivity. our data represent a first step towards a molecular picture of ca 2+ and proton regulation of clc-k channels and suggest that it is of physiological relevance. the extracellular matrix molecule hyaluronic acid modulates l-type voltage-dependent ca2+ channels e. dvoretskova 1 , g. kochlamazashvili 2 , o. bukalo 2 , c. henneberger 3 , d. rusakov 3 , m. schachner 2 , a. dityatev 1 1 italian institute of technology, genova, italy, 2 centre for molecular neurobiology, hamburg, germany, 3 institute of neurology, university college london, london, uk we studied the effects of hyaluronic acid (ha), a major extracellular matrix molecule, on activity of l-vdccs in a heterologous expression system and in hippocampal slices. we recorded currents mediated by a major neuronal subtype of l-vdccs (ca v 1.2c, β 1 b, and α 2 δ 1 ) expressed in cho cells. a five-minute application of 0.1 mg/ml ha potentiated l-vdcc currents at -20, -10, 0 and +10 mv by approximately 50%. analysis of boltzmann curves showed that ha increased maximal conductance rather than other parameters (v 0.5 or k ). treatment with hyaluronidase removed endogenous ha in murine hippocampal slices and specifically impaired long-term potentiation (ltp) induced at ca3-ca1 synapses by repetitive theta-burst stimulation. blockade of l-vdccs reduced ltp in control slices to the levels seen after hyaluronidase treatment. a potentiation of l-vdccs with bay k 8644 fully restored ltp after hyaluronidase treatment. removal of ha reduced ca 2+ transients elicited by backpropagating action potentials in individual dendritic shafts and spines of ca1 pyramidal cells, whereas pretreatment with nifedipine fully occluded this effect. thus, ha potentiates postsynaptic l-vdccs and by this way influences use-dependent synaptic plasticity. whole-cell patch clamp recordings from a variety of human cancer cells showed that functional voltage-gated sodium channel (vgsc) expression occurred specifically with strongly metastatic cells. in addition, where studied, this was accompanied by down-regulation of outward (mainly potassium) currents. this has led to the celex ("cellular excitability") hypothesis of cancer according to which metastatic cell membranes are excitable and this promotes their hyperactivity. importantly, the vgsc genes expressed are embryonic splice variants, which are normally developmentally regulated, hence the phenomenon is 'oncofetal'. in breast cancer, where the predominant vgsc is nav1.5 the neonatal and adult forms are significantly different and this is reflected in channel activity whereby the neonatal vgsc has much slower inactivation kinetics. the double-charge change at position 211 is critical for this difference. the slow kinetics results in much greater influx of na + into cells and one consequence of this is activation of protein kinase a. this is a tonic effect and, under steady-state resting conditions, it results in a positive feedback effect promoting post-translational trafficking of vgsc protein to plasma membrane. the unique amino acid sequence of the spliced region has enabled the production of a polyclonal blocking antibody specific to neonatal nav1.5. it is concluded that vgscs represent novel biophysical targets for clinical management of metastatic disease. m. pusch cnr, istituto di biofisica, genoa, italy clc proteins form an evolutionary conserved gene-family that comprises 9 members in mammals. four of the human clcs are passive plasma membrane cl − ion channels. the other 5 clcs are expressed in intracellular organelles. clc-4 and clc-5, mutations of which lead to dent's disease, are secondary active cl − /h + antiporters, similar to the bacterial clc-ec1, and with identical 2 cl − : 1 h + stoichiometry. cl − and h + transport activity of the exchanger clcs depends on two glut residues. mutating a 'gating glutamate' (e211 in clc-5) converts the exchanger into anion conductances. neutralizing the 'proton glutamate' (e268), but not its replacement by some other titratable groups, abolishes cl − and h + transport. noise analysis indicated that clc-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 ps, indicating a very large transport turnover. no 3 − uncouples h + transport but mutating the highly conserved s168 to p, as found in the plant no 3 − / h + antiporter atclca, led to coupled no 3 − : h + exchange. clc proteins are a fascinating example of how a very similar protein architecture can be used to provide either a passive electrodiffusive permeation pathway or a strictly coupled secondary active ion transporter. (supported by telethon italy -grant ggp08064). the role of ion dynamics in zebrafish fin regeneration the specific and directional ion transport across cell membranes or tissue layers results in differential accumulation of ions and endogenous electric currents. these phenomena have been shown to be important for vertebrate organs regeneration. however, the specific ion nature of such electric currents remains unknown, as well as the role of cellular ion dynamics during regeneration and the molecular signalling pathway that transduces electric cues into cellular responses. we use zebrafish caudal fin as an adult regeneration model to unveil the specific ion composition of the currents associated with wound healing and regeneration, using a non-invasive ion-specific scanning microprobe setup. our data suggests a role for potassium (k + ), calcium (ca 2+ ) and protons (h + ) at different stages of the regeneration process. k + and ca 2+ extracellular effluxes have both been detected during the wound healing stage. h + efflux is triggered during wound healing and is maintained throughout regeneration. we are validating these data with genetic and pharmacological approaches, as well as advanced ion imaging. overall, our results suggest ion-driven mechanisms underlie adult tissue regeneration and its comprehension may open way for new therapeutic strategies, both in regenerative and developmental medicine and in cancer therapy. cardiac effects of anabolic steroids: an electrophysiological approach anabolic androgenic steroids (aas) have been used by athletes and non athletes for almost five decades in order to improve performance. however, the illicit abuse of high-doses of aas has been attributed as a main cause of several cardiovascular disorders such as arterial hypertension, lipid profile abnormalities, heart failure, hypertrophic cardiomyopathy, arrhythmia and sudden death. the aim of this study was to investigate qt interval and transient outward potassium current (i to ) changes in rats treated with nandrolone decanoate (deca). male wistar rats received weekly 10 mg/kg of deca (n=10) or vehicle (control, n=10). electrocardiogram was recorded weekly, and qt interval was measured. after 8 weeks hearts were excised and single myocytes were isolated from the ventricles of animals. i to was recorded by means of the whole cell patch clamp technique. qt interval was larger in deca group from 4 th to 8 th week (p <0.01). analysis of i to showed a decreased current density (p <0.01) in ventricular cardiomyocytes of deca group, compared to control group. in conclusion, our results show that alterations on ventricular repolarizaton may constitute an early consequence of the chronic administration of high doses of anabolic steroids in rats, and demonstrated qt prolongation and i to density reduction, which may constitute an important marker of arrhythmia vulnerability and sudden death. -ion channels in channelopathies and cancer denitrifying bacteria control no and no 2 cytosolic levels by regulating the expression of denitrification gene clusters via redox signalling of specific transcriptional factors that may act as no sensors in vivo. a protein belonging to the subclass dnr (dissimilative nitrate respiration regulator) from pseudomonas aeruginosa has been recently suggested to be a heme containing protein. very recently the three dimensional structure of the apo-form of dnr (in the absence of heme) has been determined by x-ray crystallography, whereas the holo-form (in the presence of heme) has not yet been crystallized. we have investigated the heme local structure in solution of ferric, ferrous, co bound and no bound holo-dnr by x-ray aborption spectroscopy (xas) and we added a kinetic study of the co bound form by means of a flash photolysis setup using uv-visible absorption as a spectroscopic probe. the combination of fe k-edge xanes fingerprints and kinetic study reveal a heme pocket able to bind exogenous ligands like no and co with increased plasticity, thus supporting its role as the cofactor involved in no sensing activity. molecular examination of motifs that lead to the formation of s-nitrosylated proteins i. alicea, e. r. schreiter university of puerto rico rio piedras, san juan, puerto rico physiologically, a wide range of proteins experience structural and functional modifications after the addition of a nitric oxide (no) moiety to cysteine thiol. this posttranslational modification, known as s-nitrosylation, regulates a large number of cellular processes like vasodilatation, cell signaling and others, and the products of s-nitrosylation can be involved during the development of different human diseases. however, little is known about the mechanism by which different proteins specifically bind the no moiety to their cysteine. here we show a bio-statistical analysis of some properties of cysteine that are s-nitrosylated in different proteins, including the pk, electrostatic environment, solvent accessibility of the target cysteine and identity of surrounding amino acids. we also chose model proteins (human thioredoxin and s100 protein) to make specific targeted amino acid substitutions around selected cysteines to alter the properties described above. the reactivity and stability of these mutant proteins towards s-nitrosylation will be examined. sampling the flexibility of ppar-γ s. aci-sèche 1 , n. garnier 1 , d. genest 1 , s. bourg 2 , c. marot 3 , l. morin-allory 3 , m. genest 1 1 upr cnrs 4301, orléans, france, 2 fr pcv cnrs 2708, orléans, france, 3 umr cnrs 6005, université d´orléans, france a promising approach to consider the flexibility of proteins in docking studies consists in performing multiple rigid docking on a representative set of the receptor conformations. molecular dynamic (md) simulation is one of the best adapted methods for structural sampling, but exploring the conformational diversity of a protein is computationally expensive. we present a protocol for generating a wide range of conformational states of a receptor using restrained md and a partitioning protocol to select a few representative conformations of the binding site from this md. a way to speed up efficiently md calculation is using an implicit model to represent the solute-solvent interactions. we explore a protocol using a distance-dependant permittivity function to represent solvent effect and an ensemble of controlled restraints applied on a subset of specific atoms in order to prevent artefactual structural distortions, but preserving receptor's flexibility. ten 100ns simulations have been performed using different sets of parameters and compared to a reference 30ns simulation with explicit solvent. to select a representative set of conformations, partitioning (k -means algorithm) was applied on the ensemble of simulated conformations. this methodology was applied to the ligand binding domain of peroxysome proliferator-activated receptor-γ. 4 department of biomedical sciences, university of antwerp, antwerp, belgium the complex system of cavities identified in human neuroglobin (ngb) has been postulated to be of functional significance to the putative no dioxygenase activity of the protein. the interconnected hydrophobic cavities may support this catalytic activity by acting as reservoir for reactants and providing preferential pathways assisting product removal from the active site. we thus decided to investigate co rebinding kinetics to ngb embedded in silica gels to expose ligand migration processes in the geminate phase. encapsulation of the co complexes of reduced neuroglobin, leads to a slight increase in geminate recombination after nanosecond laser photolysis. increasing the viscosity of the medium, by soaking the gels in glycerol, completely inhibits escape of the photodissociated ligand to the solvent, and highlights a complex, multiphasic kinetic pattern. this finding can be rationalized by assuming the existence of a discrete set of temporary docking sites, capable of trapping the photodissociated ligand for very long times, up to a few ms after photolysis. g. bartolommei, f. tadini-buoninsegni, m. r. moncelli bioelectrolab -department of chemistry, university of florence, italy ion pumps are integral membrane proteins devoted to ion transport through a lipid membrane phase. the ion pumps ca-atpase and na,k-atpase are prominent members of the p-type atpases family. due to fundamental physiological roles of these proteins, they are very promising drug targets. bioelectrolab has a wide expertise in the study of ion transport by these proteins [1] . our attention has been recently focused on the interaction of these enzymes with molecules of potential pharmacological interest. frequently, drugs exert an inhibitory action on the transport activity of an ion pump, usually confining it in an inactive conformation. molecules like thapsigargin and cyclopiazonic acid belong to high (nanom) affinity inhibitors of the ca-atpase, whereas clotrimazole and curcumin are medium (microm) affinity inhibitors of both ca-atpase and na,k-atpase. for each of these compounds a mechanism of action is proposed. moreover, recent results concerning ca-atpase inhibition by clotrimazole analogues will be shown: the relevance of this type of molecules is due to their potential employment as an alternative to traditional drugs against malaria parasite. financial support of ente cassa di risparmio di firenze and of miur (prin project) is gratefully acknowledged. [1] tadini-buoninsegni f., bartolommei g., moncelli m.r., fendler k. 2008. arch. biochem. biophys. 476:75-86 (review). s. asthana 1 , s. shukla 1 , g. giliberti 1 , f. luliano 1 , m. ceccarelli 2 , r. loddu 1 , p. ruggerone 2 , p. la colla 1 1 department of biomedical science and technology, università di cagliari, cagliari, italy, 2 department of physics, università di cagliari, cagliari, italy studies of protein-inhibitors interactions are helpful to elucidate the mode of action of ligands and thereby providing clues for rational drug design. bvdv, is an important target of drug discovery activities largely because it is essential for viral replication. in bvdv rdrp no specific nni binding site has been reported till now. experimental results have shown that different class of inhibitors (benzimidazole, imidazoquinolines and pyridoxyquinolines), have resistant mutations located in the finger domain of the rdrp. all the reported mutations are spatially very close to each other. thereby, indicating that binding sites of nni's may lie in the finger domain for these different class of inhibitors.herein, we have utilized docking procedure to investigate binding sites, binding modes as well as binding affinity of different class of inhibitors.we then used all atom molecular dynamics (md) simulations to investigate the stabilizing interaction between inhibitor-receptor pairs. our md results are in good agreement with experimental data and provide deep insights into the dynamical features of the high affinity inhibitorreceptor binding. thus identifying the binding modes of our inhibitors and mechanism leading to inactivity of the enzyme can help us to build a microscopically well-funded picture of the functioning of these enzymes. we present an investigation of the molecular basis of ligand binding and reactivity of heme proteins using computer simulation. a combination of classical molecular dynamics and hybrid quantum-classical (qm-mm) calculations are applied to explore distal and proximal effects on diatomic ligand binding to the heme. trends in binding energies and in the kinetic constants are illustrated through a number of selected examples. an investigation of the interplay between ligand migration and protein dynamics obtained through classical molecular dynamics techniques in combination with advanced sampling tools is also presented to yield information about free energy profiles and possible secondary docking sites. results for truncated n hemoglobin of mycobacterium tuberculosis, presented as an illustrative example, suggest that the truncated hemoglobin n has evolved a dual-path mechanism for selective/distinct migration of o 2 and no to the heme, to achieve efficient no detoxification. finally, we present also an analysis of the molecular basis of hexacoordination in human neuroglobin, which suggest that the flexibility of the cd plays a key role in determining the ligand binding properties. thermodynamic bases of nucleoplasmin-histone complexes recognition by the nuclear transport machinery i. arregi, j. falces, s. bañuelos, m. a. urbaneja, s. g. taneva unidad de biofísica (csic/upv-ehu), departamento de bioquímica y biología molecular, universidad del país vasco, spain the nuclear transport of the chromatin remodeling (nucleoplasmin) and chromatin building (histones) proteins is mediated by importins. nucleoplasmin (np) contains a classical bipartite nuclear localization signal (nls) that is recognized by importin α, while histones present multiple sequence elements (nls-like motifs) for nuclear targeting. besides, ternary importin/np/histone complexes might represent a putative coimport pathway for nuclear import of linker (h5), nucleosomal core (h2ah2b) histones and their chaperone protein np, enhancing the histone import efficiency. to better understand np and histone recognition by the transport machinery we studied the thermodynamics of complex formation of importin α (a truncated form) and importin β with histones and np, and with np/histone binary complexes by means of isothermal titration calorimetry. data show that importins interact with the two histone types and np, and that importin and histones can simultaneously bind to np. analysis of the binding energetics reveals an enthalpy driven formation of high affinity binary and ternary complexes. we demonstrate that different amount of importin molecules can be loaded on np/binary complexes dependent on the histone type, linker or core, and the amount of the bound histones. g. breuzard, i. di maïo, p. barbier, d. allegro, c. brault, v. peyrot cro2 inserm u911, ufr de pharmacie, marseille, france since a decade, our laboratory has already studied the interaction of tau variant with tubulin (tub) or microtubules (mts) and phosphorylation process on this interaction. indeed, fret assay was a powerful tool to achieve binding parameters between tau and tub in vitro: 1/ with mts stabilized by fluorescein-coupled taxol (flutax-2) as donor and rhodamine-labelled tau (rho-tau) as acceptor, and 2/ in living cells by confocal laser scanning microscopy (clsm) with tau/α-tub fused to egfp/mcherry, respectively. results revealed 47 ± 3 % energy transfer efficiency from flutax-2 to rho-tau and a donor-to-acceptor distance of 54 ± 1å. by titration, the dissociation constant of tau was determined to 1.0 ± 0.5 µm. a cleavage procedure of αβ-tub was performed to determine the influence of the c-term tails of αβ-tub on the tau-mt interaction. no difference in distances and binding parameters was observed. clsm images displayed a heightened concentration of fluorescent tau in patches along mts. fret experiments revealed in particular higher efficiencies between gfp-tau to mcherry-α tub proteins in these locations. overall, our results suggested no involvement of the hypervariable and highly acidic c-term tails of tub in mt/tau binding. a molecular model is proposed in which flutax-2 is directly accessible to tau molecules. besides, the modified distribution of fluorescent tau could be implicated in local change of mechanical properties of mts. a. boreham 1 , k. winkler 1 , c. gebhard 2 , k. rueck-braun 2 , p. henklein 3 , e. michalsky 3 , r. preissner 3 , r. misselwitz 3 , a. ziegler 3 , u. alexiev 1 1 freie universität berlin, berlin, germany, 2 technische universität berlin, berlin, germany, 3 charité-universitätsmedizin berlin, berlin, germany peptide presentation by major histocompatibility complex (mhc) molecules is crucial for immune responses. photocontrol of peptide dynamics by means of photo-switchable peptide analogs will provide insights in peptide dynamics and its dependence on mhc polymorphism (1) (2) (3) . we have designed a hemithioindigo (mhti) photo-switch bearing peptide using the viral epitope rrrwrrltv (plmp2) as a template. incorporation of mhti in the peptide backbone should result only in a minor change of the overall peptide structure given the relaxed conformation of the zisomer. indeed, the human mhc molecule hla-b*2705 can tolerate nonpeptidic elements in plmp2, as evidenced by normal peptide binding. using the autofluorescent properties of mhti we determined the stability of the hla-b*2705/mhti-plmp2 complex. photoswitching from z →e results in a decrease of hla-complex stability. based on computer modeling this decrease is due to a reduced interaction of the peptide c-terminus with hla-b*2705. truncated hemoglobins (trhbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. three phylogenetic groups (n, o, and p) have been identified in trhbs. the crystal structure of truncated hemoglobin o of b.subtilis, does not show an evident tunnel/cavity system connecting the protein active site with the solvent, a fact that cannot be easily rationalized considering the very high oxygen association rate. moreover, resonant raman results of the co bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. for these reasons we performed classical molecular dynamics simulations of the oxy, carboxy and deoxy protein, and computed the free energy profiles associated with ligand migration to the active site. our results suggest that there is a key residue, glne11, that may present an alternate conformation in which a wide ligand migration tunnel is formed, consistently with the kinetic data. the results for the co and o 2 bound protein show also that glne11 is directly involved in the stabilization of the coordinated ligand, playing a similar role as tyrb10, and trpg8 in other trhbs. our results not only reconcile the structural data with the kinetic information, but also provide additional insight about the general behaviour of trhbs. patterned functionalization of surfaces for guided transport on molecular motors tracks m. bhagawati 1 , s. ghosh 2 , t. surrey 1 , j. piehler 2 1 department of biophysics, university of osnabrueck, osnabrueck, germany, 2 european molecular biology laboratory, cell biology and biophysics unit, heidelberg, germany chelator head groups with multiple nitrilotriacetic acid (nta) moieties have been very well characterized and successfully utilized as high affinity adapters for functional immobilization of oligohistidine tagged proteins to surfaces. we have recently established a generic method for patterning nta functionalized surfaces by selective photodestruction via a light induced fenton reaction. efficiency of different transition metal ions for catalyzing this reaction was tested. functionality of the patterned protein was confirmed using the interaction between interferonα2 and its receptor. implementation of this technique in a confocal laser scanning microscope allowed us to control surface density of binding sites, providing the possibility to vary the surface concentration of immobilized proteins in a spatially resolved manner. we also applied this approach for exploring guided transport of microtubules by kinesin selectively immobilized onto trisnta patterns. rheumatoid arthritis (ra) is an autoimmune disorder, leading to pathological damage at the level of joints, associated with the hla class ii allele hla-dr4. although etiology of ra is unknown, type ii collagen (cii) is a potential antigen candidate and it is believed that t cell responses in collagendependent ra are directed towards the immunodominant pathogenic epitope cii(261-273). despite recent advances in characterization of class ii major histocompatibility complex (mhc) and t-cell receptor (tcr) contacts in this epitope, the atomic details of tcr-cii(261-273)-mhc complex are not known. here, homology modeling and molecular docking studies have been used to derive a three-dimensional model of tcr β chains, obtained from a dr4+ subject, in complex with cii(261-273)/hla-dr4. the best complex from docking was further refined using molecular dynamics simulations for 20 ns. the proposed model represents a reasonable structural basis for understanding cii(261-273)-mhcii complex recognition by tcr and for rational design of inhibitors targeting tcr-pmhc interface. probing bio-molecular bonds with magnetic force for biosensor applications a. m. de jong 1 , a. jacob 1 , x. j. janssen 1 , j. m. van noorloos 1 , l. j. van ijzendoorn 1 , m. w. prins 2 1 eindhoven university of technology, eindhoven, the netherlands, 2 philips research laboratories, eindhoven, the netherlands we investigate new technologies to be applied in next generation biosensors, which not only measure biomarker concentrations but also probe bio-molecular interactions. the concept is based on the response of ligand-receptor pairs to an applied force or torque [1, 2] . we use functionalized magnetic beads and magnetic fields to apply translational and rotational forces on the molecular bonds. in a model experiment, polystyrene surfaces were coated with anti-biotin and beads were coated with biotin. after incubation, a constant magnetic force was applied to the beads and the number of bound beads was measured as a function of time. this was repeated for a range of forces. the dissociation rate (k o f f ) is determined for each force and k o f f at zero force is extracted from these data. rotational forces were exerted on protein-g coated beads bound to igg antibodies immobilized on a surface. rotating fields revealed an oscillating behavior, which can be understood from the balance between the applied magnetic torque and the torque due to the deformation of the biological bond. studying interactions between cop1 regulatory protein and hy5 transcription factor d. s. dalafave the college of new jersey, ewing, nj 18940, usa this work addresses important questions of protein-ligand interactions and selective protein recognition. proteinligand bindings are crucial for many cellular processes. dependable methods for predicting binding sites would lead to a better understanding of proteins' selective recognition and would, in turn, help research on fighting diseases. presented here is a study of interactions between the wd domain found in a regulatory protein cop1 and the motif v-p-e/d-φ-g (φ=hydrophobic residue) found in a transcription factor hy5. cop1 and hy5 proteins have opposing roles in developmental regulation. cop1 can repress hy5 by directly binding with it. the repression involves specific interactions between the wd domain and the motif v-p-e/d-φ-g. previous experimental research showed that mutations in the motif's v-p pair resulted in a large decrease in hy5 repression. to study effects of similar mutations, residues in the motif v-p-e/d-φ-g were systematically substituted with other residues. interactions between the mutated motif and the wd domain were studied. distributions of binding sites, bond lengths, local shape complementarity, and interaction potentials were modeled for each residue substitution. the study identified binding sites critical for the cop1-hy5 binding. some hy5 residues in the vicinity of the motif were also found to be important in the binding. the significance of the results for understanding selective protein recognition is discussed. determination of protein-ligand binding thermodynamics by thermal shift assay p. cimmperman, a. zubriene, l. baranauskiene, e. kazlauskas, j. matuliene, d. matulis laboratory of biothermodynamics and drug design, institute of biotechnology, vilnius, lithuania thermal shift assay determines the effect of ligand binding on the protein thermal denaturation equilibrium. several models have been derived to describe the general cases of protein-ligand binding. first, the model describing protein stabilization and destabilization by ligand binding to the native and unfolded states. second, the split of protein melting transitions by tightly binding ligands is presented when the transition of free protein and ligand-bound protein occur separately. mathematical equations describing the protein unfolding and ligand binding thermodynamic parameters at various temperatures are presented. the protein melting temperature shift can be determined by various techniques such as differential scanning calorimetry, circular dichroism, and intrinsic or extrinsic fluorescence. the advantages of fluorescent techniques are presented. the melting temperature shift caused by ligand binding is dependent on the thermodynamic parameters of protein unfolding and ligand binding, including enthalpy, entropy, and heat capacity, thus allowing determination of binding thermodynamics. application of the models in the design of hsp90 chaperone and carbonic anhydrase inhibitors is discussed. comparison with isothermal titration calorimetry data is presented. recent reports have shown that the bacterial redox protein azurin can enter into cancer cells and induce apoptosis by stabilizing p53. the formation of a complex between the two proteins has been demonstrated, but little is known about binding features. for the first time, we show here that azurin binds to the n-terminal region of p53 with a dissociation constant in the 5-10 µm range. trp phosphorescence lifetime measurements revealed conformational changes of azurin induced by the interaction with p53(1-63). acrylamide quenching of trp phosphorescence also indicated a significant increase of the overall flexibility of azurin upon binding to p53 . no change of the fluorescence emission of p53(1-63) was detected in the presence of azurin. the latter finding suggests that w23 of p53 is not directly involved in domain binding to azurin, indicating that the binding site is distinct from that of mdm2. the present results may assist the design of novel cancer treatments based on p53 stabilization by azurin. a. eleta 1 , r. georgieva 2 , h. bäumler 2 , j. l. toca-herrera 1 1 cic biomagune, 20009 donostia-san sebastián, spain, 2 charité-universitätsmedizin berlin, berlin, germany human serum albumin (hsa) is the most abundant nonglycosilated plasma protein in the human body. this multifunctional protein has ligand-binding and transport properties, antioxidant functions and enzymatic activity [1] . bilirubin interacts with albumin in order to be transported from the blood to the liver where it is secreted. the interaction occurs specifically in hsa i-domain of its three domains [2] . in our work, we investigate the interaction between hsa and bilirubin by quartz crystal microbalance with dissipation (qcm-d) and atomic force microscopy (afm) [1, 3] . albumin was adsorbed on negatively charged silicon oxide. however, hsa was removed after rinsing with pbs. hsa adsorbed on positively charged polyelectrolyte multilayers leads to a stable layer of surface mass density of 272 ng/cm 2 . cross linked albumin with glutaraldehyde after its adsorption on nh 2 -terminated thiols is also stable reaching surface mass density of 296 ng/cm 2 . a preliminary bilirubin adsorption results on cross linked hsa substrate show that 200 ng/cm 2 is immobilized. taking into account that hsa-bilirubin stoichiometry is 1:1, the outcome demonstrates that the 70% of hsa i-domains remain active. antimicrobial peptides (amps) are short positively charged polypeptides. they are important due to their potential to provide an alternative to conventional therapy against bacterial infections. rbpi 21 is a 21 kda peptide based on the nterminal region of the neutrophil bactericidal/permeabilityincreasing protein (bpi). it was shown that this amp possesses bactericidal effects on gram-negative bacteria and higher affinity for lipopolysaccharide (lps), neutralizing its effect. the peptide use against meningitis, is in phase iii clinical trials. here, we demonstrate that rbpi 21 promotes aggregation of negatively charged large unilamellar vesicles (luv) and lps aggregates, by dynamic light scattering, while for zwitterionic phosphatidylcholine (popc) luv the size remains unchanged. the aggregation increases with peptide concentration until peptide promotes massive aggregation followed by sample flocculation/precipitation. with the rbpi 21 -lipid interaction there is a progressive change in the zeta-potential of the luv systems and lps aggregates. luv systems composed of phosphatidylglycerol (popg) and popc:popg mixtures have higher zeta-potential variations than popc luv. for lps aggregates, rbpi 21 neutralizes the surface charge and at higher peptide concentrations overcompensates it. results demonstrate that the interaction of the peptide rbpi 21 with lps aggregates and luv systems has electrostatic and hydrophobic contributions. serratia marcescens heme acquisition system: heme transport and protein:protein interactions m. delepierre unité de rmn des biomolécules cnrs ura 2185, institut pasteur, paris, france heme transport systems in bacteria are required and might be potential target for antibacterial drugs. the heme acquisition system, has, exists in pathogenic as well as in opportunistic bacteria but only for the latter one extensive studies have been conducted, constituting as such a model system. the outer membrane receptor hasr, the central component of this system, functions in synergy with a secreted high affinity heme binding protein, the hemophore hasa. hasa extracts heme from host hemoproteins and returns it to hasr. then, the energy given by a protein complex of the inner membrane is used to allow heme entrance across the bacterial membrane and to eject the empty hemophore from the receptor. reconstitution of this heme acquisition system in e coli, overexpression and purification of its various components have allowed us to obtain sufficient amount of protein to perform nmr and biophysical studies to analyse at the molecular level the different steps of heme acquisition by hasr. protein-protein interactions (ppi) are the central pillar supporting most of biological functional activity on the molecular level. a binding event between two proteins typically consists of two stages: 1) diffusional search of the binding partners for each other, and 2) specific recognition of the compatible binding surfaces followed by the formation of the complex. we focus here on the non-specific component of ppi, which refers to all physico-chemical properties of the binding partners (such as size, charge, isoelectric point, hydrophilicity etc.) that are independent of the exact details of their binding sites, but which could in turn affect their localization or diffusional search for one another. it is known that proteins co-localize due to segregation into different cellular compartments, sequestration via anchor and scaffold proteins or even chemical modifications. we suggest that the non-specific component of ppi determines in part the co-localization and clustering of the binding partners, which then directly in a non-specific fashion influences their interactions. we examine the possibility that such signature might be encoded within the experimental 3d structures of a large set of known mutually interacting proteins. we provide preliminary evidence that this indeed may be the case, and corroborate our findings by using different statistical tests to compare those features of the known interacting partners, and ascertain correlations and commonalities between them. a link between hinge-bending domain motions and the temperature dependence of catalysis in ipmdh i. hajdú, a. szilágyi, j. kardos, p. závodszky institute of enzymology, hung acad sci, budapest, hungary enzyme function depends on specific conformational motions. since conformational flexibility strongly depends on temperature, temperature dependent enzyme kinetic studies with measurements related to dynamics can give us some insight at atomic level into these functionally relevant motions. the catalytic efficiency (k cat /k m ) of 3-isopropylmalate dehydrogenase for its substrate (ipm) has unusual temperature dependence, showing a local minimum at 35 • c. in search of an explanation, we measured the individual constants k cat and k m,ipm as a function of temperature, and found that the van't hoff plot of k m,ipm shows a sigmoid-like transition in the 20-40 • c temperature range. by means of various measurements including h-d exchange and fret, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperature above 30 • c. the thermodynamic parameters of ligand binding determined by itc as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. because the binding of ipm is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with ipm binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency. a simulation approach to multiple sclerosis: study of a peptide with a pharmaceutical potential c. guardiani 1 , s. marsili 2 , p. procacci 2 , r. livi 3 1 centro dinamiche complesse, università di firenze, italy, 2 dipartimento di chimica, università di firenze, italy, 3 dipartimento di fisica, università di firenze, italy multiple sclerosis (ms) is an autoimmune disease of the central nervous system, leading to premature death. one of the potential targets of the autoimmune reaction is the myelin protein mog that has been crystallized in complex with the 8-18c5 ms-autoantibody. the analysis of contacts and buried surface area combined with an alanine scanning computation reveals the key role of mog fragment 101-108 for the interaction with 8-18c5. a docking simulation shows that the 101-108 fragment, excised from mog, and kept in crystal-like conformation, is still capable of fitting into the binding pocket of the antibody. we then studied, through replica exchange molecular dynamics simulations, the structural equilibrium distribution of the free peptide and of a number of analogs stabilized by a disulfide bond. we found that the free peptide yields a significant fraction of crystal-like conformations and the proportion of native-like structures is further increased by the disulfide bridge. when we tried to dock the centroids of the most populated clusters to 8-18c5, we discovered the existence of a docking funnel whose bottom is populated by stable complexes where the peptide occupies the same spatial region as in the crystal. we therefore conclude that the mog 101-108 fragment may be used to develop a diagnostic assay or a drug for ms. the escherichia coli membrane insertase yidc reversibly binds its substrate pf3 coat protein u. gerken, s. winterfeld, a. kuhn institute of microbiology and molecular biology, university of hohenheim, germany the membrane insertase yidc of e. coli belongs to the oxa1 family of mitochondria and plays an essential role in facilitating the insertion and assembly of membrane proteins. we have previously shown with detergent-solubilized (c 12 pc) yidc, labelled with ans, and pf3 coat that the initial step of the membrane insertion process, the binding of the substrate pf3 coat to yidc, is reversible [biochemistry 47, 6052-6058 (2008) ]. the dissociation constant k d for that particular system is about 1 µm. in order to obtain data for the native system we used in this study membrane-reconstituted (dopc and dope/dopg) yidc. the effect of the initial binding was examined in vitro by fluorescence quenching of the 11 tryptophan (trp) residues of yidc which are highly sensitive fluorescent probes for changes of the tertiary structure. quenching of the trp fluorescence after titration with a trp-free pf3 mutant indicates a change in the yidcs tertiary structure upon binding to its substrate. the binding data show a k d value in the range of 0.5-1.8 µm. the influence of different environments (lipid membranes, ddm micelles) on the secondary structure of yidc as well as on the yidc large periplasmic domain p1 was investigated by circular dichroism (cd). the cd data show that the secondary structure of yidc changes upon reconstitution into a membranes when compared to the detergent solubilized state. particularly, the p1 domain is considerably affected by the detergent c 12 pc. b. karasulu, b. erman, o. keskin koc university, istanbul, turkey histone proteins are fundamental to the cells since they are involved in cell regulatory processes, such as chromatin regulation, gene silencing and transcription, cell cycle control, and epigenetics, which are controlled via post-transcriptional modifications of the histone protein tails. these modifications are categorized under four main groups: methylation, acetylation, ubiquitination, and phosphorylation. among them methylation has been very recently proven to be reversible with the discovery of histone demethylase proteins and this modification type has been extensively studied, because the abnormal methylation rates cause the excess proliferation of the cell, which, in turn, triggers the cancer. therefore, understanding of the details of reaction mechanisms of histone methylation/demethylation dynamics provide the required knowledge basis for preventing the abnormal methylation rates by designing proper inhibitor (drug) molecules. in this study, we display possible reaction mechanisms (such as amine oxidation via lsd1) for the demethylation of specific histone tail proteins. we try to explain the role/importance of residues that take place in or near to the reaction pocket for the demethylation reaction. we also carry out md simulations and reaction path (free energy profile) analysis using free energy perturbation (fep) method for qm/mm hybrid systems in order to compare different possible reaction pathways. the sonic hedgehog (shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors patched (ptc) and smoothened (smo) , which lead to birth defects, cancer or neurodegenerative diseases. however, little is known about ptc, the receptor of the shh protein, and the way ptc regulates smo, the receptor responsible for the transduction of the signal. to develop structure-function studies of these receptors, we expressed human ptc (hptc) in the yeast saccharomyces cerevisiae. we demonstrated that hptc expressed in a yeast membrane fraction is able to interact with its purified ligand shh, indicating that hptc is produced in yeast in its native conformational state. using surface plasmon resonance technology, we showed that fluorinated surfactants preserve the ability of hptc to interact with its ligand after purification. this is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor ptc. this work will allow the scale-up of hptc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by shh signalling dysfunction. dissecting the colicin translocon c. l. johnson 1 , a. solovyova 1 , p. callow 2 , s. a. holt 3 , l. a. clifton 3 , k. weiss 4 , j. h. lakey 1 1 inst. for cell and molecular biosciences, newcastle univ., uk, 2 inst. laue langevin, grenoble, france, 3 isis, rutherford appleton lab., didcot, uk, 4 oak ridge national lab., centre for structural molecular biology, oak ridge, usa pore-forming colicin n hijacks e. coli outer membrane protein ompf and exploits it as both a receptor and translocator to cross the outer membrane [1] . it is currently a matter of debate if the translocation route is through the ompf lumen or the interface between ompf and the lipid bilayer. recent electron microscopy data from our laboratory suggests the latter route for translocation [2] . the colicin n/ompf complex in detergent has been studied by sans to examine the translocation pathway undertaken by colicin n. by using a combination of deuterated ompf and hydrogenated colicin we have been able to derive a low resolution structure of individual proteins in the binary complex. low resolution structural studies supplemented by targeted mutagenesis and screening techniques including itc, potassium efflux assays and auc have allowed us further our understanding of colicin n translocation. dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly49a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined. here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. effect of osmolytes on the dhfr activity, structure and dynamics b. legrand 1 , s. renaud 2 , m. collen 1 , c. tascon 3 , s. bonnassie 2 , e. gautier 1 , j. mellet 1 , c. blanco 2 , e. le rumeur 1 , j.-f. hubert 1 , a. bondon 1 1 rmn-ilp, 2 duals, 3 cbp, umr cnrs 6026, univ. de rennes1, france osmolytes are small molecules accumulated by a wide variety of organisms in response to hyperosmotic stress. they contribute to save the cellular integrity and to stabilize the macromolecules from environmental stress. dihydrofolate reductase activity is inhibited by several osmolytes. we studied the impact of osmolytes on the dhfr structure and dynamics by various techniques. we observe that substrate (dhf) and cofactor (nadph) diffusions are quite different in glycerol and betaine despite similar viscosities. we demonstrate that the overall structure is maintained at high osmolyte concentrations while no direct interactions can be detected with the enzyme. the k o f f of substrate analogues decreases with increasing the osmolyte concentrations. the enzyme dynamics, in various media, has been compared with the dhfr behaviour in water described in the literature. the osmolyte impact appears only partly conditioned by its viscogenic properties which reduce the molecules diffusion and the k o f f of the product controlled by the m20 loop of the dhfr. we suggest that the osmolytes decrease m20 loop mobility. comparing the results obtained with different osmolytes, we offer a better understanding of the osmolyte nature dependence of the dhfr inhibition. estrogen receptor (er) is a well characterized member of the nuclear receptor superfamily that modulates the expression of estrogen-responsive target genes in response to estradiol and other natural and synthetic chemicals mimicking the estradiol structure. in human, two ers, erα and erβ, lied on two distinct chromosomes, are known. er exhibits several functional domains: two conserved domains, a short domain c involved in dna-binding and a large domain e/f responsible for ligand-binding and hormone-dependent transcription activation, are linked by a hinge domain d; a poorly conserved a/b domain, at the n terminus, mediating interactions with the general transcription machinery, is involved in hormone-independent transcription activation. upon estrogen binding, ers can specifically bind to a dna fragment, called estrogen response element ere, and activate the transcription. the optimal ere sequence consists of two six base-pair half-sites, aggtca, organized as inverted repeats with a three base-pair spacing. in this study, we have investigated, by fluorescence methods, the effect of kcl concentrations, on the protein conformational flexibility and the thermodynamic stability of hers -eres complexes. we show, here, that electrostatic interactions, inside hers, contribute to its conformational flexibility and its thermal stability. moreover, the specific interaction between hers and eres is poorly sensitive to changes in ionic strength, in opposite to unspecific complexes. kinetic and structural explanation for the low enantioselectivity of human 3-phosphoglycerate kinase p. lallemand 1 , j. rouhana 1 , l. chaloin 1 , b. roy 2 , s. arold 3 , t. barman 1 , c. lionne 1 1 cpbs umr 5236, 4 bd henri iv cs69033, 34965 montpellier cedex 2, france, 2 ibmm umr 5247, 3 cbs umr 5048 l-nucleosides comprise a new class of antiviral and anticancer agents that are converted to pharmacologically active nucleoside triphosphates in vivo. the last step of the cascade may be catalyzed by 3-phosphoglycerate kinase (pgk), an enzyme that has low specificity for nucleoside diphosphate: ndp + 1,3-bisphosphoglycerate ↔ ntp + 3-phosphoglycerate. here we compare the kinetics of formation of the complexes of human pgk with different d-and their mirror images, l-nucleoside diphosphates, and the effect of 3-phosphoglycerate thereon. two types of experiment were carried out: equilibrium experiments allow the estimation of dissociation constants, and stopped-flow experiments the transient kinetics of the interactions. in addition, by the rapid-quench-flow technique, we compare the kinetics of the phospho-transfer and product release steps with each d-or l-nucleotide. crystallographic and molecular modelling studies allow defining the structural reasons for the low enantioselectivity of pgk. the aim of this basic work on the mechanism of action of human pgk with non-natural nucleotides is to obtain information for the optimization of therapeutic nucleoside analogues that are poorly phosphorylated by pgk. anrs is gratefully acknowledged for financial support. a new itc assay for measuring high-and lowaffinity protein-ligand interactions g. krainer 1 , j. fanghänel 2 , s. keller 1 1 leibniz institute of molecular pharmacology (fmp), berlin, germany, 2 bayer schering pharma ag, berlin, germany isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions [1] . however, reliable determination of the dissociation constant (kd) is typically limited to the range 100 µm > kd >1 nm. nevertheless, interactions characterised by a higher or lower kd can be assessed indirectly, provided that a suitable competitive ligand is available whose kd falls within the directly accessible window [2] . unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of high-and low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-affinity (kd <1 nm) and low-affinity (kd >100 µm) protein-ligand interactions. a molecular dynamics study of the cftr nucleotide binding domains interaction v. martorana 1 , r. noto 1 , o. moran 2 1 cnr-istituto di biofisica, palermo, italy, 2 cnr-istituto di biofisica, genova, italy the cystic fibrosis transmembrane conductance regulator (cftr), the dysfunctional protein in cystic fibrosis, contains two transmembrane domains, two nucleotide-binding domains (nbds), and a regulatory domain. opening of the pore have been linked to the atp-driven tight dimerisation of nbd. we have studied the nbd1-nbd2 interactions on wild type and cystic fibrosis-related mutations by steered molecular dynamics simulations (smd). a fully solvated dimer, including the two bound atps, was separated by pulling one monomer with an external, increasing force. interestingly, the force needed to break the mutated (g511d) dimer is significantly smaller than in the wild type case. the effect of a cftr potentiator, the genistein, has also been tested by repeating the smd simulations with the small molecule docked at the interface between the two nbd domains. to test the validity of our results we have repeated the separation process for different simulation lengths and force strengths. the amount of distortion on the pulled nbd domain has also been studied. this work is partially supported by the italian cystic fibrosis foundation (prog ffc #2/2008), with the contribution of "mille bambini a via margutta"onlus"blunotte", "lega italiana fc toscana" stability and aggregation studies of human septin 3 (sept3) j. n. a. macêdo, r. c. garratt, a. p. u. araújo instituto de física de são carlos, usp, são carlos, brazil the septins are a conserved family of nucleotide binding proteins firstly identified in saccharomyces cerevisae as proteins required for the completion of the cell cycle. septins are implicated in several cellular functions such as cytokinesis, vesicle trafficking, exocytosis, cytoeskeletal dynamics, cell polarity and sperm motility. furthermore, they are associated with alzheimer's and parkinson's disease. sept3 was identified in rat brain being highly enriched in presynaptic nerve terminals. it colocalizes with synaptophysin and dynamin i and is associated with synaptic vesicle. in this work, human sept3 without its n-terminal domain (sept3gc) was expressed in e. coli and purified by affinity and size exclusion cromatographies. structural stability studies were performed with recombinant sept3gc using circular dichroism spectroscopy (cd), right-angle light scattering, and fluorescence spectroscopy. the sept3gc cd spectrum showed a conformational transition from a predominantly α-helical starting structure to one dominated by β-sheet, just above physiological temperatures. the formation of irreversible aggregates, detected by light scattering, and their ability to bind thioflavin-t suggested that sept3 forms amyloidlike structures, as has been previously observed for human septins 2 and 4. our data suggest that amyloid formation by isolated septins in vitro may be a general phenomenon whose physiological relevance needs to be further investigated. pln149 is an antimicrobial peptide produced from lactobacillus plantarum nric149. analog pln149 (pln149a) and a ser-derived (pln149s, tyr to ser replacement) were synthesized on solid phase and their interactions with biomembrane model systems and inhibitory property on s.aureus and p.aeruginosa were investigated by cd, leakage assays, fluorescence spectroscopy, and differential scanning calorimetry. both peptides share the same unordered structure-like cd spectrum in aqueous solution, but a helicoidal induction in the presence of negative vesicles were observed, however pln149s showed lower helical content than pln149a. the ser-derived peptide presented 30% decrease of its leakage activity in different liposome compositions, a threefold increase in the dissociation constant from the liposomes than pln149a, and close to 40% reduction for the inhibitory activity against p. aeruginosa growth. we can conclude that besides the electrostatic contacts between the amphipathic &alpha-helix, formed by the cationic residues from pln149 and negatively charged phospholipids, the pln149-membrane binding is a process driven for the hydrophobic interactions from non-polar residues. in this case, there was a significant contribution of the tyr residue, which must be allocated in a lipid interface, described as the preferential position to this residue in membrane proteins. supported: fapesp label free detection using deep-uv laser-based fluorescence lifetime imaging microscopy q. li, s. seeger physikalisch-chemisches institut, universität zürich, winterthurerstrasse 190, ch-8057 zürich, switzerland label free detection based on native fluorescence excited at uv region shows great potential for the life sciences. it offers simple, low-cost and fast method for sensitive detection of important biological analytes without modification. in this contribution we present a deep uv fluorescence lifetime imaging microscopy system (duv-flim) based on a picosecond deep uv laser using time-correlated single-photon counting method. the described setup is well-suited for biological applications for ultrasensitive detection. we have showed single uv dye (bmq) and single protein (ecβ gal ) molecules detection using duv-flim. further, the label free detection of protein interaction between ecβ gal and monoclonal anti-ecβ gal has been demonstrated by means of steady-state and time-resolved fluorescence spectroscopy. we achieved detection sensitivity for the ecβ gal/ anti-ecβ gal pair down to the nanomolar concentration range. we also extended this method to study the interaction of therapeutic drug porphyrin with bsa protein. fluorescence resonance energy transfer between protein and alexa fluor 350 has been investigated using duv-flim. the intrinsic fluorescence and fluorescence lifetime changes of donor biotin β-galactosidase have been measured. energy transfer efficiency and donor acceptor distance have been obtained. fluorescence images of acceptor af 350 due to fret have been observed when excited at 266 nm. the monomeric heme-containing indoleamine 2,3dioxygenase (ido) catalyses the oxidative cleavage of the indole moiety of l-tryptophan (l-trp). enhanced l-trp degradation contributes to various physiological disorders including depression or failure of the immuno-regulating system. the regulation of ido by inhibitors is extensively studied. however, the catalytic mechanism of ido on the molecular level is still unknown. in addition to l-trp, a wide range of substrates are degraded including d-tryptophan, melatonine or tryptamine. in contrast, indole or histidine do not function as substrates for ido. to understand the determinants for substrate specificity, we investigate the interaction of the heme iron, the heme-bound ligand and the substrate. we use (time-resolved) uv/visible and fourier transform infrared (ftir) spectroscopy over a wide temperature range (4 -300 k) to monitor the binding of diatomic ligands to the heme iron and the binding of different substrates to coligated ido. changes in the spectra upon addition of l-trp are analyzed and compared to those induced by other substrates or inhibitors. archaeal protoglobin 3d-structure: novel ligand diffusion paths and heme-reactivity modulation protoglobin (pgb) from methanosarcina acetivorans c2a, a strictly anaerobic methanogenic archaea, is the latest entry in the hemoglobin superfamily. our previous crystallographic studies on pgb have shown that protoglobinspecific loops and a n-terminal extension completely bury the heme within the protein matrix (1) . access of o 2 , co, and no to the heme is granted by protoglobin-specific apolar tunnels reaching the heme distal site from locations at the b/g and b/e helix interfaces. here we report structural and kinetic data on pgb mutants engineered to probe the protein structural and kinetic properties. six crystal structures (pgb mutants: ∆20 (missing 20 nter residues), y(b10)61→a, y(b10)61→w, f(b12)63→w, f(g7)145→w, i(g11)149→f) show that the mutations engineered essentially restrict access to ligand tunnel 1. an accurate molecular level description of the signaling mechanism in ca 2+ transducers necessitates the knowledge of the kinetics and energetics of conformational changes associated with ca 2+ binding to various calcium binding proteins. with this in mind, we have developed an approach that combines the laser-induced photolysis of photolabile "caged" ca 2+ compound, dm-nitrophen, with the photothermal beam deflection (pbd) technique to determine thermodynamic profiles associated with the ligand binding to calcium chelator, edta, and ca 2+ sensor, calmodulin. this approach allows us to monitor time profiles of volume and enthalpy changes on the microsecond to millisecond timescale. the initial pbd study of ca 2+ photo-relase from ca 2+ loaded dm-nitrophen reveals the presence of two phases. the first step takes place within first 20 µs upon and is associated with a volume decrease of -7 ml mol −1 and enthalpy change of 66 kcal mol −1 . on the longer timescale (τ = 200 µs), the second event with a positive volume change of 7 ml mol −1 and enthalpy change of 8 kcal mol −1 was detected. on the other hand, ca 2+ photorelease in the presence of calmodulin is accompanied with an additional phase with a distinct lifetime and volume and enthalpy changes that reflects the metal binding to calmodulin and concomitant structural changes. antimicrobial peptides: linking partition, activity and high membrane-bound concentrations antimicrobial peptides (amps) have been intensively studied at micro and macroscopic levels for over twenty years. knowledge from these two domains has, however, contributed little to a comprehensive understanding of amp action; rather, in vivo amp performance has been only remotely correlated to biophysical properties. we focus on the assessment of peptide accumulation on bacterial membranes as an example of this separation: amp-bilayer interactions have been subject to extensive biophysical characterization, but conversion of that information into educated estimates of in vivo membrane-bound amp concentrations is lacking. this has led to the overlooking of important factors for activity. using simple partition models we were able to analyze available information on amp activity and interaction with membranes to show that unexpectedly high membranebound peptide concentrations are likely in vivo and may, in some cases, be required for triggering bacterial death. e. e. schäfer 1 , s. schuy 2 , a. janshoff 1 1 georg august-university göttingen, germany, 2 johannes-gutenberg-university mainz, germany retrovirus entry into cells occurs through fusion of the lipid bilayers that surround the virus and the lipid bilayer of the host cell. fusion proteins, present on the surface of the virus membrane play an essential role in the early stage of virus entry. understanding of the molecular mechanism is important for the design and function of modern fusion inhibitors. in this project we analyze the fusion of active conformation of the envelope gp41 from the human and simian immunodeficiency viruses (hiv and siv). during the infection process gp41 undergoes a sequence of conformational changes where the n -terminal nhr develop a trimer pre-hairpin intermediate. afterwards the three chr fold back towards the central nhr and a six helix bundle is formed. this rearrangement forces the viral and the host membrane into close contact and fusion pores may induce membrane fusion. this decisive molecular step in retroviral fusion has been modeled by rational design of lipopeptide assemblies that mimic a coiled-coil structure serving as a receptor for potential antagonists. for this purpose, sslbs were functionalized in an in situ coupling reaction with peptides originating from the nhr (n-peptides) of siv and hiv to monitor the interactions with the specific chr peptides (c34 and t20). binding of antagonists to surface confined coiled-coil structures has been quantified by ellipsometry, quartz crystal microbalance and was visualized by atomic force microscopy. a. rupprecht 1 , e. sokolenko 2 , e. e. pohl 1 1 institute of cell biology and neurobiology, charité -universitätsmedizin, berlin, germany, 2 frumkin institute of physical chemistry and electrochemistry russian academy of sciences, moscow, russia the production of reactive oxygen species (ros) in mitochondria is very sensitive to the proton motive force and can be decreased by mild uncoupling, mediated e.g. by uncoupling proteins (ucp) 1 . in contrast, the activation of uncoupling proteins by ros as a negative feedback loop is a highly controversial hypothesis. ucp activation in mitochondria by 4-hydroxy-2-nonenal (hne, aldehydic product of lipid peroxidation) was first demonstrated by echtay et al. 2 . here we investigate the ability of hne to activate and/or to regulate the expression of ucp in two different systems: (i) in lipid membranes reconstituted with recombinant ucp and (ii) in primary neuronal cells. total bilayer conductance was enhanced in the presence of hne, but this effect was independent on ucp1 and ucp2. the results concerning the hne-mediated ucp expression after induction of ros-production and/or after exogenous addition of hne are discussed for three brain-associated proteins (ucp2, ucp4, ucp5) in view of their possible functions. 1. beck, v et al. 2007 . faseb j. 21:1137 -1144 . 2. echtay, k. s. et al. 2003 . t. rudack, j. schlitter, k. gerwert ruhr university, department of biophysics, nd 04 north, 44780 bochum, germany the gtpase ras p21 which is linked to the membrane via a lipid anchor, is a crucial switch in the cellular signal transduction processes that control cell growth and proliferation. docking simulations can be seriously hampered by the great difficulty to accurately estimate the ligand-protein binding affinity constant k, which is usually derived via the computation of the binding free energy ∆g. unfortunately, due to the involved logarithmic relationship, errors of less than 1.5 kcal/mol in the computation of ∆g result in about one order of magnitude inaccuracy on k. this can hinder computational methods from discriminating micromolar from nanomolar compounds. to improve the reliability of docking prediction, we make use of enhanced sampling methods, ranging from steered molecular dynamics 1 to metadynamics 2 . we also test several descriptors, such as the recently developed path collective variables 3 , to identify the most suitable reaction coordinates accounting for binding and unbinding processes. using these approaches we investigate ligand docking and undocking and we attempt to describe at an atomistic level the kinetics of binding, which we intend to exploit for drug design purposes. here, an example of application in drug design is reported. 1. isralewitz, b. et al.; j. mol. graph. model. 2001, 19 , 13-25. 2. laio, a. and parrinello, m.; pnas 2002, 99 , 12562-6. 3. branduardi, d.et al.; j. chem. phys. 2007, 126 , 054103. prediction of protein-protein complex structures using wang-landau simulations a. solernou, barcelona supercomputing center, jordi girona 29, barcelona, spain protein-protein interactions are essential in the majority of life processes, so they have keen interest in several knowledge areas. however, experimental data on protein complexes is being produced at a quite low pace in comparison to that of the individual components. thus, in recent years attention has focused into computational approaches to the proteinprotein docking problem. a variety of docking protocols have been recently reported, sharing usually the following strategy: fast rigid-body search of the interacting subunits, followed by scoring and refinement of the interfaces. although this kind of strategy has proven some good results in the capri blind test it has two main limitations. on one hand one should include full flexibility on the protein structures. on the other, the evaluation should be made with free energy calculations instead of using a scoring function. in this work we propose to search the native complex structure in the minimum of the free energy landscape. we use the coarse grained potential unres to get the potential energy of the possible conformations. they are generated changing the dihedral angles, the side chain rotamers, and lastly the mutual orientation using a new sampling protocol we have developed (rotation-based uniform sampling; rotbus). finally, the free energy calculations are performed using an omp parallelization of the wang-landau algorithm. s. shukla 1 , s. asthana 1 , g. giliberti 1 , f. luliano 1 , m. ceccarelli 2 , r. loddu 1 , p. ruggerone 2 , p. la colla 1 1 department of biomedical science and technology, università di cagliari, cagliari, italy, 2 department of physics, università di cagliari, cagliari, italy the virus encoded rdrp has emerged as a prime target in the search for specific hcv and other flaviviridae antivirals. recently, the determination of the hcv rdrp structure in complex with certain benzimidazoles has been reported, these nni's bind to the surface of the thumb domain, thereby disrupting its interaction with the finger domain, which is necessary for catalytic activity. on the other hand, we have found that, in bvdv, the mutations conferring resistance to same class of inhibitors lie in the finger domain of rdrp, indicating that the mode of inhibition of benzimidazole class of compounds is different in both hcv and bvdv rdrp. herein, we have applied docking approaches (binding orientation), molecular dynamics (md) simulations combined with metadynamics to elucidate the microscopic mechanism of the interactions between the ligand and the receptor in order to identify features barely seen in experiment. the recently designed algorithm overcomes the time scale problem by accelerating properly defined reaction coordinates. it mimics the real dynamics of a ligand staying or leaving the receptor and in doing so reconstructs the free energy surface, which in turn gives an idea of the residence time of the inhibitor in the cavity.finally we identified the binding modes and different mechanism of inhibition of benzimidazole class of compounds in rdrp of two closely related rna viruses. . c. seifert 1 , w. stacklies 2 , f. graeter 2 1 protein mechanics and evolution, bioquant, inf 267 bq0031, 69120 heidelberg, germany, 2 ag graeter, picb, 320 yueyang lu, shanghai 200031, china we use a new method that detects force distribution in proteins. based on molecular dynamic simulations, changes in inter-atomic forces are calculated, here caused by different ligands. these changes will then be analyzed to detect a signal transfer through a protein initiated by the binding of a ligand to the protein. chaperones are ubiquitous proteins, which help other proteins to fold into a native conformation. they are able to refold misfolded proteins with a great variety of mechanisms. in this project, our group focuses on molecular dynamic simulations of htpg, an e. coli homolog of the human hsp90 (heat shock protein 90kda). the full structure of htpg was published by shiau et al. in 2006, but the mechanism of how htpg performs its function is still not understood. the folding mechanism is an atp driven reaction cycle, in which the functional entity is a homodimer of htpg. we separate the cycle in three main states: apo, adp-and atpbound state. conventional molecular dynamics simulations are used to build a stable simulation system and provide structure trajectories and primary information about the behavior of htpg in its different states of the folding process. experiments indicate that the molecular movement is atp driven. we use force distribution analysis to elucidate how atp effects htpg conformation and dynamics. the small size of myoglobin makes it the preferred candidate to investigate the structure-function paradigm. in its interior five docking sites have been identified and for long time these xenon cavities have been classified as packing defects. recently, it was shown that they might be involved in ligands migration path. however, some questions regarding its role as oxygen carrier no scavenger remain yet open as well as the microscopic mechanism regulating these biological functions. in this work we made use of standard md simulations of solvated myoglobin to characterize internal cavities. our principal results is that we have found several secondary cavities showing volume and occurrence at least comparable to that of xenon cavities. in order to analyze and rationalize internal cavities we applied special cluster-analysis: we classified all cavities with respect to the position, size and occurrence ascribing them to different clusters. this analysis highlights possible intrinsic migration paths for small ligands within the protein matrix controlled by spontaneous fluctuations of the protein itself. moreover, we identified some key residues playing a fundamental role in controlling internal pathways. our suggestion that the secondary cavities constitute the preferred path for ligand escape is also supported by explicit metadynamics simulations of ligand escape. a. varga 1 , p. lallemand 2 , j. szabó 1 , p. závodszky 1 , m. vas 1 , t. barman 2 , l. chaloin 2 , c. lionne 2 1 institute of enzymology, brc, hungarian academy of sciences, budapest, hungary, 2 cnrs-université montpellier 1 -université montpellier 2, institut de biologie, umr5236, montpellier, france 3-phosphoglycerate kinase (pgk) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. pgk is a key enzyme in glycolysis; it catalyses the reversible reaction 1,3-bisphosphoglycerate + adp ↔ 3-phosphoglycerate + atp. here we explored the catalytic role in human pgk of the highly conserved lys 215 that has been proposed to be essential for pgk function, by a transient and equilibrium kinetic study with the active site mutant k215a. by the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type pgk and mutant k215a. by the use of a chemical sampling method (rapid-quench-flow) under multi and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild type pgk follows product formation, whereas with the mutant it is the phospho-transfer step itself that is rate limiting. these data are supported by saxs measurements which showed no direct role of lys 215 in the domain closure of the enzyme i.e. the isomerisation step of the reaction. the results are explained by the structural data of the enzyme. characterization of different recombinant nrp1 proteins and interactions with heparin k. a. uniewicz, y. ahmed, d. g. fernig school of biological sciences and centre for glycobiology, biosciences building university of liverpool, l69 7zb liverpool, u.k. neuropilin-1 (nrp1) is a mammalian membrane glycoprotein involved in tip sprouting processes like angiogenesis and neurogenesis. it has been shown that the interaction of nrp1with heparin/heparan sulfate is implicated in its enhancement of growth factor signalling, however the mechanism is not yet known. commercially available extracellular domain of nrp1 is available either as a truncated his-tagged human protein (hnrp1) or as the rat protein fused to histagged human fc and expressed as a dimer (fcrnrp1). biochemical properties such as kinetics of heparin binding and structural requirements for sugar binding together with biochemical tests of protein properties were performed in order to characterise these commercial proteins. fc rnrp1 shown high affinity to heparin (kd=2.5 nm) and required a minimum of dp10 to effectively compete with fc rnrp1 binding to immobilised heparin. competition experiments with various modified sugars show that interaction of fc rnrp1 with heparin is highly ionic and dependent on the position of sulfate groups along the heparin chain. the hnrp1 did not bind to heparin immobilised via nhs-biotin, though it did bind to some heparin affinity chromatography matrices. organic compounds of tin are among wide spread environmental pollutants. due to physical and/or chemical actions, poly-substituted alkyltins speciate into less substituted, more toxic species. tetra-or tri-alkyltins show a marked delay in their toxic action with respect to the corresponding di-and mono-alkylated analogs. it has been hypothesized that the delayed toxicity may results from the progressive dealkylation of alkyltins in more toxic di-and mono-derivatives, which bind and inhibit essential enzymes. it has been proposed that alkyltins preferentially target enzyme sulphydryl groups. previously, we showed that a nine amino acid linear peptide (i 1 lgcwcylr 9 ) containing a cxc motif is able to bind and dealkylate tri-substituted alkyltin compounds into the corresponding dialkyl derivatives. here, we investigated the time dependence of the degradation of the most common alkyltin derivatives by this peptide. we monitored the reaction kinetics using the intrinsic fluorescence of the tryptophan residue in position 5 of the peptide. we found that all of the alkyltins analyzed are progressively degraded to dialkyl derivatives, following a pseudo-enzymatic reaction mechanism. the end-point of the reactions was the formation of a covalent complex between the disubstituted alkyltin and the peptide. these data agree with the speciation profiles proposed for polysubstituted alkyltins in the environment and reveal a possible biotic degradation pathway for these toxic compounds. parkinson's disease (pd) is a multifactorial neurodegenerative condition characterized by the progressive loss of dopaminergic neurons in the substantia nigra and by the presence of intracellular inclusions, composed predominantly of fibrillar alpha-synuclein (as). post-mortem studies indicate the presence of oxidative damage in the nigral neurons. dopamine oxidation, which leads to the formation of highly reactive quinones (daq), may account for the specificity neurodegeneration observed in pd. daq have many potential protein targets for chemical modifications. among them, we focused our attention on dj-1, of which mutated forms have been found in familial cases of pd. a possible function of dj-1 is its redox-dependent chaperone activity that could prevent as aggregation and fibril formation. in the present work, we analyzed the structural and functional modification induced on dj-1 by daq. 15 n-hsqc spectra of dj-1 were recorded in the presence of different amounts of daq and chemical shift perturbations were used to identify the dj-1 residues target of daq and their relative reactivity toward daq. aggregation assays were also performed to evaluate the functional effects of the daq modifications on the chaperone activity of dj-1. rabies virus (rabv) infects neurons exclusively and causes lethal encephalitis. pathogenic rabv strains favor neuronal survival, whereas non-pathogen strains lead to neuronal apoptosis. the use of recombinant rabv showed: 1/ the g protein determined the induction of the survival or death phenotypes; 2/ the last 4 cooh amino acids of the g protein cytoplasmic domain (cytog) are critical. these residues form a binding site for pdz domain (pdz-bs). one of the 6 amino acid differences between survival and death gproteins are located in this pdz-bs. results of two-hybrid experiments showed that cytog survival interacted pdz domain of ser/thr kinases (mast), while cytog death interacted also with 3 additional pdz containing host proteins. to understand the fine structural basis for the specificity of the pdz-cytog complexes, we determined the 3d structure and the dynamics of the mast-cytog complexes by nmr. the structures, as well as the affinities and constant kinetics, of the mast2 pdz complexes with both cytog are similar. we conclude that difference by one aa in the pdz-bs of the two strains cannot drastically modify the interaction with mast2-pdz, in agreement with the two-hybrid data. preliminary results suggest that the interaction of cytog death with one additional cellular partner blurs the pro-survival signals engaging the infected cells through apoptotic trails. functional protein immobilization on glass-type surfaces s. waichman, m. bhagawati, y. podoplelova, j. piehler institute of biology, department of biophysics,university of osnabrück , barbara st.11 osnabrück, germany the immobilization of membrane proteins onto solid supports enables protein interactions and conformational changes to be probed by spectroscopic techniques under highly defined conditions. here, we present a novel method for covalent protein immobilization on glass-type surfaces using a bottom-up approach. in this approach the 4'phosphopantetheinyl group of coenzyme a (coa) was transferred to the acyl carrier protein-derived ybbr tag of the target protein by means of the phosphopantetheinyl transferase sfp. the glass-support was rendered biocompatible by coating it with an ultrathin layer of peg (polyethylene glycol), followed by functionalization with coa through maleimide chemistry. immobilization of ybbr-tagged proteins in presence of sfp was followed in real time by label-free detection using reflectance interference spectroscopy. the immobilization procedure was thus systematically optimized by means of binding specificity, enzyme activity and functionality of the immobilized protein. this approach was employed for immobilizing the type i interferon ifnα2 in order to probe ligand recognition by ifnar1 and ifnar2 and ligand-induced ternary complex formation. the versatility of this technique was further enhanced by its combination with photopatterning methods. this immobilization technique can provide a beneficial tool for bioanalytical and biophysical applications at the single molecule level. r. vijayan, p. c. biggin department of biochemistry, university of oxford, south parks road, oxford, ox1 3qu, uk ionotropic glutamate receptors mediate excitatory synaptic transmission in the brain and are heavily implicated in memory and learning as well as in numerous neuropathological conditions. one family of iglurs, the kainate receptors, show unusual sensitivity to changes in external ion species resulting in an apparent requirement for both sodium and chloride ions for activation. our recent work revealed the location and selectivity of the cation binding sites. despite this progress, it is still unclear how the cation binding sites confer sodium selectivity and how apparent affinity for chloride is influenced by the presence of cations. we have attempted to address these questions by performing extensive free energy calculations using all-atom molecular dynamics simulations. the rank order of cation binding obtained from relative binding free energy calculations is in agreement with experimental measurements of apparent affinity. these calculations also reveal that the pair of cation binding sites in the dimer interface act independently. binding free energy calculations performed using a reduced model of the binding site show that cation selectivity can been attributed to both the rigidity and high charge density of the binding sites. finally, a potential of mean force derived from umbrella sampling simulations indicate that the presence of cations stabilize the anion binding site considerably. the effect of toxins on the inorganic phosphate release during the actin filament formation a. vig, t. kupi, g. hild, m. nyitrai university of pécs, faculty of medicine, department of biophysics, szigeti str. 12, 7624 pécs, hungary actin can be found in monomeric (g-actin) and filamentous (f-actin) form in eukaryote cells under physiological circumstances. the first step of actin polymerisation is the formation of actin nuclei by atp-binding actin monomers. the next step in is the elongation when monomers are associated to the previously formed nuclei or to the ends of the growing filaments. after the association of monomers the bound atp is hydrolysed to adp.p i , and with first order kinetics the release of inorganic phosphate occures. the rate constant of the release is 0,006 s −1 , which is a slower process than the hydrolysis itself (0,02 s −1 ).phalloidin, a cyclic peptide from amanita phalloides can bind to the filaments and stabilizes their structure. jasplakinolide is another cyclic peptide from marine sponge (jaspis johnstoni) which binds actin filaments. the aim of this study was to investigate whether the binding of toxins to the newly created filaments has an effect on the kinetics of the inorganic phosphate release or not. we used absorption photometry measurements to measure the rate of phosphate release. phalloidin decreased the rate of the release substantially. although the effect of jasplakinolide was weaker, the results showed that the binding of these toxins to the actin can modify the rate of the release of inorganic phosphate from the filaments. these observations are in agreement with the molecular mechanisms by which these toxins stabilise the actin filamens. fluorescent proteins (fps) are invaluable fluorescent markers in cell biology. however, their use is often limited by photobleaching of the chromophore, notably in single-molecule, time-resolved or super-resolution imaging studies. we will present the crystallographic studies at near atomic resolution of a photo-activatable fluorescent protein irisfp that has been observed in a transient radical state en route to photobleaching. we took advantage of x-rays to populate the radical, which, under illumination with visible light, presumably forms with low probability from the triplet state. the combined x-ray diffraction and in crystallo spectroscopic data (from uv-vis and raman spectroscopies) reveal that radical formation in irisfp involves strong but reversible distortion of the chromophore, suggesting a transient loss of pi-conjugation. these results will help unravel the mechanisms of blinking and photobleaching in fps, which is of importance to rationally design variants of higher photostability. optimizing photoactivatable fluorescent proteins for live-cell imaging recently, novel fluorescent proteins (fps) have been reported which perform spectral changes in response to irradiation with light of a particular wavelength. reversibly photoactivatable proteins switch between a bright and a dim fluorescent state. this process is accompanied by photoisomerization of the protein chromophore. irreversible photoactivation results from photochemical processes within the protein, e.g., photolysis of an amino acid side chain, or an extension of the alternating π-electron system of the chromophore by a β-elimination reaction. fps have became valuable tools in live-cell imaging because they allow intracellular protein labeling by using them as fusion tag, and photoactivatable fps are powerful tools for application in novel subdiffraction imaging techniques. however, the available fps still offer potential for improvement in various ways. they frequently show a tendency to aggregate or oligomerize, incomplete chromophore maturation, fluctuating emission and low photostability. here, we will present our recent progress towards engineering the 'perfect' fp. simultaneous intracellular chloride and ph measurements using gfp-based sensor in ldcv d. arosio 3 , f. ricci 1 , l. marchetti 2 , l. albertazzi 1 , f. beltram 2 1 italian institute of technology, udr pisa, italy, 2 nest, scuola normale superiore, pisa, italy, 3 nest, infm cnr, pisa, italy chloride ion participates in many physiological functions including control of neuronal resting potential, charge balance during endosome acidification, and regulation of cell volume. as a consequence dysfunctions in regulating membrane chloride permeability lead to severe diseases including motor disorders, cystic fibrosis and epilepsy. at present processes regulating intracellular chloride ion concentrations are still widely unexplored mainly as a consequence of limiting methods to quantify chloride fluxes in living cells. in the present work a highly specific, genetically encoded sensor is developed for detecting simultaneously intracellular ph and chloride concentration. the sensor is obtained by fusion of a red fluorescent protein (dsred-monomer), insensitive to chloride and ph, to a gfp variant containing a specific chloride-binding site (gfp-chl). dsred-gfp-chl binds the chloride ion following a fluorescence static quenching mechanism, which allows measurements of intracellular ph in a chlorideindependent manner. the sensor has been successfully tested in different living cells, in a ph range 5-8 and chloride concentration up to 200 mm. for the first time, to the best of our knowledge, it allowed to measure the chloride concentration of dense core vesicles in the secretory pathway. applicability to high-throughput screening, range of validity and accuracy of time-lapse maps will be discussed. we aim to understand the basis of the photophysical changes in fluorescent proteins (fp) induced by reactive oxygen species (ros). indeed, ros might be involved in photochemistry of fp, leading to their photobleaching or photoconversion. in addition, fp may be used to investigate cellular events like phagocytosis or mitochondrial activity, where ros are naturally produced. in the latter cases, an accurate analysis of fp's fluorescence signals requires the full knowledge of reactions between ros and fp. in the future, this work may help in developing either photoresistant or photoswitchable fp and improving their use for imaging under oxidative stress conditions. using γ-radiolysis as a quantitative source of ros, we investigated the reactions of oh and o 2 − radicals on the cyan fluorescent protein (cfp) and the modifications of the cfp's photophysical properties by oh radicals were explored in detail (submitted). in order to address the corresponding chemical changes in the protein, we devised a mild proteolysis protocol that for the first time offers a peptide mass fingerprint almost covering the cfp sequence (alvarez et al. biochemistry 2009). then, we achieved the meticulous characterization of the cfp oxidation products by mass spectrometry and proposed a mechanism to account for their formation by pulse radiolysis. since the cloning of the green fluorescent protein from aequoria victoria, numerous screens have been performed to improve the brightness of this protein, its spectral variants and fluorescent proteins from other species. the improvement is evaluated by comparing fluorescence intensity of individual bacteria or colonies. in this way also expression level, folding and maturation efficiency, and thickness of the bacterial colony contribute. here we report a screening method that, in addition to fluorescence intensity, quantifies the excited state lifetime of a fluorescent protein, which is independent of intensity or expression level, and provides a direct measure for the quantum efficiency of the fluorescent protein. the novel approach was used to screen a library of cyan fluorescent protein (cfp) variants randomly mutated at position 65, 148 and 224, yielding an improved bright cyan fluorescent protein named, mposeidon, with a markedly increased fluorescence lifetime and quantum yield, increased photostability and improved fluorescence intensity in vivo. it is shown that mposeidon is the brightest cyan fluorescent protein in mammalian cells. in addition, several lifetime variants were identified that can be used for lifetime unmixing. it is demonstrated that three cfp variants can be separated and their distribution quantified in a single detection channel. a. r. faro 1 , p. carpentier 2 , d. bourgeois 3 , e. rosny 4 1 irtsv, cea, ufj, grenoble, france, 2 ibs, cea, cnrs, ufj, grenoble, france, 3 ibs, cea, cnrs, ufj, grenoble, france, 4 ibs, cnrs, ufj, grenoble, france enhanced yellow fluorescent protein (eyfp) is extensively used as a fluorescent marker. like other photo activatable fluorescence proteins (pafp), it exhibits photo-switching properties. however, the mechanism by which fluorescence can be swiched on or off upon light irradiation is not fully understood at the molecular level. the bright to dark conversion involves a protonation step and structural rearrangements of the chromophore, but it is not clear which of these two steps is the triggering event. to answer this question, we carried out photo-switching experiments at cryotemperatures. our data suggest that a photo-induced protonation step (probably in the triplet state) is the primary event in the bright to dark conversion. our results may bear relevance to other pafps, such as iris, eos, dronpa, padron or kaede. how misfolding and aggregation of proteins constitutes a toxic insult to neurons remains largely unknown. in order to obtain insight into the molecular biology of neurodegenerative disease, we have developed a number of gfp-based biosensors for the detection and quantification of cellular clearing mechanisms for aggregated proteins. the high load on these protein quality control mechanisms, and their failure to meet normal physiological demand ultimately results in a "de-compensation" condition from which the nerve cell cannot recover. our fret/flim-based bioassays visualize protein ubiquitination and degradation; proteasomal activity; foldase activity using a folding-impaired gfp mutant which gains fluorescence conditional on the upregulation of chaperone activity; chaperone binding to unfolded proteins; and autophagosome formation/lysosomal integrity via the targeted and sensitive fret-based measurement of ph changes. these sensors are employed in cellular model systems for parkinson's and alzheimer's disease, and amyotrophic lateral sclerosis (als) to delineate the molecular pathway of cellular demise, and to gain a mechanistic understanding of the toxicity of protein aggregates and the basis for the vulnerability of neurons. millisecond photo-switching dynamics of e222q gfp mutants for sensor and imaging applications m. collini 1 , v. quercioli 1 , l. d'alfonso 1 , g. baldini 1 , b. campanini 2 , s. bettati 2 , g. chirico 1 1 dipartimento di fisica, università di milano bicocca, italy, 2 dipartimento di biochimica e biologia molecolare, università di parma, parma, italy e222q mutants of the green fluorescent protein are known to possess photo-chromic properties: the anionic emission, primed by a pump 488 nm laser beam, can be switched between two levels of different brightness by irradiation with a blue, ∼ = 400 nm, probe laser light. we have studied here the amplitude and the dynamics of the brightness enhancement of the e222q mutant of gfpmut2. the fluorescence emission increases almost threefold, under saturating probe laser excitation, for pump excitation intensity in the linear regime. two characteristic activation times, estimated by means of modulated two colour fluorescence correlation spectroscopy, are detected in the 1-30 ms range, independent of solution temperature and viscosity. the brightness enhancement factor and the characteristic activation times depend markedly on the solution ph. these results indicate that this mutant can be used as a high sensitive intracellular marker for local proton concentration and for modulated excitation imaging. the advent of fluorescent proteins (fp) gave researchers the opportunity to study proteins in situ. fluorescence resonance energy transfer (fret) benefited from this. cell fixation is a commonly used approach when working with microscopy. however, we have found that fret efficiency (e) in cells transfected with cerulean and venus chimeras could not be reproducibly measured after fixation. to evaluate this problem in detail, we measured e of 3 cerulean-venus standard constructs by acceptor photobleaching fret, intensity-based ratiometric fret and flim-fret. the constructs were produced as standards (biophys j, 2006, 91, 99) with 43, 38 and 31% e values, comprising donor-acceptor separations of 5, 17 and 32 amino acids, respectively. transient transfection of the fusion plasmids was performed into hela cells and e was measured in live and pfa or methanol fixed cells. literature e values were reproduced when measuring live cells. conversely, cell fixation caused a deviation of e values. methanol fixed cells showed e between 1-5% for all the constructs. the effect of pfa fixation on both fluorescence intensity and fret varied vastly among independent experiments regardless of the measurement modality. thus, fixation should be avoided due to the effects it has on fp's fluorescence and consequently on fret efficiency. to explain the improvement in the fluorescence properties of cerulean when compared to ecfp, we have determined the x-ray crystallographic structures of these two proteins at physiological ph, and performed molecular dynamics simulations. both proteins exhibit a structural heterogeneity in the nterminal half of their seventh strand, which forms a specific set of van der waals interactions with the chromophore. the critical h148d mutation present in cerulean induces a modification of these interactions, and allows the chromophore to be more planar and better packed, albeit only intermittently. as a consequence, the probability of non-radiative decay is significantly decreased. our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins, and provide insights which should allow the design of mutants with enhanced optical properties. the fluorescence lifetime of green fluorescent protein g. jung biophysical chemistry, saarland university, 66123 saarbruecken, germany biotechnological design of the green fluorescent protein (gfp) and the discovery of other proteins boosted the development of the life sciences in the past decade. tracking protein movements and high resolution microscopy are only a few recent applications which were realized by fluorescent protein technology. among these examples, the switching between two chromophore state is exploited. our aim is to establish fluorescent proteins for bioanalytical fluorescence lifetime measurements. despite the progress in other fields, quantification with gfp still imposes practical problems [1] . in the past, we observed that the uv-light driven decarboxylation of the nearby aminoacids glu222 distorts the fluorescence lifetime of gfp [2] . we found out that this chemical reaction also occurs under excitation of the anionic chromophore state with a high quantum yield [3] . recently, we could show by time-resolved spectroscopy that, indeed, the more susceptible state for this kind of photoconversion is the anionic chromophore state [4] . suppression of this reaction therefore enables the design of autofluorescent proteins which can be used e.g. for the quantification of ions and which are beneficial as donors in fret applications. the fluorescent properties of tryptophan residues (w) in proteins are highly dependent on their immediate protein environment. however, the multi exponential decay of single w proteins is not completely understood. the most cited hypothesis contributes a multi exponential decay to the existence of several micro conformations (rotamers) of the w residue within the protein matrix. to determine rotamers we apply a method based on dead-end elimination (dee) and molecular dynamics simulations (md). low energy rotamers are calculated by dee while dynamics and further refinement is accomplished using md. the method was applied on several test cases including the protein mutant bc-csp l66e from bacillus caldolyticus, which contains a solvent exposed w residue. as resolved by x-ray crystallography, this w residue occupies two conformations. using dee and md we were able to retrieve the w conformations found in the x-ray structure. the results demonstrate the ability of the method to obtain valuable w rotamers, both for solvent shielded as exposed residues. the determined conformations were compatible with the findings based on a method using replica exchange simulations. k. nienhaus 1 , h. nar 2 , r. heilker 2 , j. wiedenmann 3 , g. u. nienhaus 4 1 inst. of biophysics, universität ulm, ulm, germany, 2 dept. of lead discovery, boehringer ingelheim, biberach/riss, germany, 3 national oceanography center, university of southampton, southampton, uk, 4 inst. of applied physics, universität karlsruhe (th), karlsruhe, germany eqfp611 is a red fluorescent protein (rfp) with the chromophore in a co-planar trans orientation, whereas the cis isomer is preferred by most other rfps. by using x-ray crystallography, we determined the structures of the dimeric variants d1eqfp611 and d2eqfp611 at high resolution (up to 1.1å). for d1eqfp611, we had previously seen a redshifted species upon irradiation with 532-nm light. concomitant changes in the raman spectrum were interpreted as evidence of a trans-cis isomerization of the chromophore. here we have combined x-ray crystallography and site-directed mutagenesis to assess whether we can create a stable fluorescent, red-shifted eqfp611 variant with a cis chromophore. in a first step, we introduced the n143s substitution. this variant, d2rfp630, is highly fluorescent, with the absorption (emission) maximum red-shifted by 24 (19) nm. with an additional s158c mutation, the chromophore is found completely in the cis form. the variant, rfp639, is highly fluorescent, with excitation and emission maxima at 588 and 639 nm. still further red shifts appear to be in reach. k. nienhaus 1 , v. adam 2 , d. bourgeois 2 , g. u. nienhaus 3 1 of biophysics, universität ulm, ulm, germany, 2 esrf, grenoble, france, 3 institute of applied physics, universität karlsruhe (th), karlsruhe, germany dendra2 is an engineered, monomeric gfp-like protein that belongs to a sub-class of fluorescent proteins undergoing irreversible photoconversion from a green-to a red-emitting state upon exposure to purple-blue light. this process occurs in the neutral state of the chromophore and is known to result from backbone cleavage accompanied by an extension of the delocalized π-electron system. we have measured the x-ray structure of green dendra2 and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. the structure, which is very similar to those reported for the closely related proteins eosfp and kaede, revealed a local structural change next to the chromophore, involving mainly arg66 and a water molecule. we propose that this structural change explains the blue shift of the absorption and emission bands, as well as the markedly higher pks of the hydroxyphenyl moiety of the chromophore. the 20-fold enhancement of the neutral species in dendra2 at physiological ph accounts for the observed higher photoconversion yield of this protein in comparison to eosfp. photochromic green fluorescent protein mutants: chromophore states unveiled by raman spectroscopy s. luin, v. voliani, g. lanza, r. bizzarri, r. nifosì, p. amat, v. tozzini, m. serresi, f. beltram nest, scuola normale superiore, cnr-infm and italian institute of technology, pisa, italy the most widespread genetically-encodable fluorescent markers used for studies in living cells and tissues belong to the green fluorescent protein (gfp) family. reversibly switchable fluorescent proteins (rsfps) were developed that can undergo repeated transitions between different states, e.g. bright and dark forms. this property makes rsfps particularly attractive as active labels in biological systems for selective photolabeling applications or subdiffraction imaging. we shall present pre-resonant raman results unveiling the photophysical mechanism underlying the observed photochromic behavior. the variation of spectral properties before and after photoconversion of chemically-synthesized isolated chromophores under different protonation and/or isomerization have been analyzed, and compared to results obtained for the case of complete folded proteins comprising the same chromophores. experimental results have been analyzed within a time-dependent density functional theory, allowing us to assign all relevant vibrational modes. these results make it possible to discriminate between the effect of cis-trans isomerization and of diverse protonation states of the chromophore in the photoproducts of these proteins. s. luin et al, j. am. chem. soc. 131, 96-103 (2009 ). r. bizzarri et al., anal. bioanal. chem. 393, 1107 -1122 . a combined study of the interaction of outer membrane proteins with cephalosporin antibiotics m. lovelle, i. barroso, m. j. feio, p. gameiro requimte , fac. ciências, universidade do porto, portugal gram-negative bacteria characteristically are protected by an outer membrane that serves as a selective permeation barrier. most of the β-lactam antibiotics appear to penetrate the outer membrane through these non-specific channels, and it becomes important to understand the possible interactions between β-lactams and the porin. fluorescence techniques have been largely used to characterize both the conformation and the dynamic behavior of large biological structures such as membranes and proteins. the fluorescence of the tryptophan residues is quenched in the presence of the different cephalosporin antibiotics. this reaction between the excited state of the fluorophores and the drug can be described as a formation of a non-fluorescent complex. the dependence of the fluorescence intensity upon quencher concentration for static quenching is proportional to the binding constant for complex formation. since β-lactam susceptibility is closely related to the presence of these non-specific porins, minimum inhibitory concentration (mic) by micro-broth dilution in microplate were used to assess the bactericidal activity of cephalosporin antibiotics upon on escherichia coli strain bl21(de3 ) and a series of bl21(de3) mutated in different outer membrane proteins. this combined study of the interactions at single molecular level and at in vivo level provides new insights for a better understanding of the antibiotic translocation. when used in combination with e.g. a cyan fluorescent protein, keima offers the unique opportunity to perform dual color fluorescence cross-correlation spectroscopy using a single laser line to excite both fluorophores. the molecular determinants of the large stokes shift of mkeima have been characterized structurally by combining x-ray crystallography with in crystallo uv-visible absorption, fluorescence and raman spectroscopy. our results reveal a ph-dependant "reverse chromophore protonation" of mkeima, driven by the key residue asp157. moreover, the chromophore protonation state is shown to be coupled with different chromophore conformations, cis conformation at ph 3.8, and mostly trans conformation at ph 8.0. these results will help unravel the mechanisms of fluorescence in fps, which is of importance to rationally design and develop new fluorescent markers. recently reversibly switchable fluorescent proteins (rsfps) have become a new branch of the green fluorescent protein (gfp) like family. the rsfps may be reversibly switched between a fluorescent and a non-fluorescent state by irradiation with light of distinct wavelengths. the key process of this switching behaviour is a light induced change of the chromophore between a cis-and a trans-isomeric state. the proteins are becoming increasingly important for diverse applications like protein tracking, sub-diffraction resolution microscopy and data storage. based on the switching mechanism, we created novel rsfps with unique and improved characteristics. padron and bs-dronpa are two of these new switchable proteins. padron features a reversed switching mechanism as compared to all other green rsfps known to date while bsdronpa exhibits a very broad absorption spectrum extending into the uv. utilizing the characteristics of both proteins, we performed multi label single detection colour microscopy as well as dual colour sub-diffraction microscopy, the latter with a resolution of 20 nm. further, we recently introduced the first monomeric rsfp exhibiting red fluorescence: using a semi rational mutagenesis approach on the non-switchable mcherry, we generated the switchable monomeric protein rscherryrev. the use of this protein in single molecule switching microscopy allowed us to record time-lapse live-cell images of the endoplasmic reticulum with sub-diffraction resolution. a spectroscopic approach to the study of chromophoric dissolved organic matter (cdom) in the sea c. santinelli, r. lavezza, l. nannicini, a. seritti cnr-ibf, via moruzzi 1, 56124 pisa, italy dissolved organic matter (dom) in the ocean represents the largest reservoir of reactive carbon on the earth. it is produce at each level of the marine food web and it represents the food for heterotrophic bacteria, which can use the different pools of dom (labile, semi-labile, refractory) with a large range of turn-over times (from minutes to millennia). dom plays a central role in the global carbon cycle and it has a high ecological significance. chromophoric dom (cdom) is the fraction of dom capable of adsorbing light at uv and visible spectral regions. it determines the underwater light availability in open and coastal regions with important implication on primary production and biological activity. it is photodegraded to co 2 , co, with a significant impact on the role of the ocean as source and/or sink of atmospheric co 2 and to highly reactive compounds, dangerous for marine organisms. in its pool "humic-like" and "protein-like" fluorophores have been individuated. cdom optical properties (absorption and fluorescence) together with doc data collected in some key regions of the mediterranean sea will be presented and discussed, in order to (i) investigate the powerful of cdom optical properties to get information on cdom "quality" (i) asses the role of dom in carbon export at depth, (iii) underline the main unresolved question on dom and cdom dynamics in the ocean, with particular emphasis to their biological lability. mechanism and applications of photo-and redox-switchable fluorescent proteins s. j. remington, j. n. henderson, x. shu, j. lohman institute of molecular biology, university of oregon, u.s.a. photoswitchable fluorescent proteins have significant advantages over conventional fluorescent labels, and in a revolutionary application, now allow cell biologists to exceed the diffraction limit in light microscopy by a factor of ten. atomic resolution crystal structures and time resolved spectroscopy of both reversible (mtfp0.7) and irreversible (pa-gfp) photoswitchable fluorescent proteins in the light and dark states explain the long term stability of either state, as well as how illumination at the appropriate wavelength causes the molecules to switch between states. the photoswitching mechanisms will be discussed in terms of photochemistry, light induced chromophore isomerization and excited state proton transfer (espt). mutagenesis of espt pathways provide insight into the nature of the ratedetermining steps in proton transfer and lead to practical applications, such as redox-sensitive gfp biosensors. k. i. willig, b. hein, s. w. hell max-planck-institute for biophysical chemistry, goettingen, germany stimulated emission depletion (sted) nanoscopy is a light microscopic technique offering a resolution far beyond the diffraction limit: the excitation beam is overlapped with a doughnut-shaped, red-shifted sted beam, which switches off the ability of the molecules to fluoresce in the outer region of the excitation spot. scanning the nanosized focal spot through the sample renders sub-diffraction images with a sub-second frame rate. we used the yellow fluorescent protein citrine to image individual structural elements of living mammalian cells: vimentin and the tubular network, structures of the cytoskeleton, were recorded with a lateral (x,y) resolution < 50 nm inside the living cell, corresponding to a 4-fold improvement over that of a confocal microscope. also, time lapse sted imaging of dendritic spines of yfppositive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. as an alternative to fluorescent proteins we used a genetically encoded protein tag which can be labelled with modified organic dyes within living samples. thus nanoscale imaging of structures in the interior of living cells greatly expands the scope of light microscopy in cell biology. pulling membrane tubes from solid-supported lipid bilayers with atomic force microscopy j. w. armond 1 , j. v. macpherson 2 , m. s. turner 1 1 department of physics, university of warwick, u.k., 2 department of chemistry, university of warwick, u.k. information on the elastic and dynamic properties of membranes is essential for a thorough understanding of biological processes such as exocytosis, endocytosis, cell division, and pore formation. we use an atomic force microscope to pull on solid-supported lipid bilayers and observe that an energy barrier must be overcome prior to the formation of membrane tubes. we have modified elastic models of lipid bilayer vesicles to describe the free energy of a planar lipid bilayer in adhesive contact with a surface. from this model we are able to extract force-distance curves for the formation of a membrane tether, including the associated energy barrier. the experimental data can thus be understood in a quantitative fashion. this work enables the atomic force microscope as a quantitative instrument for measuring membrane pulling mechanics, and in future work will allow two-dimensional mapping of elastic properties under tension. from pores to micelles -a peptide-membrane interaction study t. l. andresen, j. r. henriksen technical university of denmark, dtu nanotech, frederiksborgvej 399, b124, 4000 roskilde, denmark. email: thomas.andresen@nanotech.dtu.dk. research in the partitioning of peptides into lipid membranes has been intense for several years. along with scattering techniques and nmr, isothermal titration calorimetry (itc) has proven to be a powerful tool for thermodynamic characterization of peptide-membrane interactions. we have studied two antimicrobial peptides, mastoparan-x and melittin, and found that these peptides induce a range of structural transitions of popc and popc/popg membrane systems at different peptide-lipid ratios. we have found that itc can be used to elucidate the threshold where transitions occur, including the threshold for pore formation and micellation. this has been achieved using a thermodynamic model based on gouy-chapman theory, which provides the partitioning constant of the peptide-membrane interaction and thereby the concentration of peptide on the membrane surface. the structural changes have furthermore been visualized by cryo-tem. we have further investigated the pore formation in detail and found that the thermodynamic parameters of pore formation can be fully characterized using a system specific temperature where the enthalpy of peptide partitioning becomes zero. this allows for an exclusive study of the pore formation process. lactoferrin is a glycoprotein with two globular lobes, with of two domains each. since its discovery, the research on antimicrobial properties has been extended to peptides derived from this protein. the largest family studied so far is known as lactoferricin b, obtained from the protein by digestion with pepsin. more recently, a new family of antimicrobial peptides derived from lactoferrin was discovered by bolcher et al and named lactoferrampin (lfampin). the original sequence of lfampin contained residues 268-284 from the n1 domain of lactoferrin. we studied 3 peptides derived from lfampin obtained by extension and/or truncation at the c-or n-terminal sides, keeping the essential characteristics, in order to unravel the main structural features responsible for antimicrobial action. the peptides were tested against model membranes. the ability to adopt helical conformation was followed by cd, the perturbation of the membrane phase transition by dsc, the energetics of interaction by isothermal titration calorimetry (itc), the partition of the peptide to the membrane by trfs and the importance of charge effects assessed by zeta potential measurements. the results are discussed and compared to the antimicrobial and hemolytic activities obtained by microbiology techniques. defensins are small cysteine-rich cationic proteins or peptides found in both vertebrates and invertebrates. they can be active against bacteria, fungi and many enveloped and non-enveloped viruses; thus, being also classified as antimicrobial peptides (amps). in the present work a comparative study between psd1 (a plant defensin with antifungic properties obtained from the garden pea pisum sativum) and hnp1 (a human neutrophils defensin) was conducted, in order to shed some light on the mechanism of action at the molecular level of these defensins. large unillamelar vesicles with different lipid compositions were used for this purpose as biomembranes model systems; namely, popc/cholesterol (characteristic of the outer leaflet of vertebrates cell membranes) and popc/ergosterol (fungal) mixtures. changes on the intrinsic fluorescence of the tryptophan residues present in these peptides upon membrane binding/insertion were followed by fluorescence spectroscopy. experimental results show the affinity of both defensins for mammalian and fungal sterols. the partitioning behavior of psd1 showed a high selectivity for ergosterol rich membranes, while hnp1 has preference for cholesterol rich membranes. preliminary characterization of atomistic computer models of galactolipid bilayers k. baczynski, m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university, krakow, poland the main lipid component of thylakoid membranes are galactolipids, which constitute more than 75% of its lipid composition. the most common types of galactolipids found in thylakoid are monogalactosyldiacylglycerol(mgdg), whose headgroup consists of a single molecule of beta-d-galactose and digalactosyldiacylglycerol(dgdg), whose headgroup consists of two galactose molecules: alpha-d-galactose and beta-d-galactose linked by o-glycosidic bond majorty of thylakoid galactolipid have gamma-linolenic acid both in the sn-1 and sn-2 position. atomistic computer models of mgdg and dgdg molecules have been constructed and parametrized in the opls-aa force field. the molecules were used to construct three bilayer systems for molecular dynamics (md) simulations:(1) composed entirely of dgdg molecules,(2) composed entirely of mgdg molecules,(3) composed of a mixture of dgdg and mgdg molecules the ratio 1:2. the systems have been md simulated using the gromacs 3.3.1 package. the generated trajectories were analysed to determine a number of structural parameters among them: membrane thickness, average area per lipid, electron density profile accross the bilayer, order parameters, organisation of the bilayer/water interface as well as several dynamic parametrers like diffusion coefficients, lifetimes of conformational states, lifetimes od lipid lipid interactions. single mixed-lipid guv method reveals interaction of viper venom with lipid membranes n. m. ayvazyan, n. a. zaqaryan, n. a. ghazaryan dpt. biophysics, yerevan state university, armenia studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including bilayer lipid membranes (blms), small and large unilamellar vesicles. giant unilamellar vesicles (guvs) with a mean diameter of 30 µm have a minimum curvature and mimic cell membranes in this respect. guvs are ideal for studying lipid/lipid and lipid/protein interactions using microscopy techniques with membrane fluorescence probes. guvs were formed from the total lipid fraction from bovine brain by the electroformation method, developed by angelova and dimitrov (1987) . vipera lebetina obtusa venom was added to the sample chamber before the vesicles were formed. the membrane fluorescence probes, ans and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. ans and pyrene allows us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. the presence of viper venom in guvs media reveals a noticable decreasing of membrane fluidity compare the control, while the binding of fluorophores with guvs modified by venom lead to appearance of channel activity. it was recognized early that the vipers' venom components preferred an organized lipid substrate near the lipid's phase transition and were particularly active against micellar lipids. these studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom. the attenuated total reflection fourier transform infrared (atr-ftir) spectroscopy is ideal for highly absorbing samples such as water suspensions or even bulk water due to the small light penetration depth. it is also suited for experiments on lipid membranes in excess water. for example, we have studied the hydrogen bonding (h-bonding) between cholesterol (ch) and phosphatidylcholine (pc) or sphingomyelin (sm), which could be important for the stabilization of the cholesterol-rich lipid domains. the atr-ftir method enabled comparison of the carbonyl band shape for pc/ch samples in excess h 2 o or d 2 o, and has confirmed similar behavior for both [1] . secondly, we were able to analyze the amide ii band for sm/ch samples in excess h 2 o. the observations confirm the presence of h-bonds between ch and n-h group in sm [2] . another example is the study of the interlamellar water structure, which could influence the water-mediated phenomena in membranes. h-bonds in interlamellar water in partially hydrated lipid multibilayers are stronger with respect to bulk water. in contrast, we show by atr-ftir spectroscopy that the h-bonds are weaker in multibilayers in excess water [3] . the bactericidal activity of antimicrobial peptides is linked to their ability to perturb the permeability of bacterial cells. they often show α-helical conformation, and many peptides have a kink in the middle of this structure, caused by pro or gly. in order to understand the role of the kink we designed various analogues of p5, in which the central pro residue was moved from its central position, or removed altogether (in analogue p5f). displacement of the pro residue caused a decrease of the antimicrobial activity, and an increase in the toxicity against erythrocytes, with the less active and more toxic peptide being p5f. fluorescence studies suggest that both p5 and p5f bind on the membrane surface. fluorescence experiments show that the activities of the two analogues correlate with their affinity for different kinds of lipid bilayers: the kinked p5 peptide has a dramatically higher affinity for negatively charged vesicles (mimicking the composition of bacterial membranes) than for neutral liposomes (similar to mammalian cells), while analogue p5f exhibits comparable affinities for both membranes. therefore, our data suggest that the central pro-induced kink is involved in selectivity, inhibiting peptide binding to the membranes of eukaryotic cells. d. behn 1 , h. hoffmeister 2 , r. witzgall 2 , c. steinem 1 1 institute for organic and biomolecular chemistry, university of göttingen, germany, 2 institute for molecular and cellular anatomy, university of regensburg, germany polycystin-2, encoded by pkd2, is an integral membrane protein with a size of 110 kda and 968 amino acids. the protein, which is known to be a ca 2+ permeable, non selective cation channel, interacts with several proteins such as polycystin-1, pigea14 or pacs-1/-2 etc. the interaction takes place through a proposed coiled-coil domain of polycystin-2 located at the c-terminus of the protein.if mutation of either pkd2 or pkd1 (gene product of polycystin-1) occurs, the interaction between the proteins is disturbed leading to increased formation of renal cysts. this autosomal polycystic kidney disease (adpkd) is one of the most common genetic diseases causing renal failure due to the enormous cyst formation. in this study, the interaction of the c-terminus of polycystin-2 with its postulated specific interaction partners has been investigated in terms of its biological relevance. binding affinities as well as kinetic parameters of the interaction were determined. in particular, the interaction of c-polycystin-1 and pigea14 immobilized on solid supported membranes with c-polycystin-2 has been quantified by means of the quartz crystal microbalance (qcm) method. a dissociation constant of about 100 nm was obtained. the results are compared with those obtained by surface plasmon resonance (spr) technique using a different immobilization strategy. correlation of the lateral structure of lipid bilayers and monolayers using two photon excitation fluorescence microscopy l. a. bagatolli membrane biophysics and biophotonics group/memphys -center for biomembrane physics, bmb, university of southern denmark most of the reported fluorescence microscopy applications on langmuir lipid films are focused in obtaining fluorescence intensity images using particular fluorescence probes. in this type of experiments the probes are generally utilized to obtain "contrast" between membrane regions (lipid domains) displaying dissimilar physical properties. the obtained information largely depends on the partition of the fluorescent probes for the lipid domains and the obtained fluorescence intensity pictures are not providing any information about the local physical properties of the lipid film. local physical properties of these distinct regions can be determined by exploring fluorescent probe related parameters such lifetime, emission shift, polarization. however these fluorescent probe's properties are almost unexplored in this type of experiments. this presentation will focus in describing a new experimental setup that includes a specially designed film balance on top of a custom built multiphoton excitation fluorescence microscope. this setup allows obtaining laur-dan gp images (1) many studies on phase separations of lipids in bilayers and their leaflet asymmetry make use of fluorescence techniques. we used a dithionite assay, steady-state fluorescence anisotropy and fluorescence resonance energy transfer (fret) for characterization. dithionite assay was performed to measure the fluorophore distribution in the inner and outer leaflets of symmetric membranes. for low concentrations, the fluorophore is distributed almost homogeneously, whereas for concentrations > 0.5 mol % it accumulates in the outer leaflet. we discuss these effects taking into account the presence of multilamellar liposomes and dynamic effects produced by higher local bending elasticities. the results point out possible artifacts in the use of fluorophores to characterize bilayers under the assumption of their homogeneous distribution. dithionite relative bilayer permeability is discussed. the results won by fret and steady-state fluorescence anisotropy regarding lipid phase transitions are in good coincidence to each other and to results reported in the literature. the photosensitizing properties of three chlorins are compared in solution and when incorporated in dioleoylphosphatidylcholine vesicules. in solution, they possess a similar efficacy to generate singlet oxygen and a similar ability to induce the peroxidation. however, the role of the photosensitizer localization within the lipidic bilayer is strongly highlighted, when chlorins are incorporated in liposomes, both by the changes in order of peroxidation efficacy but by the measurements of the photoinduced permeation of the liposomes. the results are discussed in relation with the technology of photochemical internalization, pci. then, using giant unilamellar vesicles, we asymmetrically oxidize the membranes. we observed different shape transitions, such as budding, typical of membrane curvature modifications. the asymmetry of the shape transitions are in accordance to a lowered effective spontaneous curvature of the leaflet targeted. this effect is interpreted as a decreased preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidation. permeabilization of guv by photooxidation is interpret as the opening of a pore above a critical membrane tension due to the budding of vesicles. the effective spontaneous curvature of photosensitized vesicle at lysis was estimated. additionally photooxidation was shown to be fusogenic. influence of polyphenol extracts from fruits on biological and model membranes d. bonarska-kujawa, h. pruchnik, j. sarapuk, j. oszmiański, h. kleszczyńska univ. of environmental and life sciences, wroc law, poland biological activity of polyphenol extracts from apple, strawberry and chokeberry was studied. polyphenols were shown to be good antioxidants and to act as antyhemolytic agents. both the activities are the result of polyphenols incorporation into biological membranes. to check potential changes they induce in membranes some experiments were performed with the use of erythrocytes, and lipid membranes. it was found that the extracts studied induced shape transitions of erythrocytes. they were classified according to the bessis-brecher scale. strawberry extract induced mainly discocytes and discoechinocytes. populations of discocytes, echinocytes and some discoechinocytes were found on applying apple extract, while chokeberry induced mainly the formation of echinocytes and spheroechinocytes. the results evidence that the polyphenols incorporate into the external monolayer of lipid bilayer of the erythrocyte membrane. the results of the fluorimetric experiments showed that all the extracts changed fluidity in the hydrophilic part of rbc membrane. the changes observed were biggest for chokeberry extract and smallest for strawberry one. incorporation was also followed by changes in electrical resistance of black lipid membranes and in shifting the temperatures of main transition (t m ) and pretransition (t p ) in liposomes. this work was sponsored by ministry of science and education, scientific project no. n n305 337034. a. boll 1 , n. czudnochowski 2 , m. geyer 2 , c. steinem 1 1 institue of organic and biomolecular chemistry, university of göttingen, germany, 2 mpi for molecular physiology, dortmund, germany hiv-1 tat belongs to the accessory proteins of hiv and has regulatory functions. tat is concentrated in the nucleus and nucleolus of infected cells. the protein is composed of 86 amino acids with a molecular weight of 11 kda. tat is a transcriptional activator protein, which stimulates rna polymerase ii-mediated transcription elongation. therefore, tat interacts with cyclin t1 and binds to the tar rna element. tat has different domains. with respect to the interaction with lipid membranes, the most important structural motif is its basic region, including 6 arginine and 2 lysine residues. the peptide derived from this basic region belongs to the cell-penetrating peptides and is able to translocate through lipid membranes. the major aim of this study is to investigate the influence of full length hiv-1 tat on artificial lipid membranes. as a starting point solid-supported membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (popc) immobilized on silicon dioxide were used. the influence of the lipid head groups on the interaction with tat was analysed by using membranes composed of the negatively charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (pops) in a mixture with popc. fluorescently labelled tat was used to localise the protein in the membrane. the impact of tat on lipid membranes was investigated by fluorescence and atomic force microscopy, showing that it is capable of perturbing lipid membranes. g. bocchinfuso 1 , a. palleschi 1 , b. orioni 1 , g. grande 1 , f. formaggio 2 , c. toniolo 2 , y. park 3 , k. s. hahm 3 , l. stella 1 1 univ. of rome tor vergata, dept. of chemistry, italy, 2 dept. of chemistry, univ. of padova and cnr, italy, 3 chosun univ., rcpm, south korea. most antimicrobial peptides exert their activity by perturbing the permeability of bacterial membranes, but the molecular details of this process are still debated. here, we compare fluorescence experiments and molecular dynamics simulations regarding the interaction of two antimicrobial peptides (pmap-23 and trichogin ga iv) with lipid membranes, showing that their mechanisms of bilayer perturbation are significantly different. pmap-23, a cationic peptide member of the cathelicidin family, associates to the membrane close to its surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. on the other hand, trichogin ga iv, a neutral peptide member of the peptaibol family, undergoes a transition from a surface-bound state to an inserted orientation. in the first arrangement it does not cause any strong membrane perturbation, while in the second orientation it may span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane. lipopolysaccharide (lps) is the main component of the outer membrane of gram negative bacteria. lps is also known as endotoxin because of its potency to induce sepsis, a serious source of mortality in many clinical cases. among lps-neutralizing agents, polymyxin b (pxb) is considered as the "gold standard" due to its high efficiency of binding and detoxification of endotoxin. in this work, we have further explored the interaction of pxb to lps from the rough mutant of salmonella minnesota (re-lps) both in the gel and in the liquid crystalline phase, using isothermal titration calorimetry (itc) and fluorescence based techniques. the effect of pxb binding on lps-membrane integrity was determined with a fluorescence quenching assay treating vesicles of lps labeled with nbd-pe with dithionite. thermodynamic parameters associated with the binding process as well as binding stoichiometry were obtained from itc experiments. finally, itc was conducted with enterococcus faecalis and salmonella typhimurium, as representative examples of a gram negative and a gram positive bacterium respectively. pressure jumps -an accessible trigger for biomolecular transformations high pressure can be used to induce many structural changes in biological systems: from triggering phase changes in model membranes to causing protein unfolding, in fact any change involving a volume reduction is promoted by pressure. as well as being broadly applicable, pressure changes can be applied very quickly both up and down in contrast to other structure change triggers such as temperature jumps. fast pressure jumps allow the trigger to be decoupled from structural changes, so with fast structure probe techniques such as time resolved x-ray diffraction, the out-of-equilibrium evolution of these systems can be monitored. despite great advantages, high pressure remains under-utilised primarily due to its technical difficulty. in response to this technology vacuum a high pressure user facility based around a pressure jump cell for small and wide angle x-ray diffraction has been commissioned at beamline i22, diamond light source, uk and will be freely available to the user community. the cell is highly robust requiring virtually no user intervention during an experiment and the pressure system is computer controlled with a graphical user interface and is integrated with the beamline. pressures between 0 and 0.5 gpa are accessible and jumps can be carried out in approximately 5 ms at temperatures from -10 to 120 • c. sample changing has been made simple and fast with a dedicated sample loading port and modular sample holders allow optimisation for a broad range of samples. the transformation of vesicle and lateral distribution of mobile membrane inclusions b. bozic institute of biophysics, faculty of medicine, university of ljubljana, lipičeva 2, si-1000 ljubljana, slovenia membrane inclusions such as membrane embedded peptides or proteins exhibit curvature dependent interaction with the surrounding lipid matrix due to the mismatch between their intrinsic curvature and the local membrane curvature. this interaction causes an inhomogeneous lateral distribution of the inclusions and a corresponding adjustment of the vesicle shape. by taking into consideration that the membrane free energy includes elastic energy of the lipid bilayer and a contribution due to an inclusion-membrane interaction, the configurations of lipid vesicles with mobile inclusions have been studied theoretically. the variational problem to calculate equilibrium vesicle shapes is solved by applying a ritz method based on an expansion in spherical harmonics. in general, vesicle shapes adjust to the presence of inclusions by increasing regions with favorable curvature and decreasing regions of unfavorable curvature in such a way that the lateral distribution of inclusions becomes inhomogeneous. if the number of inclusions or the inclusion-membrane interaction exceeds a certain value, the prolate shapes become globally stable. investigating the structure of pores formed by antimicrobial peptides using epr spectroscopy m. bortolus 1 , k.-s. hahm 2 , a. l. maniero 1 1 universita' di padova, padova, italy, 2 chosun university, kwangju, south korea spin label electron paramagnetic resonance (epr) is a spectroscopical technique effective to study the molecular mobility of membrane components and the membranepeptide interactions, as the timescale of epr is optimally matched to the rotational motions of lipids in membranes. we applied epr to study the pore-forming mechanism of two antimicrobial peptides (amp) that create pores of different dimensions when interacting with liposomes. hp (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) is derived from the n-terminus of helicobacter pylori ribosomal protein l1, and hpa3 is an hp (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) analogue where gln and asp at positions 17 and 19 were substituted by trp. we studied the interaction of the two amp with zwitterionic and negatively charged vesicles, doped with phospholipids spin labelled in the lipid head or at the c5 or c10 positions; the different phospholipids allow us to study the peptide-membrane interaction at different depths relative to the membrane surface. we studied the interaction of the amp with vesicles following the influence of amp on label mobility as a function of temperature and membrane depth. we also prepared spin-labelled dmpc/dhpc bicelles, doped with lanthanide ions (dy 3+ /tm 3+ ) that allow us to macroscopically orient the system using the magnetic field of the epr spectrometer. we studied the interaction of amp with the oriented bicellar system monitoring the effect of amp on the order parameter of the phase. a. chattopadhyay centre for cellular and molecular biology, hyderabad, india we addressed the organization and dynamics of the human serotonin 1a receptor fused to enhanced yellow fluorescent protein (serotonin 1a r-eyfp) expressed in cho cells. serotonin 1a receptors are prototypical members of the gprotein coupled receptor superfamily and represent a prime target for therapeutic actions of several anxiolytic and antidepressant drugs. interestingly, we observed significant retention in fluorescence of serotonin 1a receptors upon triton x-100 treatment of intact cells at low temperature demonstrating their detergent insolubility. we analyzed the role of cholesterol in the plasma membrane organization of the serotonin 1a receptor by fluorescence recovery after photobleaching (frap) measurements with varying bleach spot sizes. our results show that lateral diffusion parameters of serotonin 1a receptors are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin 1a receptors in the plasma membrane. interestingly, results from frap measurements performed under conditions of mild cytoskeletal destabilization suggest that receptor signaling is correlated with receptor mobility, in agreement with the 'mobile receptor hypothesis'. our current work is focused on exploring the oligomerization of the receptor using photobleaching anisotropy measurements and indicates the presence of constitutive oligomers of the serotonin 1a receptor in live cells. expression and reconstitution of connexin43 in pore-suspending membranes c. carnarius 1 , s. kaufmann 2 , m. tanaka 2 , c. steinem 1 1 institute of organic and biomolecular chemistry, university of göttingen, tammannstrasse 2, 37077 göttingen, germany, 2 institute of physical chemistry, university of heidelberg, im neuenheimer feld 253, heidelberg, germany the intercellular communication and electronic coupling between adjacent cells of vertebrates are mediated by gap junctions. these proteins are composed of two connexon hemichannels, whereas each connexon consists of six connexin subunits. each subunit is characterized by two conserved motives: two extracellular loops and four transmembrane α-helices. in this study, we focused on cx43. to obtain large amounts of this protein, we expressed cx43 and cx43+gfp in a rather new expression system: dictyostelium discoideum. in contrast to human tissue cultures, the system allows for high cell densities up to 30 million cells per ml and the cells can be cultivated by fermentation. cx43+gfp was successfully visualized in d. discoideum by confocal laser scanning microscopy, where it was preferentially found in the plasma membrane.after the cells were harvested, plasma membranes were prepared and both proteins (cx43 and cx43+gfp) were verified by western blot analysis. the proteins were solubilized by addition of 5% n-octyl-β-d-glucopyranoside and purified by ion metal chelate affinity chromatography. the activities of both proteins were confirmed by a cytochrome c assay. after the purification of both proteins, they were reconstituted in µm-sized pore-suspending membranes. in the near future, we plan to determine the mobility of cx43 and cx43+gfp in these membranes by fluorescence recovery after photobleaching. o. cañadas, c. casals dpt. biochemistry and molecular biology i, and ciber enfermedades respiratorias, complutense university of madrid, madrid, spain inhaled bacterial lipopolysaccharide (lps) may incorporate into the lung surfactant monolayer. in this study, the effect of smooth lps (s-lps) on the surface activity of lung surfactant was evaluated. to that end we investigated the behavior of dppc films containing s-lps, with and without surfactant protein a (sp-a) in the subphase, using epifluorescence microscopy combined with a surface balance. our data show that s-lps injected into the subphase incorporated into dppc films forming mixed dppc/s-lps monolayers. cospread s-lps fluidized the dppc monolayer as demonstrated by epifluorescence images and changes in the compressibility modulus of the monolayer as a function of s-lps molar fraction (x s−lps ). the presence of low amounts of s-lps in the monolayer promoted early collapse, preventing high surface pressures to be reached. moreover, s-lps hampered the re-spreading of dppc molecules during dynamic compression at s-lps concentrations as small as x s−lps = 0.02. such inhibitory effects could not be relieved by repeated compression-expansion cycles or by adding surfactant protein a. however, sp-a facilitated the squeeze-out of s-lps from dppc/s-lps mixed monolayers, suggesting that sp-a is an s-lps scavenger. cholesterol displaces ceramide from its tight packing with sphingomyelin in the absence of ld phase j. v. busto, j. sot, j. requejo-isidro, f. m. goñi, a. alonso unidad de biofísica (csic-upv/ehu) and departamento de bioquímica, u. país vasco (upv/ehu), spain a set of biophysical approaches have been applied to study the phase behaviour of palmitoylsphingomyelin (psm)/cholesterol (chol) model membranes upon palmitoylceramide (pcer) addition. fluorescence spectroscopy of di-4-aneppdhq-stained psm/chol vesicles reveals no segregation of large liquid-ordered (l o ) microdomains. in contrast, the formation of disperse, compositionally homogeneous l o psm/chol (3:1) nanodomains over a psm gel (l β ) phase is proposed. dsc measurements show that pcer addition to vesicles with coexisting l o and l β phases (low [chol] ) induces the formation of large gel-like psm/pcer microdomains, coexisting with a l o psm/chol phase. the ∆h for the psm/pcer phase at high psm/(chol+pcer) ratio is close to that of the binary mixture in the absence of chol, supporting immiscibility, but no displacement, between chol and pcer-rich phases. on the contrary, both confocal microscopy of guvs and the dsc data coincide in showing that a rise in pcer increases the gel-like phase to a lower extent than in the pure psm/pcer mixture, revealing some cholinduced restriction. in the presence of a pure l o psm/chol phase (high [chol] ), pcer addition is unable to induce the formation of large psm/pcer microdomains. the present data support the role of chol as the key determinant in controlling its own displacement from l o domains by ceramide. hydroquinones modifying lipid membrane morphology c. di vitta 1 , l. marzorati 1 , v. rebbin 2 , s. s. funari 2 1 iqusp, university of são paulo, são paulo, brasil, 2 hasylab, hamburg, germany quinones are important molecules in cells. flavina, ubiquinone and coenzyme q, coq, are associated with electron transfer. coq, having a hydrophobic tail, is soluble in the internal lipid membrane of mitochondria. their reduction leads to the equivalent hydroquinones. we synthesized novel alkylthioquinones aths, to investigate their interaction with phospholipids. one or more long hydrophobic chains attached to the quinone ring alter their hydrophobicity and electron availability. we aimed at their influence on the structure of membranes. different phs highlights the charge/polarity effect on the interaction and on the morphology of the bilayer. for that, pope and popc, lipids with different charges, but identical chains were selected. x-rays diffraction on pope/2,6bath, 100/1 at different temperatures and ph, showed cubic phases coexisting with the usual phases formed by simply hydrated pope, at different phs. for investigation of the charge/polarity, we turned to the popc/2,6bath system (smaller charge/polarity). despite decrease in temperature of phase transition and dimensions of the lattice, the structures were the same as in hydrated lipid, illustrating a less significant effect than on the similar lipid pope. another additive, 2,5ath, mixed with pope showed the effect of multiple thioalky chains. no morphology change was seen, compared to pure lipid. interestingly, despite both additives differ by one thioalkyl chain only, their influence on the pope matrix is so different. cross-linking of phospholipid membranes by calcium-sensitive synaptotagmins synaptotagmins are vesicular proteins implicated in many membrane trafficking events. they are highly conserved in evolution and the mammalian family contains 16 isoforms. we now show that the tandem c2 domains of several calcium-sensitive synaptotagmin isoforms tested, including drosophila synaptotagmin, rapidly cross-link phospholipid membranes. in contrast to the tandem structure, individual c2 domains failed to trigger membrane cross-linking in several novel assays. large-scale liposomal aggregation driven by tandem c2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic lightscattering and both confocal and negative stain electron microscopy. high-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem c2 domains results in a constant distance of approximately 9 nm between the apposed membranes. our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem c2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. membrane proteins can be challenging samples to work with and as such often require the use of multiple techniques. here we present an insight into the structure of the antimicrobial peptide melittin in lipid membranes using various techniques. we explore the conformational changes of membrane-bound melittin and its interaction with model membrane lipid systems with a series of spectroscopic methods that can be used in parallel. the techniques used include linear dichroism and ft-ir for orientation information and circular dichroism for conformation information. we use dynamic light scattering for molecular sizing, fluorescence emission for information on peptide environment, thin-layer chromatography for lipid identification, analytical ultracentrifugation to identify oligomerisation state and calorimetry to investigate the thermodynamics. we observe how the physical properties of both the peptide and the membranes affect the insertion kinetics of the peptide in the membrane. phospholipid membranes dynamics: molecular dynamics vs neutron scattering v. conti nibali 1 , m. tarek 2 , u. wanderlingh 1 , g. d'angelo 1 1 department of physics, faculty of science, university of messina, italy, 2 unité mixte de recherche cnrs/uhp 7565, université henri poincaré, nancy, france collective dynamics and single-particle dynamics of hydrated multilamellar phospholipid bilayers (1,2-dimyristoylsn-glycero-3-phosphatidylcholine, dmpc) have been studied by means of all-atom molecular dynamics simulations. here we report results of a gel phase bilayer at 283 k and of a liquid crystal phase bilayer at 303 k. coherent and incoherent dynamic structure factors and meansquare displacements have been calculated from the trajectories for both the inplane and out-of-plane lipid dynamics. moreover, the results have been compared to recent quasi-elastic and inelastic neutron scattering data. optical tweezers allow trapping of particles of different types in a wide range of sizes [1] . among these, unilamellar vesicles are of interest as they are known to be effective vectors in drug delivery and they are also studied as models for cell membranes. this work focuses on the interactions between optically confined unilamellar vesicles and their cross-linking by proteins [2] . synaptotagmins are vesicular proteins implicated in many membrane trafficking events having an accelerating effect on the membrane fusion. calcium-sensitive synaptotagmins are thought to confer calcium sensitivity to the fusion of secretory vesicles with target membranes [3] . the novel optical assay reported here allowed us to visualize the cross-linking of 600 nm liposomes mediated by synaptotagmin and calcium. the use of the optical tweezers approach to investigate the function of other fusogenic proteins related to exocytosis (i.e., snare proteins) is discussed as well. antimicrobial peptides (amps) have received increasing interest as the search for new potential antibiotics has become imperative due to increasing bacterial multiresistance. acylating the natural occurring polymyxin b (pmb) significantly enhances antibacterial activity towards two representative gram-negative bacteria e. coli and k. pneumonia compared to the nonacylated pmb. the aim of the presented work was to study various biophysical parameters, such as partitioning coefficients, zeta potentials and effective charges of a selected amp, mastoparan-x (mpx) and a propanoylated (pampx) and octanoylated (oampx) analogue, respectively. fluorescence spectroscopy, isothermal titration calorimetry and ζ-potential measurements were the main techniques used for the measurements. we employed 2 different luv model systems; a partially charged (popg/popc 1:3) and a neutral system (popc). for the neutral luvs there was an increase in partitioning with increasing length of the acyl chain, whereas partioning into the partially charged luvs was governed by a balance of hydrophobic and electrostatic contributions. the selectivity for the partially charged luvs over the neutral luvs was in the order pampx, mpx and oampx. the modeled effective charge for the peptide followed the same trend as the partitioning coefficient for the three peptides for the respective luv systems. does the lysosomal membrane need triglycerides? a spectroscopic study of a simple model membrane l. duelund 1 , k. pakkanen 2 , m. vuento 2 , j. h. ipsen 1 1 memphys -center for biomembrane physics, university of southern denmark, odense, denmark, 2 nanoscience center, university of jyväskylä, jyväskylä, finland lysosomes are intracellular organelles in which proteins and other macromolecules are degraded. morphological and functional changes in different compartments of the endocytic pathway are connected to several diseases. a crucial step in understanding biogenesis of lysosomes and their role in disease conditions, is to characterise the properties of the lysosomal membrane. by the use of tlc we have found that lysosomes contain non-neglible amounts of triglycerides (tg). to investigate how the presence of tgs could influence the lysosomal membrane, we have investigated the properties of a mixture of popc and triolein, as a simple model for the lysosomal membrane. we found the system to form two types of popc-rich membranes. these were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. by using epr and fluorescence spectroscopy the physical properties, including order, fluidity and water penetration, of these phases were found to differ markedly despite of their almost identical composition. the results suggest that presence of tgs on lysosomal membranes could have a crucial effect in the barrier functions and thus, the integrity of the organelle. model membranes with the capacity to align in magnetic fields such as bicelles and magnetically sensitive vesicles are of high interest, since their macroscopically alignment allows for the determination of structure, dynamics and topology of molecules within the membrane [1] . we performed 31 p solid state nmr on a magnetically sensitive lipid possessing a large positive magnetic anisotropy introduced in form of a biphenyl unit during lipid synthesis in one of its acyl chains (1-tetradecanoyl-2-(4-(4biphenyl) butanoyl)-snglycero-3-phosphocholine (tbbpc)). the phosphorus lineshapes of tbbpc mlvs studied at various magnetic fields (7.1t, 9.4t, 11.7t, 16.4t), revealed a drastic change in shape upon exposure to fields > 9.4 tesla: resonances resulting from phospholipid molecules oriented perpendicular to the magnetic field decrease whereas resonances resulting from parallel oriented molecules increase. this is a sign for magnetically induced vesicle deformation from vesicle to oblate ellipsoid shape. factors influencing this drastic deformation, such as magnetic anisotropy, membrane elasticity, lipid chain length and field dependency are discussed based on existing theories [2] . hepatitis g virus (gbv-c/hgv) is an enveloped viruses belonging to the flaviviridae family. clinical findings have suggested that in people co-infected with gbv-c/hgv and human immunodeficiency virus (hiv), delayed progression of aids has been observed (1). the mechanism by which this virus may inhibit the progression of aids remain to be elucidated. enveloped viruses acquire their lipid membranes by budding through host cellular membranes (2) , in this process; fusion peptides play an important role. study of the interaction of hgv-c peptides with lipid membranes could lead us useful information about the mechanism that takes place. in this work we present a study on the effects of e1 peptides on the fluidity of and polarizability of model membranes (dmpc and dppc liposomes) by dsc and fluorescence polarization. both techniques showed that the presence of the studied sequences in phospholipid mixtures already affected the thermotropic properties of the gel to liquid-crystalline phase transition. systems were thermodynamically characsmall angle neutron scattering (sans) studies have been performed to study the structural changes induced in membranes of vesicles prepared from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations of the anti-fungal drug, amphotericin b (amb).the vesicles, sonicated to a mean size∼100nm, were prepared using dimyristoylphosphatidylcholine(dmpc) or dmpc-cholesterol or dmpc-ergosterol mixtures -with both of the mixed systems involving 30mol% sterol. analysis of the sans data show that when the concentration of amb added is just above the drug's cmc (∼1µm) there is an increase in the membrane thickness of both the dmpc-chol and dmpc-erg vesicles (both cases + 4å), but the thickness of the pure dmpc vesicle membranes remains the same as in the absence of amb. when amb is added at a concentration in excess of its cmc (∼10µm), the mixed-sterol vesicles show the same changes in membrane thickness as observed with the lower amb concentration, and the pure dmpc vesicles again remain unaffected. on the basis of these studies, therefore, there appears to be no difference in the structural changes induced by the insertion of amb into the model fungal cell membranes (mimicked by dmpc-erg vesicles and those resulting from its insertion into the model mammalian cell membranes (mimicked by dmpc-chol vesicles). the third transmembrane helix of bacteriorhodopsin, also known as phlip, is a unique model system for studying the interactions of a natural transmembrane domain with lipid membranes: depending on ph, the water-soluble peptide either adsorbs superficially or inserts as a transmembrane helix on addition of lipid vesicles [1] . published values for the free energies of these processes were based on a stoichiometric model invoking two distinct sets of binding sites [2] . however, discrepancies between data obtained from different experimental techniques and inconsistencies between experimental and expected temperature dependencies suggest that these values should be taken with caution. we therefore reassessed membrane interactions of phlip using titration calorimetry and fluorescence spectroscopy. if electrostatic effects at the membrane surface are taken into account, the data can be described quantitatively by a partition equilibrium, but not by a stoichiometric binding model. the thermodynamics of membrane partitioning differ substantially from those determined previously [2] and draw a different picture of peptide-lipid interactions. beyond deepening our insights into the first step of the two-stage model of membrane protein folding, this also sheds light on the ability of phlip to drag cargo molecules across lipid membranes. adsorption of monolysine and polylysines at the surface of lipid membranes of varied composition was studied by two methods. both methods -the electrokinetic one, measured the surface potential of liposomes, and the intramembranouse field compensation sensitive to boundary potential of planar bilayer lipid membranes -detected the positive changes of the potentials for membranes with negatively charged component (cardiolipin, cl). neither monolysine, nor polylysines adsorbed at neutral membranes (phosphatidylcholine, pc). pentalysine show the difference between these potentials for the membranes composed from cl/pc mixtures. this difference is attributed to dipole part of boundary potential and indicates the changes in lipid packing. polylysines show high affinity to the membrane and saturation with plateau. these saturation levels correspond to surface charge densities 0.005 and 0.016 c/m 2 for oligomers with 5 and 12 units, and 0.032 c/m 2 for polymers with 130 and 1435 units. these values do not depend on the ionic strength of background electrolyte but proportional to the content of negatively charged components in the lipid bilayer. the polylysine layer at the mica surface was studied by atomic-force microscope (afm) technique. it was shown that pentalysine molecules cover the surface by the layer of 0.8 nm thickness and polylysines of high molecular weight by the layer up to 4 nm. effects of lysolipids on the mechanical stability of lipid membranes j. r. henriksen 1 , l. feldborg 1 , j. h. ipsen 2 , t. l. andresen 1 1 technical university of denmark, dtu nanotech, frederiksborgvej 399, b124, 4000 roskilde, denmark., 2 university of southern denmark, department of physics and chemistry, memphys center, campusvej 55, 5230 odense m, denmark lysolipids (lpcs) and fatty acids (fas) are the natural products formed by the hydrolysis of phospholipids. lpcs and fas form micelles in solution and thus act as detergents in the presence of lipid membranes. in the present study we investigate the detergent strength of a homologous series of lysolipids (lpcs) on popc lipid membranes by use of isothermal titration calorimetry (itc) and vesicle fluctuation analysis (vfa). the membrane partition coefficient (k) and critical micelle concentration (cmc) are determined and found to obey an inverse proportionality relation (cmc x k ∼ constant). the partition coefficient and critical micelle concentration are used for the analysis of lpc's effect on the membrane bending rigidity. the dependence of the bending rigidity on the lpc membrane coverage has been analyzed in terms a phenomenological model based on continuum elastic theory which yield information about the curvature inducing properties of the lpc molecule. the results reveal: (i) an increase in the partition coefficient with lpc acyl-chain length and (ii) the degree in acyl chain mismatch between lpc and popc determines the magnitude of the membrane mechanical perturbation per lpc molecule on the membrane. patch clamp electrophysiology remains the gold-standard for ion channel research because of the information richness of the data produced. automated planar patch clamp devices, with their higher throughput and high data information content, have made the technique accessible to a wider audience. nanion technologies offers two planar patch clamp workstations combining higher throughput with high data quality. the port-a-patch records from a single cell at a time and the patchliner from up to 8 cells simultaneously with high success rates (typically 60-80%). both, the port-a-patch and the patchliner are bench top patch clamp rigs which uses a planar borosilicate glass chip for obtaining a giga-seal for electrophysiological recordings. suction applied from the underside of the chip is used to attract a single cell to the recording site without the need for optical visualization. both workstations have been successfully used for whole cell, perforated patch, and cell attached recordings as well as for guv-bilayer recordings. due to the versatility of nanion`s products it is possible to study a wide variety of ion channels including nav, kv, cav, ligandgated, herg or reconstituted proteins such as ksca, cx26, ompc. special features unique to nanion products, including but not limited to internal solution exchange and temperature control, expand the experimental possibilities. waves on lipid monolayers j. griesbauer, s. boessinger, a. wixforth, s. f. matthias univiversity of augsburg, experimental physics i, biological physics group, augsburg, germany " for the sake of illustration we shall try to provide a physical basis for the equations, but must emphasize that the interpretation given is unlikely to provide a correct picture of the membrane." [ hodgkin&huxley, 1952 ] to explain the occurrence of reversible heat production during nerve pulse propagation it has been suggested by multiple authors [wilke,kaufmann,heimburg] that travelling sound waves and not ion channels may offer a better explanation than the hodgkin&huxley theory. therefore, if sound waves are an essential feature of the nerve membrane they should also appear on lipid monolayers where ion conductivity is evidently absent. here we demonstrate that sound waves can be excited on a lipid monolayer by using a set of planar electrodes incorporated into the monolayer and driven by an alternating voltage. not only do our results indicate propagating waves on lipid monolayers in accordance with their thermodynamic predictions, but importantly, no significant attenuation is detected proposing an adiabatic phenomenon. in order to provide evidence that our physical explanation provides a correct picture of the membrane, direct detection of the waves was done, whereas a clear transduction of signals was shown. finally, the impact of toxins, neuropharmaca and anaesthetics needs to be integrated in our physical picture of the nerve membranes, what can easily be done now and could deliver a new approach for understanding their physical mechanism. our earlier studies showed that organometallic compounds (otc) in the presence of uvc radiation enhanced the degree of phosphatidylcholine (pc) liposome oxidation, whereas quercetin effectively protected the membrane. the present investigation is concerned with the effect of uvb and otc (chlorides of diphenyl-and dibutyltin, triphenyl-and tributyltin), both in combined action and separately, on oxidation of erythrocyte proteins, pc liposome and albumin. the degree of oxidation of proteins and liposomes induced by otc and radiation, also in the presence of selected antioxidants (trolox, quercetin) was determined on the basis of changes in the number of sulfhydryl groups, carbonyl groups and malone dialdehyde, respectively. the studies indicate that uvb induces pc liposome and erythrocyte oxidation, whereas in the case of albumin it causes both an increase in the number of c=o groups and free sh groups (most probably, braking sulphonic bridges). otc compounds interact with membrane biomolecules both as weak pro-and antioxidants, also in combined action (uvb plus otc). the prooxidative effects are markedly diminished by application of antioxidants. the action of quercetin results from its ability to incorporate into membranes and formation of complexes with otc (liposome>erythrocyte>albumine). this work was supported by grant n n3364 34. a phospholipase c/ sphingomyelinase from pseudomonas aeruginosa has been assayed on giant unilamellar vesicles (guv) consisting of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol, at equimolar ratios. the enzyme activity modifies the chemical composition, thus the physical properties of the bilayer, and conversely the latter influence the enzyme activity. biochemical assays of enzyme activity, together with confocal fluorescence microscopy examination of guv provide novel information about the system. the original lipid composition in the absence of enzyme gives rise to lateral phase separation of liquid-ordered and liquid-disordered domains in the guv. the two enzyme end-products, diacylglycerol and ceramide, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase. morphological examination of individual guv shows that the enzyme binds preferentially the more fluid (or more disordered) domains, and that, in most cases, it causes the fluidification (disordering) of the other domains.. cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. to this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures (33-75 mol% cholesterol) have been measured and compared with the original mixture before lipid hydration. there is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the bilayers is much lower than expected. the maximum solubility of chol in bilayers containing saturated pc or pc with less than four unsaturations is 50-60mol%, while in polyunsaturated pc, e. g. dic18:4, and in sphingomyelin the maximum chol contents is 23mol%. a quantitative analysis of the vesicles is thus required before any experimental study is undertaken. membrane fusion assay based on poresuspending lipid bilayers i. höfer, c. steinem institute of organic and biomolecular chemistry, university of göttingen, germany membrane fusion has attracted significant interest due to its biological relevance. thus in recent years a great variety of fusion assays has been developed. since the process of membrane fusion is not yet fully understood, our aim is to develop a new fusion assay based on pore-spanning membranes, which have been proven mechanically stable, highly insulating and rather tension free. the application of these so-called micro-blms should allow simultaneous monitoring of lipid mixing, content release as well as electrical readouts. furthermore both membrane sides can be addressed individually to apply transmembrane potential or fusion modulating compounds. first results show that the micro-blms provide the opportunity to investigate lipid bilayer fusion by means of fluorescence microscopy. the formation of porespanning membranes is achieved by the painting technique of dphpc doped with oregon green dhpe dissolved in ndecane. the addition of large unilamellar vesicles doped with texas red dhpe allows direct observation of single fusion events. lipid mixing during fusion leads to a decrease of the donor fluorescence (oregon green) and an increase in acceptor fluorescence intensity (texas red) in the plane of the planar bilayer due to fret. in future work simultaneous monitoring of lipid-mixing and content release combined with electrical measurements are planned to gain further insight into different intermediate steps of membrane fusion. the kinetics of membrane-peptide folding and orientation m. r. hicks department of chemistry, university of warwick, uk. understanding interactions of peptides with lipid membranes is essential if we are to be able to design new and better antibiotic peptides. there are different steps in these interactions and following the kinetics of these processes requires that we combine the information from different biophysical techniques. here we present data on the kinetics of peptidemembrane interactions using circular dichroism to report on conformation and linear dichroism to report on orientation of the peptides in/on membranes. these are combined with other techniques such as dynamic light scattering and fluorescence spectroscopy to elucidate the mechanisms of action of the peptides. one of the most important aspects of membrane-active peptide design is that of specificity. we investigate specificity for different types of membranes by using libraries of lipids with different properties of charge, chain saturation and curvature stress. in this way one can test for effects of e.g. negatively charged head groups found in bacterial membranes or cholesterol found in animal and human membranes. using these approaches it is proposed that we will be able to modify peptide sequences, test what part of the kinetic processes are affected and subsequently use this information to design new and better antibiotics. k. kubica, h. misiak wroclaw university of technology, wroclaw, poland reversible membrane electroporation, that can be observed as a effect of electric field influence on biological membranes, finds application in cell delivery of biologically active compounds. the membrane susceptibility for creation of stable pores, depends on electric field parameters applied to the membrane as well as on the membrane environment (ionic strength, ph, temperature) and membrane molecular composition. we have already shown that the changes of van der waals lipid chain interaction effect the process of pore formation. there are known factors which increase or decrease the membrane respond to electric field. because pores appear in lipid membrane matrix it make sense to study this phenomenon using model planar lipid membranes. as lipids are very receptive on oxidizing factors we decided to study the relation between lipid oxidation and membrane reaction on electric field. with the use of four electrode galvanostat we are examining the influence of factors initiating lipid oxidation (uv, fe 2+ ) and the efficiency of some antioxidant compounds of lipid protection on electric properties of lipid membrane. the product of lipid oxidation is determined by characteristic reaction with tiobarbituric acid. we want to determine the role of polyunsaturated fatty acids on membrane properties also. results of our work will be useful in optimization of medicine distribution, aided by electric field, into cells. budding of giant phospholipid vesicles induced by β2-glycoprotein i j. kovacic, b. bozic, s. svetina institute of biophysics, faculty of medicine, university of ljubljana, 1000 ljubljana, slovenia β 2 -glycoprotein i (β 2 gpi) is classified among amphitropic proteins: in its inactive form it is dissolved in plasma and in its active form it is bound to membrane. in comparison to other amphitropic proteins it exhibits a distinct membrane interaction behavior. a patch of positively charged amino acid residues contacts anionic phospholipids via electrostatic interactions, and a hydrophobic loop anchores the protein into the outer leaflet of the membrane via hydrophobic interactions. the binding constant depends on physical properties of the membrane such as membrane lipid composition, surface potential, lipid packing density and curvature. the binding of an amphitropic protein alters the membrane's spontaneous curvature as well as the difference between the equilibrium areas of membrane leaflets. theoretical model has been developed to predict vesicles shape transformations realized by the formation of buds. it was shown that the effects are stronger for flaccid vesicles which were therefore chosen for our experimental investigations. vesicles were composed of 20 % pops and 80 % popc, and flaccidity was achieved by an osmotic adjustment. the solution containing β 2 gpi was subsequently injected to a chamber with flaccid vesicles by a syringe pump. observation took place under a phase-contrast microscope. experiments showed a concentration dependent occurrence of buds. their number and size were related to the degree of vesicle flaccidness. the interplay between detergent cohesion and peptide adsorption on the structure and dynamics of a glycophorin a tm-detergent complex j. khao, j.-p. duneau, j. n. sturgis lism, cnrs -aix marseille university, marseille, france in spite of the major interest of membrane proteins at functional, genomic and therapeutic scales, their biochemical and structural study remain challenging as they require, solubilization in detergent micelles. the complexity of this task arises from the structural dependence of membrane proteins on their anisotropic environment and in particular by a delicate balance between different physico-chemical properties. in order to study these properties in a small protein detergent complex, we have used molecular dynamics simulations on the transmembrane part of glycophorin a (gpatm) solubilized in micelles of the detergent di-hexanoyl-phosphatidylcholine(d6pc). we show that the molecular aggregates organizes to give distinct populations of detergent molecules. those molecules which loosely interact with the peptide are preferentially involved in highly cohesive inter-detergent interactions that impose a global bilayer structure to the micelle. interaction profiles of other detergents with gpatm depend upon the nature of residues along the surface of the peptide. this topology dependence leads to different modes and strength of interactions that ultimately constrain the orientation of the micelles around the peptide. this simple model illustrates how differential detergent selectivity for faces and strong constraints coming from purely environmental features could influence transmembrane helix packing, membrane protein structure and assembly. in this paper we present a study of a new family of bolaamphiphiles. these amphiphiles are unsymmetrical bolalipids having one sugar polar head and the second glycine betaine polar head. they are potentially useful for pharmaceutical or cosmetics applications as vectors for drugs. therefore it is important to investigate their self-assembled properties. the chemical variations that we introduced in this new family concern the length of the main chain that connects two polar heads as well as the length of the side chain placed on the position 1 of the sugar moiety. another variation concerns the introduction of a diacetylenic unit into the main chain in order to rigidify it. we have performed the saxs (small angle x-ray scattering) measurements on the dehydrated compounds as a function of temperature and observed the lamellar liquid crystalline structures. we also measured the saxs spectra of aqueous solutions of these compounds that have shown lamellar l α structure in all cases. these measurements are compared with polarised optical microscopy measurements that confirmed our interpretation. formation of membrane tubular structures induced by phase separation in giant vesicles y. li, r. lipowsky, r. dimova max planck institute of colloids and interfaces, science park golm, 14424 potsdam, germany tubular membrane structures (also known as tethers) exist widely in eukaryotes. abundant work has been done on tube extrusion from cells and model membranes under the application of external forces. we present a novel system allowing tube formation in the absence of external forces. aqueous solutions of two chemically dissimilar polymers, polyethylene glycol (peg) and dextran are encapsulated in giant vesicles, a cell-size model system. the exposure of vesicles to hypertonic solutions induces phase separation of the internal aqueous polymer solution. the excess membrane area created by this vesicle deflation, engages in the formation of tubular structures. membrane tube formation and phase separation are coupled processes. hydrodynamic flows and changes in the membrane spontaneous curvature during phase separation might be the driving forces for tube formation. the tubes are rather stable: without external perturbation, they can exist for several days. they prefer to be located in the peg-rich phase at low polymer concentration. at high concentration, they are absorbed at the interface of the liquid phases to lower the surface energy of the system by decreasing the contact area between the liquid phases. the membrane tubes can be retracted back to the vesicle surface by increasing the membrane tension via vesicle aspiration. membrane tubes, which can form and be retracted easily, might be relevant to lipid storage in cells. insight into the antimicrobial mechanism of a de novo auto-assembling peptide b. legrand 1 , m. laurencin 2 , e. duval 3 , c. zatylny 3 , j. henry 3 , m. baudy-floc´h 2 , a. bondon 1 1 rmn-ilp, umr cnrs 6026 -univ. de rennes1, france, 2 icmv, umr cnrs 6226 -univ. de rennes1, france, 3 pemm, umr ifremer 100 -univ. de caen, france a short (14 residues) de novo antimicrobial peptide (k4) composed of a cationic polar head and a hydrophobic tail was studied. it exhibits a broad spectrum of antimicrobial activity on bacteria. no haemolytic activity or cytotoxicity on eukaryotic cells are observed at mic. when bacteria are lysed by the k4, spherical objects are observed on the sem micrograph. k4 was structurally studied using various membrane mimetic media such as different micelles and small unilamellar vesicles (suv) of different composition. cd revealed that k4 adopt various structures (random, β-turn, α-helix) in the presence or the absence of detergents or phospholipids. nmr structures confirmed the α-helical structure of k4 hydrophobic tail in presence of sds. k4 self-assembles at high concentration as observed by sem and dls. we suggest that k4 may act as a surfactant building mixed microsomes composed of peptide and lipids. this destabilization mechanism of the bacterial membrane support the "detergent like model" previously described in the literature. oxidative stress and the membrane dipole potential; modulation with tocopherol s. le-nen-davey 1 , b. m. davis 2 , j. l. richens 2 , k. a. vere 2 , m. w. tilley 2 , p. g. petrov 1 , c. p. winlove 1 , p. o'shea 2 1 biomedical physics group, university of exeter, uk, 2 cell biophysics group, university of nottingham, uk tocopherol, a component of vitamin e well know for its antioxidant properties, has recently been thought also to influence the structure of cellular membranes. tocopherol treatment reduces hyperglycaemia induced oxidative stress and the associated endothelial dysfunction which is a precursor to the vascular complications of diabetes. tocopherol and insulin interactions are also modified by hyperglycaemia. it is unclear whether these clinically important effects arise from the anti-oxidant and/or structural properties of tocopherol. we have therefore measured the dipole and surface potentials of phosphatidylcholine vesicles containing different amounts of cholesterol, ketocholesterol and tocopherol. we have also investigated the effects of hyperglycaemia, ketocholesterol and tocopherol, alone and in combination, on microdomain formation and interactions of insulin within the membranes of cultured endothelial cells. both sets of experiments indicate that tocopherol causes significant modifications of the membrane dipole and surface potentials. the physiological significance of these changes will be discussed. t. d. lazzara 1 , a. janshoff 2 , c. steinem 1 1 georg-august university göttingen, institute for organic and biomolecular chemistry, tammannstr. 2, 37077, germany, 2 georg-august university göttingen, institute for physical chemistry, tammannstr. 6, 37077, germany cell penetrating peptides (cpp) have been shown to penetrate cellular membranes. they have been of interest for their ability to translocate not only themselves through cellular membranes, but also carry along with them, cargo as large as iron nanoparticles. the exact entry mechanism remains unclear, but has been shown to vary with peptide sequence. their translocation properties have been demonstrated through different experiments involving vesicles, cells and living animals. we plan on using nano-black lipid membranes (nano-blm), which span the pores of nanoporous materials as a model system to study the interaction between cpp and lipid membranes. the nanopores can be used as cellular containers whose interior surface can be functionalized with receptors for biotin-streptavidin recognition. we hope that this system will provide greater control over experimental variables, such as the type of cpp and lipid used, as well as provide kinetic data that can be used to evaluate cpp activity and the kinetics of cargo transport. ethanol induces phospholipid acyl chain interdigitation. while much is known, important issues remain unclear, such as the role of lipid domains. the main purpose of this study was to follow, in real time, the changes induced by ethanol on supported lipid bilayers with nano to microdomains by in situ atomic force microscopy. to this goal, a pure lipid in the fluid phase (dopc), a pure lipid in the gel phase (dppc), binary phospholipid mixtures with gel/fluid phase coexistence, and cholesterol-containing, raft -forming mixtures (dopc/dppc/cholesterol and dopc/sphingomyelin/cholesterol) were investigated. from the height differences observed upon ethanol addition to pure lipids (dppc and dopc), and to dopc/dppc mixtures, it is shown that in the binary system the interdigitation of the fluid phase occurs prior to the gel phase. however, for the lipid rafts mixtures the simultaneous interdigitation of both raft and non raft portions of the membrane is observed both in mica and silicon substrates. for all compositions studied, domain formation or rearrangement accompanied by lipid bilayer expansion occurs as a consequence of interdigitation. these results show the ability of ethanol to influence the bilayer properties in different ways according to membrane composition. ethanol may exert its biological effects by reducing bilayer thickness, and also by changing membrane proteins conformation and lateral distribution, as a consequence of the altered properties of the lipid bilayer. interactions between non-steroidal anti-inflammatory drugs and a pc/cholesterol bilayer m. markiewicz 1 , t. librowski 2 , p. serda 3 , m. pasenkiewicz-gierula 1 1 faculty of biochemistry, biophysics and biotechnology, jagiellonian university " 2 department of pharmacodynamics, medical college, jagiellonian university " 3 regional laboratory, jagiellonian university, krakow, poland. the non-steroidal anti-inflammatory drugs (nsaids) are among the most frequently prescribed and used drugs [1] . there are several side effects connected with frequent use of nsaid, mainly gastrointestinal ulcers and bleedings. a plausible molecular mechanism of these effects are direct interactions of nsaids with gastric phospholipids [2, 3] . the influence of three well-known non-steroidal antiinflammatory drugs with a diverse gastric toxicity (aspirin, ketoprofen and piroxicam) and three newly-synthesized xanthone derivatives (belonging to the nsaids) on the structure and dynamics of lipid bilayers was studied using small angle x-ray scattering and molecular dynamics simulations. the results showed some correlation between nsaid toxicity and its binding to the lipid polar groups. this binding increases membrane fluidity by reducing its density due to an increased membrane surface area. a reduced lipid packing in the membrane most likely increases gastric mucosa permeability, which can result in a decreased resistance of the gastric mucosa to luminal acid. we studied the partition of the anionic amphiphile 1pyrenesulfonate (psa) into mlv and luv (produced by extrusion), composed by zwitterionic popc and zwitterionic/anionic mixtures with pops (until 10 mol %). psa is an anionic amphiphile that mimics several xenobiotics (e.g. pharmaceuticals, pesticides) and endogenous substrates that interact with biological membranes. we found increasing b. lorenz, s. schuy, a. janshoff georg august university, göttingen, germany cell-cell and cell-virus interactions are ubiquitous in living organisms. the analysis of the forces acting between two cells or a cell and a virus gives insight into details of these interaction processes such as their stochastics, cooperativity, reversibility, and energy landscape. using colloidal probe microscopy in conjunction with solid-supported lipid bilayer techniques, we mimic the contact between two membranous compounds displaying sponge glycolipids or viral peptides on their surfaces. after spreading functionalized lipid bilayers on both -a colloidal probe and a silicon wafer surface -the molecular interactions are quantified by means of force-distance curves. by probing the dynamic interaction strength between the viral peptides n36 and c34 we aim for a deeper understanding of the role of these peptides in the complex process of the formation of the prehairpin intermediate as the key step in retroviral fusion. as far as the cellular interaction of marine sponge cells is concerned, we intend to investigate the strength, specificity, and the ca 2+ dependency of the self-recognition between sponge glycans. the systems advantages are the flexibility of the membrane composition and the control over the distribution of receptor molecules in the membranes. since adhesion in biological systems relies heavily on cluster formation within the biomembrane, we plan to mimic the clustering by "printing" interaction domains and comparing the results to homogeneous samples. development of video-rate imaging microscope using laurdan and its applications to lipid raft t. ohba, k.-i. muto, t. kiuchi, k. ohki department of physics, graduate school of science, aobaku, sendai, japan 980-8578, ohki@bio.phys.tohoku.ac.jp various biological functions are located on cell membranes, and the biomembranes maintain heterogeneity as microdomain even in its dynamic structure. existence of such domain was reported as phase-separated 'rafts' in a cell. and the bio-functions of membrane protein are affected by physical property of its surrounding lipid bilayers. laurdan is a useful fluorescence dye to monitor membrane fluidity, and we have developed an instrument to image spatial and temporal change in membrane fluidity by use of laurdan. in order to examine role of 'micro-domain' in biomembrane, we applied this imaging instrument to a giant liposome, cho cells and pc12d cells. fluorescence microscope was equipped with a home-build dual-view optical unit. microscopic image of membranes stained with laurdan is separated into an image at 440 nm and an image at 490 nm by the combination of monochromatic filters and dichroic mirrors. the each image is focused on an image plane of a ccd camera side by side. and generalized polarization (g.p.) image is calculated from those images by personal computer according to the definition of g.p. the g.p. imaging at video rate was applied to a giant liposome of composed of dmpc and dmpe in order to observe phase separation. it was also examined that specific interaction of sphingomyelin and cholesterol in living cho cells and neuritis protrusion from raft region of pc12d cells stimulated by neuron growth factor. current fluctuations in biological lipid membranes from human cell lines s. nuschele, a. wixforth, m. f. schneider university of augsburg, experimental physics 1, univer-sitätsstrasse 1, d -86159 augsburg, germany lipid membranes can undergo phase transitions at physiological temperatures. not only do single lipid component membranes show melting behaviour but also biological lipid membranes from eukaryotes as well as from prokaryotes. performing calorimetric and monolayer studies we are able to detect phase transitions in extracted membranes from different human cell lines (e.g. keratinocytes and melanoma cells). close to and in the melting transition regime we measured distinct channel like current fluctuations. the opening times of these current fluctuations can be predicted based on the lateral compressibility measured from the monolayer isotherm and agree well with the experimental data [*]. the applied method of extraction excludes the presence of functional proteins in the membrane rendering the lipid bilayer as the source of the observed current fluctuations. the molecules are composed of single hydrophobic tail and two hydrophilic aldonamide-type groupings (gluconyl c12-dga or lactobionyl c12-dla) linked by the propylene chain at the nitrogen atom. the micellization processes of c12-dga and c12-dla were studied by means of itc. the critical micelle concentrations, the enthalpies (∆h m ) and the entropies (∆s m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (∆g m 0 (hy)) were calculated. qspr analysis was also used to predict cmc of studied compounds. the interactions of c12-dga and c12-dla with model membranes (dppc and dppc/chol. bilayers) were studied by means of dsc. using quantum computations some basic molecular properties were calculated. the conformational space was explored using molecular mechanics. obtained results were compared with those for analogical compounds with single head groups, e.g. with also synthesised by us n-alkanoyl-nmethyllactitolamines (c n mela) and common sugar-based surfactants c 12 gluc and mega-10. enfuvirtide (t-20) was the first hiv-1 fusion inhibitor peptide approved for clinical use. t-1249 is an inhibitor still under evaluation. previous studies, based on tryptophan intrinsic fluorescence, showed that the peptides interact differentially with membrane model systems (luv) with different lipid compositions. studies with human blood cells were necessary to further establish the role of membranes on these peptides mode of action. an experimental strategy was applied based on the membrane dipole potential, as measured by the fluorescent probe di-8-anepps. human erythrocytes and peripheral blood mononuclear cells (pbmc) were successfully labeled. for both systems, a fusion inhibitor concentration-dependent decrease on di-8-anepps fluorescence excitation ratio (a measure of the spectral shift and dipole potential) was observed. the quantitative analysis of these variations indicated that t-1249 has an approximately ten-fold higher affinity towards erythrocyte and pbmc, when compared with enfuvirtide. this is in agreement with the previously known adsorption of t-1249 on cholesterol-rich membrane domains and with its higher partition constants. hiv associates with erythrocytes in vivo, which can constitute a route to deliver peptide to the viral membranes (also rich in cholesterol). lymphocyte membranes can concentrate and accelerate the drug interactions with its molecular target, gp41 in its exposed conformation. oxidation of low density lipoprotein (ldl) is known to be a key step in atherogenesis, leading to inflammation, proliferation and apoptosis of cells of the arterial wall. these effects are largely exerted by oxidatively fragmented phospholipids, which are highly exchangeable between cells, tissues and lipoproteins. in particular, pgpc has been identified in minimally modified ldl and has been reported to elicit a wide range of pathophysiological responses in vascular cells, e.g. the activation of apoptotic signaling pathways. we investigated here the behavior of the fluorescent oxidized pgpe-alexa647 compared to dhpe-bodipy in artificial supported lipid bilayers with different cholesterol contents. the two labeled lipids differ in the type of membrane insertion: while dhpe-bodipy is anchored to the membrane via two fatty acids, pgpe is incorporated with only one fatty acid. the second chain is an acyl fragment in sn-2 position, which represents the oxidation product of an unsaturated acyl chain. with increasing cholesterol content we observed a decrease in the diffusion coefficient for both lipids. interestingly, the diffusion of the oxidized lipid was reduced in a higher degree compared to that of the non-oxidized lipid. the calculated ratios of the diffusion constants of pgpe-alexa647 and dhpe-bodipy suggest a different type of interaction with cholesterol. membrane proteins account for a third of all proteins encoded for by the human genome, and play a vital role in a number of cellular processes. few membrane protein structures have been determined to date in comparison to soluble proteins. this discrepancy is due to experimental difficulties in preparing membrane protein samples for structural analysis. traditional techniques for studying membrane proteins by x-ray crystallography or solution nmr use detergent solubilised proteins which can differ from their native confirmations. solid-state nmr allows the study of transmembrane proteins in lipid bilayers, representing a more native like environment in which to obtain biologically relevant structural information. we have been working on the development of reliable methods for reconstitution of transmembrane peptide into liposomes using glycophorin a as a model tm protein. using reconstitution methods based on the removal of detergent by bio-beads, we have used electron microscopy to screen the ideal conditions for insertion of gpa into liposomes in preparation for mas nmr experiments. em has allowed us to identify conditions favourable for insertion of peptide into lipid vesicles and those that result in aggregation. in order to confirm the secondary structure and insertion of gpa into lipid vesicles, techniques such as cd, ocd, ftir and dls have been used to provide quantitative information in addition to the visual results from em. nonesterified fatty acids regulate a broad spectrum of metabolic activities and are involved in many physiological, pathological and/or pharmacological processes in living cells. they spontaneously transfer between donors and acceptors such as fatty acid binding proteins and lipid membranes. we focus on protein-lipid dispersions of human serum albumin (hsa) and sterically stabilized liposomes (ssl) composed of dppc and appropriate amount of peg:2000-dppe in which stearic acids (sa) are inserted either in the protein or in the ssl. exploiting the fact that hsa has a single tryptophan residue and that the intrinsic trp-fluorescence emission signal is quenced by the presence of sa, the kinetics of sa transport between hsa and peg:2000-grafted dppc membranes is studied by means of fluorescence. it is found that the transfer of sa between hsa and ssl is a first-order process and the kinetics of transfer depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers), and on the grafting density of the peg-lipids at the protein/lipid interface. indeed, in the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer and it is faster for the passage from dppc bilayers to hsa. the presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer-chains, especially for lipid membranes in the fluid phase. a. orth 1 , w. römer 2 , l. johannes 2 , c. steinem 1 1 institute for organic and biomolecular chemistry, university of goettingen, germany, 2 laboratoire trafic et signalisation, institut curie, paris, france shiga toxin (stx) from shigella dysenteriae is an ab 5 -class bacterial toxin. infections with stx lead to the haemolyticuraemic syndrome, which is known to be a major cause for renal failure at an early age. the interaction of the homopentameric b-subunit (stxb), which is responsible for binding and intracellular transport of the holotoxin, with its cellular receptor, the glycosphingolipid gb 3 , is the first step for endocytosis of the toxin. one b-subunit can bind up to 15 gb 3 -molecules to form stxb-gb 3 -clusters causing negative curvature of a membrane. the binding of stxb to giant unilamellar vesicles, composed of dopc, cholesterol and gb 3 induces tubular membrane invaginations, which were also found in experiments with energy-depleted hela-cells. in recent studies, protein and lipid reorganization processes after attaching stxb to solid supported membranes, composed of dopc/sphingomyelin/cholesterol/gb 3 have been investigated. the compaction of gb 3 -molecules led to an additional stxb-gb 3 -enriched phase, which was also observed in lipid monolayers at the air/water interface. in this study, the influence of stxb on model membranes will be investigated combining the advantages of free-standing lipid membranes with those of ssms. the impact of stxb on pore-suspending membranes will be followed by confocal laser scanning mi croscopy and atomic force microscopy. a. robaszkiewicz 1 , c. spickett 2 , p. sicinska 1 , g. bartosz 1 , m. soszynski 1 1 department of molecular biophysics, university of lodz, lodz, poland, 2 strathclyde institute of pharmacy and biomedical sciences, glasgow, u.k. hypochlorite generated in vivo under pathological conditions is a known oxidant, able to initiate lipid peroxidation process, which affects the stability of biological membranes. the aim of this study was the analysis of the products formed during the reaction of hypochlorite with phosphatidylcholines containing unsuturated fatty acid residues (1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, 1stearoyl-2-arachidonylphosphatidylcholine) and their effects on the human erythrocytes. using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in the decrease of the rotational correlation time and rotational motion freedom of liposomes estimated by epr using spin probes (16-and 5doxylstearic acid). unilamellar chlorohydrin liposomes had lower diffusion coefficient for calcein than liposomes made of parent lipids. flow cytometry demonstrated fast incorporation of uni-and multilamellar chlorohydrin liposomes labeled with nbd-pe into erythrocytes. this effect was accompanied by the formation of the erythrocyte subpopulations of higher volume, decrease of the rate of fluorescein diacetate hydrolysis, estimated by flow cytometry, and increase of affinity and maximal velocity of the membrane enzyme acetylcholinesterase. extensive bilayer perforation coupled with the phase transition region of an anionic phospholipid k. a. riske 1 , l. q. amaral 2 , m. t. lamy 2 1 depto. biofisica, universidade federal de sao paulo, sao paulo, brazil, 2 instituto de fisica, universidade de sao paulo, sao paulo, brazil at low ionic strength dimyristoylphosphatidylglycerol (dmpg) exhibits a broad phase transition region characterized by several superimposed calorimetric peaks. peculiar properties, such as sample transparency, are observed only in the transition region. we use differential scanning calorimetry, turbidity and optical microscopy to study the narrowing of the transition region with the increase of ionic strength. upon addition of salt, the temperature extension of the transition region is reduced and the number of calorimetric peaks decreases until a single cooperative event is observed in the presence of 500 mm nacl. the transition region is always coupled with a decrease in turbidity, but a transparent region is detected within the melting process only in the presence of up to 20 mm nacl. optical microscopy of giant vesicles shows that bilayers first rupture when the transition region is reached and subsequently lose optical contrast. fluorescence microscopy reveals a blurry image in the transparent region, suggesting a different lipid self-assembly. overall sample turbidity can be related to the bilayer optical contrast. our observations are discussed in terms of the bilayer being perforated along the transition region. in the transparent region the perforation is extensive and the bilayer completely loses the optical contrast. financial support: fapesp. label-free imaging of biological membranes using surface imaging techniques j. l. richens, k.-a. vere, p. o'shea cell biophysics group, university of nottingham, nottingham, uk surface plasmon resonance (spr) is a detection technique traditionally used for specific protein detection, which is now exploited routinely as a generic label-free sensor. spr responds to changes on a metal surface conditioned to sense the binding of analytes. thus, the composition of the external medium and metal surface will be fundamental to the signal output. here we use a model phospholipid membrane system to investigate the effect of altered buffer and surface compositions on spr signals. comparisons are undertaken between continuous gold surfaces and gold nanoparticles. we demonstrate that surface electrostatics and the salt composition and molarity of a buffer all have significant impacts on spr output. the association of respiratory syncytial virus matrix protein with membrane microdomains the association of the matrix protein from respiratory syncytical virus with membranes has been characterised by tensiometry, brewster angle microscopy, and atomic force microscopy following deposition of langmuir monolayers onto modified silica substrates. association of the protein with monolayers containing phosphocholines and cholesterol leads to the formation of materials with new properties that differ from those of either of the pure components. the behaviour of the protein in monolayers rich in cholesterol and sphingomyelin exhibits a significant concentration dependence. at low concentrations, the protein exhibits a simple partioning behaviour. at higher concentrations, lipids are extruded from the monolayer below a critical surface area. these findings are discussed in relation to the recently published structure of the protein (pnas, 2009, 106, 4441-4446) , the documented formation of viral filaments during key stages of the infection cycle and the isolation of the protein from detergent-resistant membrane fractions. structure and dynamics of closed melting membranes v. m. rondelli 1 , s. santorsola 1 , p. brocca 1 , e. del favero 1 , l. cantù 1 , m. zimbone 2 1 dep. of chemistry, biochemistry and biotechnologies for medicine, university of milan, segrate, italy., 2 dep. of physics, university of catania, catania, italy. we studied the dynamical and structural properties of large unilamellar vesicles (≈ 120 nm luvs) of phospholipids (dmpc, dc 15 pc and dmpc : dc 15 pc = 1 : 1 molar mixture) in the temperature range around the chain-melting transition, ≈ 3 • c wide, with 0.1 • c resolution and 0.01 • c accuracy. small-angle (saxs) and wide-angle x-ray scattering (waxs) measurements show that across the transition the vesicle behaves as an 'evolving membrane', passing through several different states, each of them being characterized by different proportions of coexisting fluid-and gelchains molecules. noteworthy, no kinetics has been detected. on the same samples, a unique and very sensitive laser light scattering technique allows to determine the characteristic times of thermally induced shape fluctuations, connected to the elastic properties of the membranes. results indicate a clear softening of the membranes in correspondence to the chain-melting transition, as indicated by a manifold increase of the corresponding fluctuation characteristic time. meanwhile the overall size of the vesicle is not sensibly changed. this softening is likely to be due to the presence of structural defects, eventually driving to local morphological modifications. thermodynamics characterization of isolated lung surfactant assembled as lamellar bodies e. x. rodriguez 1 , r. alvarez 1 , r. barrio 2 , c. irles 3 , a. ortega 1 1 biochemistry depto., school of medicine, 2 inst. of physics unam, 3 nat. inst. of perinatology, mexico city, mexico lungs are a large extension of ∼80m 2 of one layer cells, which is structured as compacted sacs called alveoli, where lung function takes place: the gas exchange. alveolar endotelium is formed by two kinds of cells: neumocyte type i (nti), which covers ∼97% of the alveolar surface where gas exchange occurs and, ntii where lung surfactant (ls) is produced. ls are a mixture that lay over a water film in the luminal surface of the alveoli and it is thought to decrease the surface tension to avoid alveolar collapse during expiration. ls are made of phospholipids and proteins, which are determinant for the structure of ls under particular conditions of pressure and temperature. ls inside the cell is packed in organelles called lamellar bodies (lb), and is released to the water/air interphase in other conformation. in the present study we isolate lb from pig's lung and study lb thermodynamic characteristics by microcalorimetry in the presence and in the absence of structural proteins. phase transition profile of lb with and without proteins is basically the same; while ls in the alveolar lumen have a higher transition temperature (tm). mayor changes in tm are observed between lb and ls from alveolar lumen. although ls lipid composition in and outside the cell is assumed to be the same, the ground for the differences in tm under these two conditions is unknown. m. rodrigues 1 , g. rádis-baptista 3 , b. g. de la torre 2 , m. castanho 1 , d. andreu 2 , n. c. santos 1 1 instituto de medicina molecular, portugal, 2 pompeu fabra university, spain, 3 federal university of cearà, brasil nucleolar-targeting peptides (nrtps) have been recently designed by structural dissection of crotamine, a toxin from the venom of a south america rattlesnake (radis-baptista et al. 2008. j med chem 51:7041) . at µm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work is to decipher the molecular mechanism for the translocation of nrtps into cells. quantification of partition into membranes was carried out, based on intrinsic tyrosine fluorescence. the role of the bilayer phase, anionic lipids, reducing agents and peptide concentration on the extent and kinetics of partition were studied. both nrtp1 and nrtp2 exhibited high partition to popc (neutral) lipid vesicles (k p ≈3×10 3 ), which was enhanced by the anionic lipid popg for nrtp1, but not nrtp2. the peptides showed a decrease in partition for popc:cholesterol (liquid ordered state) or dppc (gel) membranes. depending on the lipid composition, the peptides either increased or decreased their quantum yield upon membrane insertion. quenching experiments with acrylamide showed no peptide aggregation in solution. once the translocation mechanism is fully understood we will test nrtps as carriers of relevant cellular cargos, evaluating their potential clinical application in drug delivery or gene therapy among other applications. the ingestion of trans fatty acids (tfa) formed during the partial hydrogenation of vegetable oils has been linked to a detrimental impact on health by an, as yet, unknown mechanism. we synthesized deuterated analogs of 1-elaidoyl-2-stearoylphosphatidylcholine (t18:1-18:0pc) that contains a single "unnatural" trans double bond and 1-oleoyl-2-stearoylphosphatidylcholine (c18:1-18:0pc) that contains a single "natural" cis double bond. solid state 2 h nmr, complemented by molecular dynamics (md) simulations, was then employed to compare molecular organization in model membranes prepared from these isomeric molecules. analysis of spectra recorded as a function of temperature reveals a higher chain melting temperature for the trans isomer, indicating tighter molecular packing in the gel state. in the liquid crystalline, however, the difference between the trans and cis isomers is subtle. order as probed by the perdeuterated [ 2 h 31 ]18:0 sn-2 chain, and corroborated by computer simulation, coincides within <5%. only in the conformation of the double bond is an appreciable difference implied. thus, our results contradict the conventional view that tfa resemble saturated fatty acids, which is >20% more ordered. (supported by acs, prf 43281-ac7.) photooxidation and lateral membrane diffusion of dipole molecules v. s. sokolov 1 , e. a. sokolenko 1 , a. a. lents 2 , p. pohl 2 1 a.n frumkin institute of physical chemistry and electrochemistry, russian academy of science, moscow, russia, 2 institut fuer biophysik, johannes kepler universitaet linz, austria the photodynamic oxidation of phloridzin, of the styryl dye di-8-anepps and of unsaturated lipids was monitored "online" by measuring the collapse of the dipole potential which has been introduced to the membrane by these molecules. their photodamage occurred at different rates when the target molecules and the singlet oxygen generating photosensitizer (phthalocyanin) were adsorbed to the same or to opposite sides of the planar lipid bilayer. the difference in the oxidation rates were attribute to singlet oxygen transport through lipid bilayer and therefore we were able to estimate the permeability of lipid bilayers to singlet oxygen. however, the apparent permeabilities derived from experiments with different targets were differ from each other. therefore, we tested the hypothesis that the lateral membrane diffusion of target molecules and oxidation product may have biased our analysis. in line with this anticipation we found that the apparent permeability is dependent on the size of the planar bilayer. the development of a new mathematical model, which takes the mobility of all reacting species in the aqueous and lipid environments into account, allowed estimation of the real membrane permeability to singlet oxygen. it appeared to be very close to that of oxygen in the ground state. a number of complex three-dimensional lyotropic liquid crystal phases are already known, such as the bicontinuous cubic phases, but so far only a single example has been found -a cubic phase of spacegroup fd3m -of a structure based upon a complex close packing of inverse micelles (1) . we now report the discovery (2) of a novel lyotropic liquid crystal phase, of space group, p6 3 /mmc, whose structure is based upon a hexagonal close packing of identical quasi-spherical inverse micelles. the model membrane system consists of a hydrated mixture of dioleoylphosphatidylcholine, dioleoylglycerol, and cholesterol. this novel phase has a number of unique features which may render it useful for a range of applications. firstly, it is the only known self-assembled lyotropic phase whose structure consists of a periodic close packing of identical inverse micelles. secondly, it is stable in excess aqueous solution, which is very important for potential biological or biomedical applications. references (1) v. luzzati, v., r. vargas, a. gulik, p. mariani, j.m. seddon, and e. rivas, biochemistry 31, 279-285 (1992) . dystrophin is a rod-shaped muscular subsarcolemal protein. its deficiency is one of the root causes of duchenne muscular dystrophy. dystrophin rod domain contains 24 homologous repeats, where the sub-domain constituted by the repeats 11 to 15 (r11-15) was reported to bind actin and membrane lipids. we analyzed the interaction of r11-15 with lipid monolayers. to better understand the assembly mechanism of this protein with lipids, we studied its adsorption behavior at the air-liquid and lipid/liquid interface using ellipsometry, surface pressure, and atomic force microscopy (afm). using two different mixtures of phospholipids, ellipsometry and pressure surface data show that r11-15 interacts with the lipid monolayers, but is inserted into the monolayer formed by dopc-dops while it lies below the monolayer of dopc-dope. this indicates that r11-15 interacts more strongly with dopc-dops than with dopc-dope. afm images show that the pressure of lipid monolayer influences the organization of r11-15. when the lipid surface pressure is 30mn/m, r11-15 forms a striking network, indicating a protein-protein interaction in addition to the protein-lipid interaction. this unique behavior of one part of the central domain of dystrophin may explain its key role in muscle cell. studies on polyphenol extracts from helichrysum l., fagopyrum mill., crataegus l. and hypericum l. were performed in order to find if they may be applied as a natural free radical scavengers protecting biological membranes against peroxidation. ghosts erythrocytes were used in the experiments. they were suspended in tris-edta solution, ph 7,4, then.irradiated with a bactericidal lamp without (control) or with a proper amount of the extracts studied. the product of lipid peroxidation was malonic dialdehyde (ma). the colour reaction of ma with thiobarbituric acid (tba) enables to determine the concentration of ma spectrophotometrically. it was found that peroxidation increased with the irradiation time. however, it significantly decreased when concentrations of poliphenols increased. the best antioxidtive property was found for hypericum l. the antioxidative efficiency sequence of the plant extracts studied was the following: hypericum l. > crataegus l. > fagopyrum mill. > helichrysum l. the results obtained indicate that polyphenol extracts exhibit excelent antioxidative properties that make them good free radical scavengers for efficient protection of biological membranes. this work was sponsored by ministry of science and education, scientific project no. n n305 337034. interaction of cationic porphyrins with neutral and negatively charged liposomes i. voszka 1 , g. corradi 2 , p. maillard 3 , h.-j. steinhoff 4 , g. csík 1 1 institute of biophysics and radiation biology, semmelweis university budapest, hungary, 2 research institute for solid state physics and optics, hungarian academy of sciences, budapest, hungary, 3 institute curie, section de biologie, orsay, france, 4 fachbereich physik, universitat osnabrück, germany porphyrin derivatives are used in photodynamic therapy of tumors. the knowledge of the photosensitizer location within the cell is important. the effect of porphyrin derivatives containing cationic side groups was examined on neutral and negatively charged liposomes by esr. the esr signal was first examined as a function of temperature and porphyrin concentration in the dark. a significant change related to the appearance of quasi free spin labels was obtained for spin probes at the 12 th carbon atom and was more expressed for the asymmetrical derivative. illuminating the samples the esr amplitude decreased for all positions of the spin probe but to different extent. the effect was more expressed in case of the symmetrical derivative, especially for label positions 5 and 12. for the asymmetrical derivative the effect changed from moderate to weak from the 5 th to the 16 th position. this indicates that the asymmetrical derivative is incorporated nearer to the lipid head groups while the symmetrical one may be located deeper in the membrane. under oxygen-free conditions both derivatives showed weaker but still pronounced effects.. single channel recording of α-hemolysin in nanopore-spanning tethered bilayer lipid membranes n. t. thet 1 , i. pfeiffer 2 , w. knoll 1 , i. köper 1 1 max planck institute for polymer research (mpi-p), ackermannweg 10, 55128 mainz, 2 austrian research centers gmbh -arc, tech gate vienna, 1220 vienna, austria artificial lipid bilayer membranes mimic biological cell membranes in many aspects and can be used to study functional processes such as ion channeling, signal transducing, transport of nutrients etc. as most of the functions of a cell are accomplished by membrane proteins, research has been ongoing in studying and characterization of membrane proteins embedded in model lipid bilayer membranes. black lipid membranes (blms) were studied for decades but their potential for practical applications is hindered, mainly due to their lack of stability and limited lifespan. recently, tethered bilayer lipid membranes (tblms) proved to have long life span of up to several months without significant changes in membrane resistance and capacitance. in order to combine advantages of blms and tblms, we have designed a membrane system which is freely spanned across a single nanopore in a silicon nitride membrane. this system not only mimics cell membranes, but also it allows control over the chemical composition of buffer with unlimited ionic reservoirs on both sides of the membrane. this freestanding tblm maintains structural stability and lifetime of up to 120 hours without significant decrease in its structural integrity and electrical sealing. for the validation of the tblm formation, we have inserted well known α-hl pores. we are able to control the amount of α-hl pores insertion and measure single channel ion transport across the tblm by α-hl pores using a capacitor feedback amplifier. the language of shape: biological reactions are dramatically affected by the shape of lipid membrane d. stamou university of copenhagen, copenhagen, denmark a plethora of biological process are taking place on the surface of lipid membranes. as a rule membranes in vivo are curved in a variety of complex geometries. here i will present a quantitave study on the influence of membrane curvature on protein-membrane and membrane-membrane interactions. to gain systematic access to a continuum of membrane curvatures we immobilized liposomes on a surface at dilute densities. using confocal fluorescence microscopy we imaged single liposomes of different size, and therefore different curvature, and monitored their interaction with a binding partner (proteins or other liposomes). i will discuss unpublished data on two important classes of biomolecular interactions that exhibited dramatic curvature dependence: a) snare-mediated docking and fusion b) anchoring of peripheral proteins. the following references provide partial information on the single-liposome assay: a. zettergren, c. gudmundsson, t. nylander, e. sparr physical chemistry1, lund university, 22100 lund, sweden there is accumulating evidence of substantial amounts of phospholipids in the cell nuclei 1 , although the function of these lipids is still not fully understood. it has been shown that the chromatin complex composed of dna, rna and proteins also includes phospholipids, and that rna colocalize with these 2 . although the rna-phospholipid interactions may have important implications to biological function, in gene therapy and in medicine, very little work has been dedicated to the characterization of rna interaction with phospholipids. the objective of this work is to investigate the adsorption behavior of short single stranded 10 bases long rna (ssrna 10 ) molecules (similar to mirna) to lipid monolayers at the air-water interface as well as to study how the presence of rna affect the domain formation in the monolayers using fluorescence microscopy. monolayer studies have shown adsorption of ssrna 10 to monolayers consisting of zwitterionic dppc as well as to monolayers consisting of cationic dodab. the adsorption behavior of these very short nucleic acids differ significantly from the adsorption process for longer nucleic acids as for example a 2000 base pairs long ds dna (dsdna 2000 ) which has been used as a reference system 3 . the presence of ssrna 10 significantly changes the compression isotherm of both dppc and dodab monolayers. plant defensins are cysteine-rich antimicrobial peptides found in various plant species. they share a common threedimensional structure, stabilized by eight disulphide-linked cysteines consisting of three antiparallel β-strands and one α-helix. most plant defensins show antifungal activity with no effect on mammalian and plant cells. expression of these peptides in plant tissue is induced by pathogen infection. the mechanism of defensin action is based on membrane permeabilization. this occurs through an interaction with high affinity binding sites on fungal membranes, resulting in alteration of membrane potential. the genes encoding for a peach ppdfn1 and a grape vvamp defensins were expressed in e. coli and purified to homogeneity. they were tested for antimicrobial activity against some fungi and showed to have an inhibitory effect on the spore germination. biophysical analysis showed that defensins were able to interact with artificial membranes. binding of defensins to membranes was dependent on lipid composition, increasing with the sphingolipids content. interaction between peptides and sphingolipids could lead to insertion of the defensins into the membrane resulting in its destabilization. extracts of the plant perilla frutescens have many uses in the asian kitchen, e.g. as a popular garnish used in sushi. the plant is also employed in eastern traditional medicine to treat a variety of ailments including colds, food allergy and depression. two of the main constituents of the plant are limonene and perilla aldehyde. by bio-oxidation, these molecules can be converted to perillic alcohol and perillic acid. these cyclic terpenes possess antibacterial and anticarcinogenic properties. the modes of action for these compounds are at present not understood, but their remarkably diverse pharmacological properties suggest that they might target the phospholipid matrix of the cellular lipid membrane. indeed, the cyclic terpenes can bind to, and alter the physiochemical properties of the lipid bilayer of the membrane, the effects of which can cascade down to several essential cellular processes. here, we use molecular dynamics (md) simulations to investigate the effect of limonene and its derivatives on the properties of lipid bilayers including the changes in acyl chain order parameters, bilayer thickness and the area per lipid. md can afford molecular-scale dynamic information, often not easily accessible from experimental measurements. this information can be used to interpret existing experimental data obtained by e.g. isothermal titration calorimetry, electron paramagnetic spectroscopy and differential scanning calorimetry. catalysis in the membrane: interfacial mechanism of phospholipase a2 h. p. wacklin 1 , r. k. thomas 2 1 institut laue langevin, grenoble, france, 2 physical and theoretical chemistry laboratory, oxford university, u.k. phospholipase a2 cleaves the sn-2 acyl chains of membrane phospholipids and performs a range of physiological functions. one of the least well understood aspects of the its mechanism is how its activity is regulated by the interaction with the substrate membrane. we have used neutron reflection to monitor changes in membrane structure during lipid hydrolysis [1, 2] . the penetration depth of pla2 depends on lipid packing and increases during the lag phase of porcine pancreatic pla2. by using a selectively deuterated lipid substrate, d31-popc, we determined the relative membrane-water partitioning of the lipid products in-situ. the lyso-lipid product partitions into the solution phase, while fatty acid accumulates in the membrane and increases the affinity of pla2 [2] . pla2 is inhibited at ph 5, which is consistent with protonation of the catalytic histidine. however, irrespective of ph, pla2 is fully activated by me-betacyclodextrin, which facilitates the release of the lyso-lipid from the enzyme-substrate complex. me-beta-cyclodextrin does not interact directly with the membrane surface or the substrate lipids, indicating that product release occurs outside the immediate membrane-water interface. diffraction limits resolution in optical microscopy, but many interesting biological problems occur on shorter (molecular) length scales. recently, methods to circumvent the diffraction limit have been presented. fluorescence photoactivation localization microscopy (fpalm) uses activation of many small subsets of photoactivatable or photoswitchable fluorescent probes (pafps) to generate images with effective resolution in the tens of nanometers. pafp molecules are photoactivated, imaged, localized, and photobleached in small numbers. the process is repeated for many subsets to build up data on thousands to many hundreds of thousands of molecules. the positions of all localized molecules are used to construct an image of the sample with resolution limited not by diffraction, but by the localization precision and molecular density. results will be shown from a variety of biological systems, including live and fixed cells expressing a variety of pafp-tagged proteins. bi-plane fpalm can image in three dimensions with demonstrated resolution of 30 nm x 30 nm x 70 nm. polarization fpalm can image both molecular positions and anisotropies simultaneously with lateral resolution of ∼20-30 nm. using these powerful capabilities, many potentially interesting biological problems can be addressed. binding of the hiv-1 ncp7 on oligonucleotides at the single-molecule level j. godet, p. didier, a. jouonang, y. arntz, y. mély laboratory of biophotonics and pharmacology, umr cnrs 7213, university of strasbourg, france the nucleocapsid protein (ncp7) of the hiv-1 is a small basic protein which plays key functions in the viral life cycle. the activity of ncp7 mainly rely on its potent rna-and dna-chaperone activities that direct the rearrangement of numerous nucleic acid molecules into their most stable conformation. two main features of nc's chaperone activity are its abilities to aggregate and destabilize nucleic acids. in addition, the rapid kinetics of ncp7 interaction with nucleic acids was recently proposed as another major component of nc's chaperone function based on the apparent correlation between an indirect measurement of the nucleic acid dissociation kinetics of ncp7 and its overall chaperone activity. but so far, no direct measurement of the on/off rates of ncp7 binding to oligonucleotides was performed. in the present work, we realized single molecule fluorescence resonance energy transfer (smfret) measurements to probe the transient interactions of a tmr-labelled ncp7 with a short cy5-labelled dna oligonucleotide confined into nanovesicles. after confirming the efficiency of the ncp7/odn complex encapsulation into the nanovesicles by fcs, the vesicles were tethered to the surface for immobilization. integrity of the entrapping vesicles attached on the surface was confirmed by afm. finally, the association and dissociation constants retrieved from these smfret measurements were discussed in the context of the dna-chaperoning activity of the protein. high-resolution spatiotemporal organization of the integrin lfa-1 m. f. garcia-parajo 1 , t. s. van zanten 1 , g.-j. bakker 1 , r. diez-ahedo 1 , a. cambi 2 , c. g. figdor 2 1 bionanophotonics group; ibec, barcelona, spain, 2 tumour immunology dept; ncmls, nijmegen, the netherlands lfa-1 is a leukocyte-specific integrin involved in different steps of the immune response. on monocytes, lfa-1 plays a key role in the regulation of monocyte-endothelial interaction during rolling, arrest and extravasation into the underlying tissue. i n-vivo experiments showed that blood borne lymphocytes can 'switch' within seconds from rolling to arrest. furthermore, tem observations of pro-active, ligandindependent nanoclusters confirmed that affinity and clustering are complementary processes required in adhesion. yet, the mechanisms leading to fast-switching remain obscure. in our group, we used a combination of single molecule fluorescence techniques to study the spatiotemporal organization of lfa-1 on monocytes. we performed optical nanoimaging of lfa-1 nanoclusters in relation to membrane rafts with a resolution of 70nm and accuracy of ∼3nm. in quiescent cells, lfa-1 nanoclusters do not associate with membrane rafts and diffuse freely on the membrane. binding of the integrin to its ligand icam-1 induces the formation of microclusters that further associate with rafts and exhibit reduced mobility, consistent with cytoskeleton interactions. our work highlights the markedly different spatiotemporal organization of lfa-1 that might explain its concerted action to form larger and stable platforms on the cell surface required for rapid and effective cell adhesion. c. ciobanasu, u. kubitscheck rheinische friedrich-wilhelms-universität bonn, germany cell penetrating peptides like the hiv1 tat peptide have the property to rapidly translocate the cell membranes and the capability to deliver a wide range of cargoes. the mechanism of the membrane translocation is still under investigation and object of considerable controversy. we applied and single-molecule and confocal laser scanning microscopy (lsm) to study peptide-membrane interactions. electron-multiplying ccd cameras yield images of single fluorescent molecules with a time resolution in the range of a few milliseconds only, which allows the tracking of fluorescently labelled peptides and lipids at bio-interfaces in realtime with a localization precision of a few nanometers. we formed giant unilamellar vesicles (guvs) from different lipid mixtures and examined their interaction with fluorescently labeled tat peptides. we found that the passive peptide internalization process depends on lipid composition, charge of the lipid bilayer, and the ionic properties of the medium. a translocation of cationic tat peptides was observed in membranes containing at least 20 mol% of lipids with a phosphatidyl ethanolamine or a high mol fraction of the phosphatidyl serine head group. in salt-free solution tat efficiently bound to guvs, however, in a physiological nacl solution tat binding was completely abrogated, but the peptides efficiently equilibrated across the guv membrane. new approaches to measure interactions in the live cell plasma membrane g. j. schütz biophysics institute, johannes kepler university linz, austria in my lecture, i will show examples how to obtain insights into the organization of the cellular nanocosm by single molecule experiments. our primary goal is an understanding of the role of such structures for immune recognition. brightness and single molecule colocalization analysis allows us to study stable or transient molecular associations in vivo. in particular, i will present results on the interaction between antigen-loaded mhc and the t cell receptor directly in the interface region of a t cell with a surrogate antigenpresenting cell. in addition, we developed a method for in vivo micropatterning of plasma membrane proteins to measure molecular interactions. the method allows identifying and characterizing interactions between an arbitrary fluorescence labeled protein ("prey") and a membrane protein ("bait") directly in living cells. cells transfected with a fluorescent fusion protein of the prey are plated on micropatterned surfaces functionalized with specific antibodies to the extracellular domain of the bait; the fluorescence copatterning is used as readout for the interaction. we applied this tool for the study of the interaction between cd4 -the major coreceptor for t cell activation -and lck, an important tyrosine kinase in early t cell signaling. in addition to the well-known zinc-clasp structure, we found strong contributions of lck membrane anchorage for the binding of the two proteins. developing a fluorescent redox sensor for monitoring metal-ion mediated catalysis in biosystems a. rybina 1 , a. kiel 1 , b. thaler 2 , a. sprödefeld 2 , r. krämer 2 , d. p. herten 1 1 bioquant and, 2 department of inorganic chemistry, heidelberg university, germany a fluorescent redox sensor is an electron photo-switching device that can be used for the characterization of the redox state of a given environment. it combines a fluorescent fragment with a redox-active unit that senses the media by a redox reaction and controls the light emitting properties of the fluorophore. such reversible sensor can help to examine the electrochemical state and changes in biological systems during biochemical processes in real time. recently a new fluorescent molecular sensor with a redox-active hydroquinoneunit covalently linked to fluorophore rhodamine b was developed. (kierat r.m. et al., bioorg. med. chem. lett., 2009 -accepted) . the reduced hydroquinone-form of the sensor is fluorescent while its oxidation to benzoquinone-derivative leads to a significant decrease of the fluorescence. although the above method shows great promise for applications in biological systems, the exact mechanism of this process is not fully understood yet. we use fluorescence spectroscopy to investigate oxidation reactions on the ensemble and single-molecule level and study kinetic rates. the proposed strategy is to use cu (ii) complex as oxidation mediator immobilized on surface via dna linker to examine the oxidation mechanism. fluorescently labeled atp as a probe of the outer mitochondrial membrane barrier: role of vdac fluorescence correlation spectroscopy (fcs) was applied for studying the distribution of fluorescently labeled atp (bodipy-atp) in isolated mitochondria. the setup and peak intensity analysis (pia) was described in our recent paper (perevoshchikova et al. 2008 biochim.biophys.acta 1778 :2182 . the binding of bodipy-atp to mitochondria was maximal in the non-energized state, whereas the addition of succinate (respiratoty substrate) or atractyloside (adenine nucleotide translocase inhibitor) led to a decrease in the binding. nadh reduced the fcs signal from bodipy-atp added to non-energized mitochondria more than nad+ did under the same conditions suggesting the control of nucleotide transport through voltage-dependent anion channel (vdac) residing in the outer membrane. konig's polyanion also decreased the bodipy-atp binding to mitochondria with the effect being reduced by alamethicin or digitonin. control experiments showed that bodipy-atp did not bind to liposomes showing minor role of unspecific binding. it was suggested that bodipy-atp in combination with fcs can be used to monitor the functional state of mitochondrial vdac which is considered to be a principal regulator of mitochondrial function. fluorescence correlation spectroscopy studies of lysozyme partition to phospholipid vesicles a. m. melo 2 , a. coutinho 2 , m. prieto 1 1 cqfm and in, ist, 1049-001 lisboa, portugal, 2 dqb, fcul, 1749-016, lisboa, portugal binding to membrane lipids has been increasingly recognized as an important step in the aggregation and cytotoxicity of several amyloidogenic proteins [1] . in addition, it has been recently reported that membranes containing negatively-charged phospholipids can also trigger rapid amyloid-like fiber formation by a variety of several nonamyloidogenic proteins, such as cytochrome c and lysozyme [2] . our study aims to elucidate the factors that govern the formation of these lipid-protein complexes. given the importance of electrostatic interactions between the proteins and the acidic phospholipids in the putative membrane-induced protein misfolding step, it is essential to first characterize quantitatively the protein partition behavior towards liposomes prepared with variable anionic lipid content. in this study, lysozyme was chosen as a model protein and fluorescence correlation spectroscopy (fcs) was used to monitor its binding to liposomes after its conjugation to alexa fluor 488. most organic dyes labelling techniques produce a mixture of populations of molecules labelled with a different number of fluorophores. the influence of this poli-dispersity of labelled molecules on the protein partition behaviour will be explored, namely the ability of the fcs technique to detect the production of non-competent membrane-binding species. t. wohland 1 , p. liu 1 , x. shi 1 , y. h. foo 1 , t. sudhaharan 2 , s.-w. chong 3 , v. korzh 3 , s. ahmed 2 1 chemistry dept., singapore nat. univ., 2 medical biology inst., singapore, 3 molecular & cell biology inst., singapore biomolecular interactions have been measured mostly under in vitro conditions because of higher accuracy and ease of measurement. however, it has become clear that the cellular environment has an important influence on these interactions. it was therefore necessary to develop new tools to allow the measurement of interactions in cells and organisms. recently, we have developed a modality of fluorescence cross-correlation spectroscopy (fccs) called single wavelength fccs (sw-fccs), which uses one-photon excitation to excite two fluorophores with overlapping absorption but separable emission spectra. sw-fccs has been used to determine e.g. dimer fractions of proteins in live cells. here we aim to extend the use of sw-fccs to cells and organisms. in the first part we determine the dissociation constants of a small rho-gtpase (cdc42) with an effector domain (crib) and two effectors (n-wasp or irsp53). in the second part we measure the binding between cdc42 and a scaffolding protein (iqgap1) in dependence of their expression levels in cho cells and in live zebrafish embryos. by using gfp/mrfp fusion proteins we can excite both fluorophores at 514 nm and detect them separately in two different wavelength channels. we quantitatively determine the dissociation constants and compare their differences in vitro, in cells, and in embryos. these experiments demonstrate that sw-fccs is a powerful biophysical tool for the quantitation of biomolecular interactions in cells and organisms. addressing plasma membrane structure at the nanoscopic length-scale s. wieser, m. axmann, g. j. schütz biophysics institute, johannes kepler university linz, altenbergerstr.69, a-4040 linz, austria there is increasing interest in a detailed understanding of the structure and dynamics of the cellular plasma membrane, primarily based on recognizing its essential role for controlling cellular signaling processes. we employed single molecule fluorescence microscopy to study diffusion of cd59, a gpi-anchored protein, in the plasma membrane of living t24 cells at sub-wavelength resolution, both on the cell body and on tunneling nanotubules connecting cells. the lateral motion of this single fluorescence labeled molecule was imaged on a millisecond time scale. within the experimental errors, no indications for confined diffusion for cd59 on the cell body in t24 cells have been found. furthermore by separating longitudinal and transversal mobility, we found isotropic diffusion behavior on the surface of tunneling nanotubules. in both studies we analyzed the mean square displacement as a function of the time-lag and the distribution of displacement steps. however, a closed analytical theory for these analysis is only available for the simplest models. to address a suspected diffusion process we reasoned that a full analytical description may not be required; it may well be sufficient to compare the experimental data with monte carlo simulations of the process. we demonstrated the working principle for this simulation based analysis for free diffusion, hop diffusion and transient binding of the tracer molecule to slowly moving receptors. n. chakroun 1 , f. fraternali 1 , m. malfois 2 , h. rezaei 3 , c. a. dreiss 1 1 king's college london, u.k., 2 diamond light source, didcot, oxfordshire, u.k., 3 inra, jouy-en-josas, france prion(prp) diseases are fatal neurodegenerative diseases affecting mammals including human and sheep.they are characterized by the accumulation of extracellular βrich fibrillar deposits of a structurally modified form (prp sc ) of the cellular prp c .despite the increasing interest for prp diseases,the mechanism of prp c /prp sc conversion is still unknown.studies on prp diseases suggest that neurotoxicity arises from small pre-fibrillar oligomers.we have used a range of biophysical techniques combined with molecular dynamics simulations (md) to resolve the oligomerization pathways of sheep prp (sprp).we have shown that under well established conditions, sprp oligomerizes into three oligomers, which form in parallel.in addition, we have now identified the minimal region of sprp leading to the same oligomerization profile of the entire sprp,namely h2h3.low resolution shapes of sprp, h2h3 and resulting oligomers have been determined by small-angle x-ray scattering.time-resolved studies have been used to follow the oligomerization of sprp and h2h3 monomers into the oligomers.the conversion of sprp sc at the molecular scale was studied by md.simulations of the h2h3 region recreating experimental conditions revealed a complete unfolding of h2 helix followed by h3 helix.these crucial steps are followed by the formation of β-sheet structures leading to a stable βrich double hairpin structure. single-molecule force spectroscopy investigation of the conformational equilibria of alphasynuclein m. brucale 1 , a. rampioni 1 , m. sandal 1 , i. tessari 2 , l. tosatto 2 , l. bubacco 2 , b. samorì 1 1 istituto di biochimica g.moruzzi, università di bologna (italy), 2 dipartimento di biologia, università di padova (italy) alpha-synuclein (asyn) is an abundant intrinsically disordered protein (idp) primarily located at presynaptic terminals. mutations in the gene encoding asyn have been linked to early-onset parkinson's disease (pd). by means of single molecule force spectroscopy (smfs) experiment, we show how the conformational equilibrium of monomeric wild type (wt) asyn shifts toward more compact structures in several unrelated conditions linked to pd pathogenicity [1] . the conformational heterogeneity of pathological alpha-syn mutants a30p, a53t and e46k has also been characterized, revealing marked differences in the conformational behaviors of the mutants with respect to wt asyn [2] . all the mutants show a distinctively higher propensity, with respect to wt, to acquire a monomeric compact conformation that is compatible with the acquiring of beta structure. the same smfs experimental methodology is then used to characterize the conformational behavior of wt and mutant asyn in a variety of conditions, in an attempt to gain insight about the multiple and contrasting parameters controlling the equilibrium. in vitro protein folding studies using chemical denaturants have contributed tremendously to our understanding of the folding thermodynamics and kinetics of water-soluble proteins. this is not the case for integral membrane proteins, which constitute about one third of all eukaryotic proteins and more than half of all validated and potential drug targets. fully reversible denaturant-induced unfolding remains limited to a few β-barrel porins, whereas the much larger and more relevant class of α-helical membrane proteins has thus far evaded this approach. we report here the first example of an α-helical membrane protein that can be unfolded completely and reversibly by a chemical denaturant: mistic, a 110-residue protein from bacillus subtilis [1] , dissociates from detergent micelles or lipid vesicles and assumes an unfolded monomeric state on titration with urea. using spectroscopic and microcalorimetric techniques, we exploited this unique property to provide (i) a quantitative comparison of membrane protein stability in different membrane-mimetic systems; (ii) an experimental test of controversial predictions [2] regarding the folding core of mistic; and (iii) a convenient setup to study the spontaneous, translocon-independent membrane insertion of this unusual membrane protein. the mechanical functioning of biological tissues is important from many viewpoints such as diseases, clothing and even food. the protein collagen makes up the greater part of these tissues, and is remarkable for its many uses in the body, however there are at least two other major components. one of the most interesting properties of these tissues is their non-linear behavior under stress. this behavior is essential to prevent a catastrophic failure such as an aneurysm. at least three major theories have been proposed within the past few years to explain this behavior, but have been impossible to verify. in order to determine a correct description of the mechanical structure of the tissue we have been using cutting edge technological solutions to address the single molecules within the extra cellular matrix. this technique combines optical methods with single molecule force spectroscopy, allowing stiffness measurements over the nanoscale as well as determining the individual protein tensions within the extra cellular matrix. the results show that this method can be used to determine the network properties even in the complicated aortic wall enabling better understanding of disease states, which in this case include marfan's syndrome and ascending aortic aneurysms. beta amyloid peptide abetapy3-42 shows a faster aggregation kinetics than abeta1-42 c. d'arrigo 1 , m. tabaton 2 , a. perico 1 1 institute for macromolecular studies, national research council, 16149 genova, italy, 2 department of neurosciences, university of genova, 16132 genova, italy we test directly the differences in the aggregation kinetics of three important beta amyloid peptides, the full-length abeta1-42 and the two n-terminal truncated and pyroglutamil modified abetapy3-42 and abetapy11-42, found in different relative concentrations in the brains in normal aging and in alzheimer disease. 1 we find by following the cd signal and the tht fluorescence of the solution in phosphate buffer, a substantial faster aggregation kinetics for abetapy3-42. this behavior is due to the particular sequence of this peptide which is also responsible of the specific oligomeric aggregation states, found by tem, during the fibrillization process which are very different from those of abeta1-42, more prone to fibril formation. in addition abetapy3-42 is found here to have an inhibitory effect on abeta1-42 fibrillogenesis, coherently with its known greater infective power. this is an indication of the important role of this peptide in the aggregation process of beta-peptides in alzheimer disease. the puzzle of the anomalously long denaturation kinetics of green fluorescent protein (gfp) mutants still is largely unveiled. in this study we have followed the effect of mutation h148g on the stability of gfpmut2 (mut2g) in the presence of guanidinium hydrochloride (gdnhcl). different techniques of fluorescence spectroscopy have been employed in order to obtain information concerning the unfolding event: time resolved fluorescence, fluorescence correlation spectroscopy (fcs), and fluorescence anisotropy. the substitution of the histidine with glycine affects protein stability versus ph: in particular mut2g kinetics is not ph dependent and at basic ph values the protein is less stable. the fluorescence properties (quantum yield, lifetime) and the rotational correlation time are unchanged during the unfolding dynamics, while the number of fluorescent proteins decreases exponentially. an extrinsic probe, bound to cysteine 48, has been employed in order to gain more insights on the unfolding process, monitoring the stability of a different region of the protein. in particular, it has been found that a softer region is present around cysteine 48 in both gfp variants, showing that the unfolding process does not follow a simple two step mechanism. recently, negatively-charged membranes were reported to catalyze amyloid fiber formation by amyloidogenic peptides/proteins and also to induce formation of "amyloidlike" fibrils by nonamyloidogenic proteins. here, we used an approach which combines steady-state and time-resolved fluorescence measurements to obtain structural information about these supramolecular assemblies and to gain insights about the factors that control their formation. by exploring a wide range of lipid concentrations, the interaction of alexa488-lysozyme with phosphatidylserine-containing membranes was found to be a complex multi-step process, critically dependent upon the protein-to-lipid molar ratio (p/l) used. upon increasing the total lipid concentration in solution, there was a balance between an increased overall protein binding to the lipid vesicles and a progressive protein dilution on the membrane surface. as the surface potential of the vesicles decreased upon increasing the protein interfacial coverage of the liposomes, the protein binding mode was found to switch from a peripheral binding of lysozyme to the anionic headgroups (at low to intermediate p/l) to a partial insertion of the basic protein into the hydrophobic core of the membrane (at a high p/l). it is hypothesized that disruption of the protein tertiary structure might be a stepwise process beginning with loosening of the structure caused by its deeper insertion in the membrane bilayer. unexpected scaling laws in the mechanical unfolding of single protein molecules m. clusel, e. i. corwin, h. lannon, j. brujic center for soft matter research, physics department, new york university, new york, ny, usa it is a question of fundamental importance to understand the response of proteins to a stretching force, particularly in the case of mechanically active proteins, such as those in muscle fibers. we aim to understand how the structure and topology of a protein affect its resilience to external forces and presumably its function. owing to recent advances in single molecule force-clamp spectroscopy using the atomic force microscope (afm), we are now able to probe the structure and dynamics of single proteins under a constant stretching force by measuring their end-to-end length over time. the probability distribution of unfolding times at a given force allows us to estimate the strength of the protein in terms of a characteristic energy barrier, while the shape of the distribution provides a window into the microscopic mechanism by which the protein breaks apart. here we show a novel scaling of the unfolding kinetics with the stretching force, which deviates significantly from the currently accepted bell model. instead, we propose a physical picture for forced unfolding that is analogous to the mechanics of fracture. v. garcía-gonzález, j. mas-oliva instituto de fisiología celular. universidad nacional autónoma de méxico. 04510 méxico, d.f. méxico. studies focused on the thermodynamic and kinetic analysis have demonstrated that transfer of neutral lipids such as cholesterol esters through an aqueous phase is a highly costly biophysical event. therefore, nature has developed a series of lipid transfer proteins such as the cholesterylester transfer protein (cetp) designed to efficiently lower the energy barrier for transfer of cholesterol-esters between lipoproteins through an aqueous environment. employing circular dichroism we evaluated the secondary structure stability of a small peptide derived from the carboxy-end of cetp (helix y ) in a wide range of ph's. the percentage of α-helix is diminished only at extreme temperatures and acidic ph's in a reversible way. we report that while a mixture of phosphatidylcholine/cholesteryl-ester forms large aggregated particles independently of ph, inclusion of helix y to the mixtures close to neutral ph's allows the formation of small micellar-like structures confirmed by dynamic light scattering and electron microscopy. these results suggest that helix y when close to physiological ph values presents the ability to organize a micellar structure around itself. this type of organization allows the process to dramatically lower the energetic barrier for lipid transfer through aqueous media, phenomenon directly related with the facilitation of lipid transfer between lipoproteins. mimicking metastasis by a novel microfluidic approach there is increasing evidence that cancer metastasis shares commonalities with thrombosis. the von-willebrand-factor (vwf), a mechanical active blood clotting protein appears to be a particular potent candidate to bridge the gap between clotting and cancer extravasation. modeling the crucial physiological conditions of the blood circulatory system, for an in situ study of blood clotting-metastasis connections is not only, absolutely necessary, but also a challenging task. here, we present acoustically driven flow as a novel microfluidics method for mimicking the blood flow. this method enjoys very beneficial advantages of possibility of handling very little volumes of fluids, together with freedom to model most of the geometries present in our microcirculatory system. one technologically challenging, yet physiologically important factor, is the hydrodynamic condition in a bifurcated vessel, where complex shear profiles arise. we present a model to mimic these conditions and discuss the impact of hydrodynamics on vwf mediated cancer cell adhesion in bifurcated vessels of our microcirculatory system. protein structural changes occurring in flows stresses inherent to viscous fluid flow have previously been associated with protein unfolding, although structural changes have not been well documented as a function of relevant hydrodynamic parameters. we have used raman spectroscopy to monitor the structure of various protein solutions in situ for multiple flow scenarios within a concentric cylinder fluidic device (1) . the flows, which ranged from circular couette to wavy taylor-couette flow, were characterised experimentally using particle image velocimetry. several proteins were observed to change conformation when exposed to these flows, although the nature of these changes was protein specific. shearing hen egg white lysozyme in water altered the protein backbone structure, while similar shear rates in a 95% glycerol, 5% water solution affected the solvent exposure of the side chain residues near the exterior of the α-domain. the solventdependent response may be due to the flow topology, viscous stress, or the surface hydration properties. comparison of spectra acquired at different time points, including before and after flow, confirmed that the observed changes are reversible and independent of fluid stress exposure time. the scripps research institute, la jolla, ca, usa. intrinsically disordered proteins are increasingly found to play major roles in cell biology and disease. we are utilizing single-molecule fluorescence methods to probe these complex and highly dynamic molecules, allowing more direct measurements of structural distributions and dynamics, while avoiding loss of information due to ensemble averaging. in one example, we investigated the structural dynamics of sup35-nm, whose regulatable amyloid formation is believed to have functional significance in yeast. using a combination of single-molecule fret as a molecular ruler, coincidence to interrogate intermolecular interactions, and correlation analysis to probe conformational dynamics, we showed that the monomeric protein populates an ensemble of compact and rapidly interconverting conformations. a particularly interesting feature of intrinsically disordered proteins is that they are relatively unstructured in isolation, but can gain stable structure by interaction with binding partners. in this context, we used single-molecule fluorescence to characterize the complex folding pathway for the parkinson's-related idp alpha-synuclein induced by binding to a lipid-mimic. this combined single-molecule fluorescence methodology provides a powerful approach for detailed studies of the coupling of folding and dynamics with interactions and biology of this important class of proteins. m. ito 1 , j. johansson 2 , r. stromberg 1 , l. nilsson 1 1 department of biosciences and nutrirtion, karolinska institutet, huddinge, sweden, 2 department of anatomy, physiology and biochemistry, swedish university of agricultural sciences, the biomedical centre, uppsala, sweden amyloid β-peptide (aβ) is a 39-42 amino acid polypeptide and known to aggregate and form insoluble amyloid fibril, which is regarded as a primary cause of alzheimer's disease (ad). the discovery of practical and effective treatments and drugs for ad has been waited eagerly. in a recent experimental study, it was suggested that stabilization of the helical conformation of the aβ middle region, which strongly favors collapsed coil formation in the extracellular environment, would reduce the aβ fibril formation. based on the experimental evidence, inhibition of the unfolding of the aβ α-helix can be a forceful strategy to repress the aβ fibril formation resulting in prevention of ad. however, the detailed mechanism of the unfolding of the aβ α-helix has remained unclear, because the x-ray or the nmr structure of the aβ α-helix in aqueous solution has not been reported due to its instability. the aim of this study is to find effective ways to inhibit the unfolding of the aβ middle region, which is a prerequisite for the aβ fibril formation. in this study, we attempted to elucidate the molecular mechanism of the aβ unfolding by molecular modeling and molecular dynamics (md) simulations. the md simulations were performed for α-helical structures of a wild-type aβ(13-26) model and a mutant aβ(13-26) model. linker average hydrophilicity as a tool to discriminate between extended and non-extended calcium binding proteins a. isvoran 1 , e. quiniou 2 , c. craescu 2 , l. mouawad 2 1 west university of timisoara, department of chemistry, pestalozzi 16, 300115 timisoara, romania, 2 inserm u759, centre universitaire paris-sud, bâtiment 112, 91405 orsay, france the ef-hand calcium binding proteins (cabps) may exist either in an extended or a compact conformation, sometimes correlated with their functions. for the cabps with know structure and function, calcium sensors are usually extended and calcium buffers compact, hence the interest to predict the form of the protein starting from its sequence. in this study we used two different procedures, the sosuidumbbell algorithm and a novel procedure that is based on the linker average hydrophilicity (lah). the two procedures were tested on 17 known-structure cabps and then applied to 59 unknown-structure centrins. the so-suidumbbell algorithm yielded the right conformations for 15 of the 17 known-structure proteins and predicted that all centrins should are compact. the lah procedure discriminated well between the extended and non-extended forms of all the known-structure cabps and it reflected well the phylogenetic classification of centrins being a simple and powerful means to discriminate between extended and nonextended forms of cabps. only few residues that constitute the linker are responsible for the form of the cabp, showing that this form is mainly governed by short-range interactions. (http://u759.curie.u-psud.fr/modelisation/lah) lipid and protein organization of hepatitis b antigen characterized by fluorescence spectroscopy v. greiner 1 , c. egelé 1 , s. oncul 1 , f. ronzon 2 , c. manin 2 , a. klymchenko 1 , y. mély 1 1 laboratoire de biophotonique et pharmacologie, umr 7213 cnrs, faculté de pharmacie, université de strasbourg, france, 2 sanofi pasteur, 1541 av. marcel mérieux, 69290 marcy l'étoile, france. hepatitis b surface antigen (hbsag) particles are 20 nm lipoprotein particles, mainly composed of the major s surface viral protein containing 13 trp residues and yeast-derived lipids. since the structure of these particles is still missing, we further characterized them by fluorescence techniques. fcs indicated that the particles diffuse mainly as monomers and contain about 80 proteins per particle. fluorescence quenching and time-resolved fluorescence experiments showed that the fluorescence signal is largely dominated by the trp residues at the protein surface. moreover, time-resolved anisotropy measurements indicate that the protein motion is restricted and that the surface trp residues exhibit both local and segmental motions. the lipid part of the particles has been studied by environment sensitive 3-hydroxyflavone probes and viscosity-sensitive dphbased probes, and compared to lipid bilayers and low density lipoproteins (ldls), taken as models. the results suggest that the lipid part of hbsag is closer to ldls than to model lipid bilayers. we present an extensive calorimetric study of bovine alphalactalbumin for various ca++ content. equilibrium dsc raw data are analyzed and the melting temperature tm, the specific heat jump deltacp, the heat of unfolding deltahm are directly extracted. the binding of calcium on the native (n) state greatly stabilizes the protein, essentially by the enthalpic difference between the unfolded (u) and n states. we show that subsequent addition of calcium in the mm range stabilizes further the n state. the equilibrium calorimetric measurements are completed with out of equilibrium stopped flow refolding kinetics by cd spectroscopy performed at different temperature and ca++ concentrations. we discuss the possible stabilization mechanisms compatible with our measurements. protein unfolding/refolding in cellular compartments: application of luciferase constructs our studies show that a reporter enzyme, firefly luciferase, can be used for evaluation of the stress-induced proteotoxicity within different cellular compartments such as the nucleus, cytoplasm or mitochondria. in transfected mammalian cells, firefly luciferase is localized in microsomes. we engineered plasmid constructs encoding luciferase with inserted specific sequences that ensure its cytoplasmic or intranuclear, or intramitochondrial localization. in addition, we fused luciferase to the green fluorescent protein (gfp) that enables to visualize patterns of the compartment-targeted product. using such gfp-labeled constructs we had a possibility to monitor protein unfolding, aggregation and refolding in the cytoplasm, nucleus and mitochondria of transfectants exposed to either stressful conditions. gfp-luciferase expressed in mammalian cells behaves as a relatively labile protein which can undergo reversible unfolding and aggregation in response to heat shock, atp depletion or action of toxic agents. in the case of cell recovery, refolding of denatured luciferase is carried out at the chaperone machine. we explored unfolding/refolding of gfp-luciferase in the cytoplasm, nucleus and mitochondria of ischemia-stressed rat cardiac cells and in several cancer cell lines treated with hyperthermia or some chemotherapeutic drugs. the results obtained have revealed intriguing correlations between the proteotoxic impact within either compartment and the viability of treated cells. amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid bilayers s. jha 1 , d. sellin 1 , r. seidel 2 , r. winter 1 1 biophysical chemistry, department of chemistry, tu dortmund university, otto-hahn str. 6, d-44227, dortmund, 2 max-planck-institute for molecular physiology, otto-hahn str. 11, d-44227, dortmund, germany the islet amyloid polypeptide (iapp), which is considered as the primary culprit for β-cell loss in type 2 diabetes mellitus patients, is synthesized in the β-cells of the pancreas from its precursor, the pro-islet amyloid polypeptide (proiapp). proiapp is co-processed in the secretory granules and co-secreted to the extracellular matrix together with insulin as iapp. here, we compare the amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. the two peptides show an enhanced amyloidogenic propensity in the presence of negatively charged membranes and similar secondary structural properties. however, proiapp shows a much less amyloidogenic propensity, probably due to the increased net charge on proiapp, compared to iapp. unlike iapp, proiapp forms small oligomeric structures at the lipid interface, having heights of ∼3.5 nm. this morphological distinction can be attributed to the presence of the pro-region, flanking the amyloidogenic iapp. the addition of proiapp to iapp marginally delays iapp fibrillation, probably by interfering with the interaction between amyloidogenic iapp cores of distinct iapp molecules. thus, it appears reasonable to speculate that the pro-region of the proiapp could serve to delay the fibrillogenesis of iapp at negatively charged lipid bilayers. the role of transmembrane domain interactions in the kinetics and folding of cpt 1 z. a. jenei 1 , k. borthwick 2 , v. a. zammit 2 , a. m. dixon 1 1 chemistry dept., univ. of warwick, coventry, uk, 2 clinicalȃsciencesȃres.ȃinst., warwick univ., coventry, uk carnitine palmitoyltransferase i (cpt 1) enzymes are polytopic integral membrane proteins in the outer membrane of mitochondria (omm). cpt 1 controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for βoxidation. the two catalytically active isoforms, cpt 1a and cpt 1b, are different in their inhibitor binding kinetics and structure (interaction between n-and c-segments, interactions of transmembrane domains (tmd)). it has been suggested that inter-and intramolecular tmds interactions are important for cpt 1a, but not for cpt 1b function. cpt 1a has also been implicated in formation of oligomeric complexes through its tm segments. the study of tm helix-helix interactions in cpt 1 isoforms could lead to a better understanding of their function and inhibitor binding kinetics, and will contribute towards the design of pharmacological strategies aimed at modulating the activities of cpt 1 enzymes in conditions such as diabetes. to investigate the ability of the tmd in cpt 1 to self-associate and the order of any oligomers formed, several biochemical and biophysical techniques have been used. we found the self-association of rcpt 1a tmds (tm1, tm2) to be different as measured using the in vivo tox-cat assay. chemical cross-linking and analytical ultracentrifugation studies demonstrated formation of both trimers and hexamers by the rcpt 1a tm2 peptide. these results provide further evidence that tm2 plays role in formation of a channel in the omm. self-assembly of transmembrane domains in cpt1: role of gxxxg(a) motif in possible channel formation z. a. jenei 1 , k. borthwick 2 , v. a. zammit 2 , a. m. dixon 1 1 department of chemistry and, 2 warwick medical school, university of warwick, coventry, uk carnitine palmitoyltransferase 1a (cpt1a), a membrane protein that controls the rate of oxidation of long-chain fatty acids, is of key importance in diabetes and has recently been reported to exist as an oligomer in vivo. we have investigated full-length cpt1a and find that the protein exists as a hexamer in liver mitochondria. using mutants of cpt1a expressed in yeast mitochondria, we have localised key protein interactions in the hexamer to the transmembrane (tm) domains of the protein. detailed study of the tm domains in isolation, in both e.coli membranes and detergent micelles, demonstrated that while tm1 shows little self-assembly, tm2 displayed significant self-association. biophysical analyses of a tm2-derived synthetic peptide revealed oligomerization behaviour identical to native cpt1a in mitochondria, providing a strong link between tm2 helixhelix interactions and cpt1a hexamer formation. this is significant in light of a recent suggestion that cpt1a oligomerization may lead to formation of a channel in the mitochondrial outer membrane through which acylcarnitine gains access to the inter-membrane space. our data supports this new theory, and we go on to demonstrate experimentally the structural determinants of hexamer (channel) formation, specifically gxxxg(a) motifs in the tm domain which pack favourably in the hexamer and stabilize the oligomer. investigation of flexible loop role in structure and thermodynamic stability of firefly luciferase p. maghami, b. ranjbar, s. hosseinkhani department of biochemistry and biophysics, faculty of basic sciences, tarbiat modares university, tehran, iran protein folding, like any chemical process, consists of two fundamental components, thermodynamics and kinetics, which determine the stability of the folded state and the pathway of folding, respectively. these processes are currently too difficult to be solved de novo by purely computational methods. experimental evidence is required to simplify the problem via protein engineering .in this research, the wild type firefly luciferase (photinus pyralis) and some of its mutants were over expressed and purified. then their unfolding thermodynamics were examined, using circular dichroism and conventional fluorescence measurements. the unfolding equilibrium constant were measured over a complete rang of denaturant conditions. the measurements were shown structural and physico-chemical changes between wild-type and mutant proteins. exploring intrinsic disorder of unstructured membrane proteins by surface polymer physics intrinsically unstructured proteins (iups) are considered as a separate class within the protein world because they lack a well-defined folded structure. because iup's function is indeed directly linked to structural disorder, they are assumed to be natively unfolded. several physicochemical techniques are available to discriminate the degree of disorder. however a clear structural classification is still lacking. in this context, polymer physics emerges as a powerful tool for getting inside on the conformational abilities directly related to structural disordered of iups. in the present contribution, surface pressure and ellipsometry experiments in conjunction with polymer physics have been used to infer structural data in terms of molecular conformation and flexibility of a membrane protein essential for bacterial division, zipa. this protein has been pointed to posses a high molecular flexibility and to adopt a random coil conformation. folding dynamics of peptides studied by timeresolved infrared spectroscopy c. krejtschi 1 , o. ridderbusch 1 , r. huang 2 , l. wu 2 , t. a. keiderling 2 , k. hauser 1 1 institute of biophysics, university of frankfurt, germany, 2 department of chemistry, university of illinois at chicago, usa peptides are ideal model systems to study protein folding mechanisms. ir techniques provide both the necessary time resolution as well as the structural sensitivity. we initiate rapid heating by laser-excited ns temperature jumps (∼10 • c) and study fast ns-to-µs relaxation dynamics [1] . the dynamics of the α-helix to random coil transition of polyglutamic acid was analyzed under reversible folding/refolding ph-conditions. the observed relaxation kinetics allowed separation of the folding and unfolding process with additional use of ftir measurements in thermal equilibrium. sitespecific dynamics were monitored by use of isotopic labeling for a β-hairpin peptide whose conformation is stabilized by a hydrophobic core. various single and cross-strand isotopically labeled variants were analyzed. the isotope-edited kinetics show variations in local structural stability of the hairpin backbone. our data support a multistate dynamic behavior, and the site-specific kinetics are consistent with a hydrophobic collapse hypothesis for hairpin folding [2] . small heat shock protein hsp22 was predicted to belong to the family of intrinsically disordered proteins. one of the features of these proteins is that they do not demonstrate cooperative thermal transitions on heating. we applied different methods (dsc, ftir and intrinsic tryptophan fluorescence) to investigate the thermal unfolding of hsp22. dsc results have shown that thermal denaturation of hsp22 begins from 25 o c and occurs, with very low cooperativity, within a broad temperature region (up to 80 o c and above). the thermal unfolding of hsp22 is fully reversible. the ftir data show that heating of hsp22 from 20 to 70 o c results in complete disappearance of α-helices (from 8-12% to 0) and the decrease in β-sheets content from 42 to 30%. studies on the temperature dependences of tryptophan fluorescence have shown a significant red shift of the spectrum. these changes occurred within temperature region from 30 to 80 o c with midpoint at ∼56 o c. probably, this transition can be explained by destruction of β-sheets around trp96, the only trp residue of the α-crystallin domain of hsp22 (other 3 trp residues of hsp22 are localized in the n-terminal domain). the data obtained confirm the suggestion that hsp22 is a protein, whose significant part is intrinsically disordered. we propose that, on heating, the α-crystallin domain containing β-sheets melts at higher temperature than the n-terminal domain containing the most of α-helices. t. rosenkranz 1 , a. katranidis 1 , d. atta 1 , j. enderlein 2 , i. gregor 2 , m. grzelakowski 3 , p. rigler 3 , w. meier 3 , j. fitter 1 1 isb-2: molecular biophysics, research centre jülich, germany, 2 institute of physics, biophysics/complex systems, georg august university, göttingen, germany, 3 institut für physikalische chemie; universität basel, basel, swizerland the protein folding mechanism is the missing link in the biological flow of information from the dna to its specific function. since most of proteins within a cell consist of more than one domain studies on this protein class are of major importance. it is a common feature of multidomain proteins to aggregate under refolding conditions, which hinders a refolding. molecular encapsulation of single molecules prevents aggregation. by immobilizing the nanocapsule the observation period in a wide field microscope will be extended, so that slow or rare folding events can be detected. a major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein folding studies [1] . polymer vesicles maintain their structural integrity even under harsh unfolding conditions. furthermore the nanocontainer proved to be permeable to guanidinium hydrochloride. using encapsulated phoshoglycerate kinase, labeled with atto-655, a dye which experiences fluorescence quenching by photo-induced electron transfer (pet) with tryptophans, we demonstrate the remarkable properties of polymeric nanocontainers. applying pet as a folding probe we detected multiple unfolding/refolding transitions of single proteins. proteins frequently become irreversibly modified by carbonylation, a process of introducing the carbonyl group (carbon monoxide) in a reaction with reactive oxygen species (ros) such as superoxide, peroxide or ozone. the main targets for carbonylation in proteins are amino-acid side chains of lysine, arginine and proline. products of carbonylation are aminoadipic semialdehyde from lysine (asa) and glutamic semialdehyde (gsa) from arginine and proline. importantly, carbonylated proteins are marked for proteolysis by the proteasome, but can escape degradation and form aggregates that can be cytotoxic. carbonylation increases with the age of cells and it is associated with ageing and age related disorders such as alzheimer's disease, parkinson's disease and cancer. we have used the molecular dynamics method to study the stability of carbonylated proteins villin headpiece and ubiquitin. simulations were run after mutations of arginine, proline and lysine into gsa and asa had been performed. in addition, we have used thermodynamic integration on lysine, arginine, proline, asa and gsa residues in order to estimate their solvation free energy (related to relative hydrophobicity and hydrophilicity). our results suggest that carbonylation markedly decreases the overall stability of proteins, and that one potential reason for that may be a disruption of the balance between hydrophilic and hydrophobic regions in the protein. a. martino, d. crane, i. m. feavers, b. bolgiano division of bacteriology, national institute for biological standards and control, potters bar, uk the sensitivity to protein's secondary structure and progress in computational calculations have made circular dichroism (cd) an attractive technique to explore the optical properties of three promising vaccine candidates to neisseria meningitidis. clinical batches of a c-term deleted form of nada (genome-derived neisseria antigen -gna1994) and the fusion proteins gna2132-1030 (fp-1) and gna2091-1870 (fp-2) were therefore studied. increases in temperature and denaturant concentration on secondary structures and folding/unfolding profile were monitored by cd in the far and near uv regions and complemented by trp fluorescence spectroscopy data. furthermore, epitope conformational changes on binding activities to immune-sera were investigated. the calculated secondary structure content was in broad agreement with the available predicted or solved protein structures. upon temperature incubation, a structural transition from a highly α-helical nada to a more unordered conformation, with a mid point at ∼40 • c, was observed. fp-1 and fp-2 maintained their conformation up to 50 • c or 6m guhcl in the case of fp-1. unfolding was not always reversible. reductions in binding to monoclonal ab titrated along with increasing unfolding. folding/unfolding studies have proven useful in better understanding the solution behaviour and extent of folding of proteins. cold denaturation of yfh1 offers the clue to understand the effect of alcohols on protein stability s. r. martin 1 , v. esposito 1 , p. de los rios 2 , a. pastore 1 , p. a. temussi 3 1 national institute for medical research, the ridgeway, nw7 1aa london, u.k., 2 laboratoire de biophysique statistique, sb/itp, ecole polytechnique fédérale de lausanne (epfl), ch-1015, lausanne, switzerland, 3 dipartimento di chimica, università di napoli federico ii, via cinthia, i-80126 napoli, italy although alcohols are well known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. here we present a study of the effects of alcohols on protein stability using yfh1. exploiting the unusual property of this protein of undergoing cold denaturation around 0 • c without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol,ethanol methanol. in all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. we suggest that alcohols, at low concentration and physiological ph, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation. biomolecular sciences and biotechnology tor vergata moro 5, 00185 rome, italy cholesterol plays an important role in regulating the structural properties of phospholipid and non-phospholipid membranes. in this study we have applied in situ energy dispersive x-ray diffraction (edxd) to investigate the effect of cholesterol on the structure of different phospholipid and non-phospholipid oriented membranes. in detail, phosphatidylcholine (pc) bilayers and niosomal membranes, made of a non-ionic surfactant centre for bioactive chemistry, department of chemistry department of chemistry this process is initiated at nuclear envelope remnants (ners) in the presence of atp and gtp. the mvs can be divided in two main populations: mv1 and mv2. mv2 has a classical lipid composition while mv1 is enriched in phosphoinositides (pips: pi, pip, pip 2 and pip 3 ). ners have an unusual lipid composition, enriched both in cholesterol and pips. physicochemical properties of the pips were investigated as a function of ph and temperature (t) using nmr, saxs and dls to map out their phase state. pips-water dispersions are observed in lamellar, hexagonal or isotropic phases depending on t and ph. in parallel, model membranes mimicking mv1 and ners lipid composition were studied by 2 h and 31 p nmr. mv1 modelling shows a complex behaviour of pips on pc membranes: they order or disorder membranes, whereas the order of pc/pi/pip/pip 2 membrane is lower than that of pc or pc/pi membranes c. manzo, t. s. van zanten, m. f. garcia-parajo bionanophotonics group, ibec-institut de bioenginyeria de catalunya, barcelona, spain membrane proteins play a fundamental role in intra-and inter-cellular functions. in particular, the proteins lateral mobility in the fluid membrane environment is crucial for the regulation of several mechanisms, as receptor-mediated signal transduction and establishment of immunological synapses. these mechanisms are controlled through protein crowding and reduced lateral diffusion, which induce macromolecular associations and limit the application of conventional single molecule fluorescence techniques. to measure proteins mobility on living cells membrane, we developed a fluorescent correlation spectroscopy (fcs) setup in which the sample illumination is obtained through near-field scanning optical microscopy (nsom) probes. the use of nsom probes is particularly suited for the observation of dynamics on the cell membrane and overcomes the drawbacks of other techniques. in fact, through a shear-force-based position control, the probe is kept at a fixed distance from the membrane and its sub-wavelength aperture (∼100 nm) reduces the illumination area, allowing the observation of highly crowded regions of the membrane. on the basis of preliminary results, the nsom-fcs is expected to provide an additional insight on the proteins trafficking at the membrane level. the technique also presents several potential developments, as the further reduction of the illumination area and two-colors correlation. single molecule fluorescence microscopy of the store-operated calcium channel subunit orai1 j. madl, j. weghuber, d. bergmair, m. fahrner, m. muik, c. romanin, g. j. schütz johannes kepler university, institute for biophysics, linz, austria store-operated calcium entry (soce) is essential for many cellular signalling processes. the essential pore forming subunit of soce channels in the plasma membrane is orai1. here we present single molecule fluorescence microscopy of orai1 which was performed in order to directly visualize the stoichiometry of mobile orai1 pores. the protein was fluorescently labeled with monomeric gfp. a novel single molecule fluorescence approach, toccsl (thinning out clusters while conserving the stoichiometry of labeling), was used for the determination of the stoichiometry. this technique allows reducing the density of fluorescently labeled molecules without affecting the stoichiometry of labeling. density reduction is achieved by completely photobleaching a defined area within the plasma membrane; nonbleached gfp-orai1 aggregates enter the bleached region subsequently by diffusion. our data indicate that there are different populations of orai1 present in the cell: most of orai1 is located in the plasma membrane. a second population of orai1-mgfp was found to be localized in intracellular vesicles. a significant fraction of the plasma membrane orai1 exhibits a diffusive movement. we found by analyzing the bleaching characteristics of single orai1-mgfp aggregates that in resting cells mobile orai1 is predominantly dimeric. a. kobitski 1 , a. nierth 2 , m. helm 2 , a. jäschke 2 , g. u. nienhaus 3 1 university of ulm, germany, 2 university of heidelberg, germany, 3 university of karlsruhe, germany rna molecules have attracted enormous attention in recent years, and various novel roles were revealed for rna in biological processes. ribozymes are a class of rna molecules capable of catalyzing chemical reactions. we have studied a diels-alderase (dase) ribozyme, a small artificial 49-mer ribozyme, which is capable of catalyzing carbon-carbon bond formation between an anthracene diene and a maleimide dienophile in multiple turnovers. single-molecule fluorescence resonance energy transfer was employed to investigate the intramolecular dynamics of this rna molecule as a function of mg 2+ ion concentration. folding into a functional state occurs via an intermediate state, and continuous fluctuations between these two states were observed on the 100ms time-scale at the midpoint concentration of mg 2+ ions. an effect of substrates binding on the folding and catalytic reaction of the dase ribozyme is in the focus of our recent research with the ultimate goal to obtain a detailed structural view of the single-molecule conformational changes that accompany the catalytic reaction. a. katranidis 1 , r. schlesinger 1 , k. nierhaus 2 , i. gregor 3 , m. gerrits 4 , g. bueldt 1 , j. numerous studies showed that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins. here we present an approach employing a two color single molecule sensitive fluorescence wide-field microscope in order to visualize surface tethered fluorescently labeled ribosomes and de novo synthesized gfp molecules in real time [1] . fluorescence of co-translational folded proteins was observed from mature fluorescent gfp molecules which carry 31 additional amino acids at the c terminus remaining linked to the ribosome. thus it was possible to co-localize fluorescence from labeled ribosomes and from gfp molecules. we demonstrate that the green fluorescence protein mutant gfp emerald is produced with a characteristic time of five minutes. the fastest gfp molecules appeared already within one minute. processes precedent to chromophore formation, such as polypeptide synthesis and protein folding, are fast and last not longer than one minute. in fluorescence spectroscopy, photobleaching is a process which leads to irreversible loss of fluorescent properties of a dye molecule, usually due to photochemical reactions. it is especially important for fcs experiments on slow-diffusion systems since for high excitation intensities it can have a strong impact on fluorescence intensity correlation function. usually it is observed as apparent shortening of the mean diffusion time of the dye molecules. the behavior of tmr-labeled fd-virus rods in water solution under various excitation conditions was investigated. the experiments were conducted for low (1:1) and high (625:1) tmr:virus ratios and for increasing laser intensities. the correlation function was measured in the experiments. the results were fitted using origin software to estimate the influence of photobleaching, and compared with computer simulations. a strong effect of photobleaching was visible for rods labeled with a single dye molecule, while rods labeled with 625 tmr molecules showed little to no bleaching. a prolongation of characteristic diffusion times for highly labeled virus rods in comparison to low-labeled ones was also observed. k. toth 1 , a. gansen 1 , a. valeri 2 , v. böhm 1 , c. a. seidel 2 , j. langowski 1 1 abt. biophysik der makromoleküle, deutsches krebsforschungszentrum, heidelberg, germany, 2 lehrstuhl für molekulare physikalische chemie, heinrich heine universität, düsseldorf, germanythe nucleosome has a central role in the compaction of genomic dna and the control of dna accessibility for transcription and replication. we studied the effect of dna sequence and selective histone acetylation on the structure, stability and disassembly of the mononucleosomes. quantitative single molecule fret measurements between dyes attached to different parts of the nucleosome permitted us to detect the equilibrium between several subpopulations of reconstituted nucleosomes in solution. we obtained that the heterogeneity and stability of the samples are correlated with each other and influenced both by the dna sequence and the histone acetylation. the path of the linker dna is more sensitive to all studied effects than the dna on the core. intermediates of the disassembly pathway were identified and characterized. j. strömqvist 1 , s. johansson 1 , y. ohsugi 3 , k. andersson 2 , l. xu 1 , m. kinjo 3 , p. höglund 2 , j. widengren 1 1 experimental biomolecular physics, kth, stockholm, sweden, 2 department of microbiology and cell biology, karolinska institutet, stockholm, sweden, 3 laboratory of molecular cell dynamics, hokkaido university, sapporo, japan dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly49a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined.here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. dynamic multiple-target tracing probes spatiotemporal cartography of cell membranes in order to decipher the non random and non homogeneity of the plasma membrane organization, we had performed fluorescence correlation spectroscopy measurements on live cells. this allowed us to establish the presence of nanoscale confining structures 1 and to demonstrate their implication in signaling process 2 . complementing these studies, we present here a new analytical method, namely multiple-target tracing (mtt) 3 which takes advantage of the high resolution provided by singlemolecule sensitivity to generate dynamic maps at high densities of tracked particles. introducing deflation by subtracting detected peaks allows detecting peaks of lower intensity. we achieved an exhaustive detection of particles with performances reaching theoretical limits, and a reconnection of trajectories integrating the statistical information from past trajectories. we demonstrate the potential of this new method of analysis by applying it to the epidermal growth factor receptor labeled with quantum dots, in the plasma membrane of live cells. this has allowed us to build up a global representation of molecular dynamics in cell membranes. dual polarisation interferometry (dpi) is a surface analytical technique capable of dynamically measuring biophysical parameters of conformational change in biomolecular interactions. the technique measures three key parameters, namely layer thickness, layer density (ri) and mass, thereby enabling the resolution of conformational changes involved during binding. a number of different applications are presented. protein-protein interactions: understanding the biophysical nature of protein interactions can deliver insights into the mechanisms by which proteins interact, thereby elucidating protein function. dpi enables correlation between binding affinity and conformational change, greatly enhancing the study of structure-function relationships. lipid layers: the birefringence mode of dpi can be used to study the formation of lipid bilayers and biomolecular self-assembly. it is possible to use a combination of bilayer refringence and mass to study phase transitions associated with protein or peptide binding to the lipid bilayer. the individual stages of adsorption, absorption and micellisation can be distinguished. carbohydrate interactions: dpi uses a planar glass surface and a wide range of coupling chemistries. a carbohydratespecific surface can be used to study a wide range of biomolecular interactions, such as lectins, acidic proteins, extracellular matrix signaling and interactions with complex membranes. the experimental characterization of the elementary conformational steps constituting the protein folding pathway remains a big challenge. quenching of the triplet state of tryptophan by close contact with cysteine has been shown to provide a new tool for measuring the rate of intramolecular contact formation -one of the most elementary events in the folding process -in peptides and proteins using only natural probes. here we show an extensive study on a stabilized mutant of the second beta-hairpin of gb1 domain. steady state fluorescence and kinetics of contact formation between a natural tryptophan and a cysteine added to the c-terminal are measured for different temperatures and solvent conditions. we separately address the contributions of different structural elements to the overall stability of the hairpin. we extract kinetics parameter for contact formations in the unfolded state, formation of the hydrophobic core and tails pairing in the folded state. by means of a fragment peptide terminated with a tryptophan and a cysteine, we also estimate the structural propensity of the turn region. the data coherently combine with a simple model previously developed to describe the dynamics of unstructured chain [biophys. j. 96, 1515 (2009)], here modified with the addition of attractive interactions between specific residues. catalytic power of partially denatured enzymes: implementation of molten-globule-like states m. shushanyan 2 , d. e. khoshtariya 1 , m. makharadze 1 , t. tretyakova 2 , r. van eldik 3 1 institute of molecular biology and biophysics, gotua 12, 0160 tbilisi, georgia, 2 department of physics, i. javakhishvili tbilisi state university, 0128 tbilisi, georgia, 3 department of chemistry and pharmacy, university of erlangen-nürnberg, germany impact of nonspecific moderate denaturants, urea and dmso, on the kinetic (functional) and thermodynamic (stability) patterns of a hydrolytic enzyme, α-chymotrypsin (α-ct) has been investigated. furthermore, an impact of urea and guhcl in combination with of temperature on the kinetic pattern of carboxypeptidase a has been examined. for the case of α-ct, in particular, we have observed about tenfold increase of the apparent mickaelis constant when going from 0 to 6 m urea (25 o c), whereas the value of catalytic constant remained almost unchanged, indicating that the protein is not unfolded even under those severe conditions. the matching microcalorimetric experiments revealed that both the temperature-induced melting temperature and transition enthalpy decrease gradually with the increase of the additive concentration. with 6 m urea the peak-shaped calorimetric feature disappears totally. however, catalytic power of α-ct was preserved owing to its catalytic constant. for other studied enzyme/substrate/denaturant arrays diverse kinetic peculiarities due to mgls have been observed. rubredoxins are a class of iron-containing proteins whose biological role on electron transfer processes and metal binding is still unclear. the unfolding dynamics of the rubredoxin mutant rda51c from the mesophile desulfovibrio vulgaris (dv) was studied on the temperature range from 25 • c to 87 • c and along time at 87 • c. resonance energy transfer (ret) was used to determine the donor (d; trp37) to acceptor (a; 1,5-iaedans) distance. from 25 • c to 87 • c the d-a distance increased 4å. however the random coil expected d-a distance was only achieved after heating the protein solution during 2.5 hours at 87 • c. from uv-vis absorption data it's clear that this protein is capable of maintaining its iron-sulfur center at 90 • c. by melting the protein at the same temperature all iron-centers disintegrate and the protein unfold after 2.5 hour. the trp37 fluorescence also shifts 13 nm to the red reflecting the partial exposition of the indole ring to the solvent. from fluorescence and anisotropy decay curves a breathing type movement of the protein structure was observed between 30 • c to 60 • c without lost of significant secondary structure. this structure flexibility should play an important role on the thermal stability of the dvrd the antimicrobial peptide novicidin (nc) -modified from the sheep self defense peptide smap-29, for reduced mammalian cytotoxicity -is a cationic peptide (net charge +∼7.5) that adopts random coil structure in solution, but an α-helix in the presence of lipid vesicles. the conformation of nc in presence of the lipids dlpc, dlpg, dmpc, dmpg, dopc, dopg, dope, and dops in different combinations reveal the lipid selectivity, affinity, and phase dependent changes with varying l/p ratios and temperatures, observed from cd spectroscopy. it is understood that the conformational change is dependent on chain length and head group of the lipid. apart from the in vivo results on the nc activity, studies using qcm-d, dual polarisation interferometry, and calcein release assay reveal the kinetics and concentration dependent activity of nc in lipid bilayers and vesicles. preliminary studies on orientation of nc in various lipid environments using ssnmr, lsnmr, and molecular dynamics simulations apparently suggest that nc may form toroidal pores/detergent effects depending on the chain length of the lipids. further experiments on nc in presence of lipids using dsc, itc, ssnmr, oriented cd, ld, and confocal microscopy to determine the structure, thermal stability, orientation in lipid bilayers, and thereof the action of nc will help in proposing a comprehensive model for its mechanism of action in model membranes. dielectric method for measuring glass transition and denaturation temperatures of hydrated proteins g. e. thomas 1 , s. bone 1 , g. drago 2 1 institute for bioelectronic and molecular microsystems, bangor university, gwynedd, uk., 2 applied enzyme technology, pontypool, uk.the flexibility of protein structures is important in allowing the variety of motions, covering a wide range of magnitudes and frequencies, essential to biological activity. high frequency dielectric measurements can be used to study the flexibility of proteins by probing the relaxation of dipolar constituents of their structures. hydrated proteins exhibit a broad dielectric loss extending over the frequency range from 1mhz to 10ghz which can be decomposed into a number of constituent dispersions. one of these dispersions, with a relaxation time of ∼18ns, has been attributed to the relaxation of protein backbone peptide groups in the protein interior. in the work reported here, this dielectric dispersion was investigated as a function of temperature for the enzyme glucose oxidase. two critical temperatures were identified as the glass transition and denaturation temperatures, both of which were found to decrease with increasing protein water content. the results were consistent with a scheme in which the hydrated glassy protein undergoes a change in structural mobility at the glass transition temperature and experiences an irreversible change in conformation at a higher denaturation temperature. both glass transition and denaturation temperatures are key indicators of protein stability and are important in the production and storage of protein based pharmaceuticals. a. szymańska, k. kacprzyk, g.ślósarek department of molecular biophysics, a. mickiewicz university, umultowska 85, 61-614 poznań, poland aggregation dynamics of proteins plays an important role in molecular biology and medicine as it permits explanation of several disease states. an interesting problem is to find out in which conditions the interactions of the protein molecules lead to formation of ordered structures and in which to disordered ones. in this study, dynamic light scattering, circular dichroism and also congo red dye experiments were performed to analyze various structural states of lysozyme induced under different concentration of ethanol solvent. at low ethanol concentration the attractive interaction between the protein macromolecules dominate. after addition of more ethanol solvent, the translational diffusion coefficient was much smaller than that for lysozyme solution at zero ethanol concentration. it can be explained by the structural transformation of the polypeptide chain leading to a partially folded conformation needed for oligomerization and fibrillation process. on the basis of the cd experiments we concluded that the ethanol solvent induces changes in secondary structure of lysozyme solution. on addition of 75% v/v ethanol solution the intramolecular hydrogen bonds were destabilized. above this ethanol concentration, β -sheet were the dominant secondary structure of lysozyme in solution. the phase diagram illustrating the formation of: monomers, oligomers at various structural states, protofilament formation state, protofilament and amyloid fibrils was constructed. key: cord-023211-kt5gt26t authors: nan title: poster session abstracts date: 2007-08-29 journal: pediatr pulmonol doi: 10.1002/ppul.20700 sha: doc_id: 23211 cord_uid: kt5gt26t nan causing mutation is ∆f508, making studies of nbd1 essential in understanding cftr function. crystal structures have been solved of wild-type (wt) and mutant cftr nbd1, including variants containing ∆f508 (lewis et al. embo j. 2004; lewis et al. j. biol. chem. 2005; thibodeau et al. nat. struct. mol. biol. 2005) . cftr nbd1 contains a regulatory insertion (ri) and a regulatory extension (re) that contain pka phosphorylation sites. although the nbd core is very similar in the various structures, the ri and re in some structures of human wt nbd1 differ by 180°rotations, indicating that they are mobile. we have used nmr spectroscopy to study wt and ∆f508 nbd1 in the phosphorylated (phospho) and non-phosphorylated (non-phospho) states, and bound to different regions of cftr. despite their similar structures, differences are observed in the nmr spectra of wt and ∆f508 nbd1. these changes, possibly due to altered dynamics or interactions of the ri and re with the nbd core, will be discussed. the inherent stability and function of nbd1 could be affected by these changes, accounting for part of the ∆f508 defect. different interactions between wt and ∆f508 nbd1 with the icls could also affect channel function. to identify residues comprising the icls, we generated a homology model of cftr based on the crystal structure of sav1866 in which nbd1 and nbd2 form a productive dimer (dawson and locher. nature. 2006) . our data on a peptide corresponding to the first intracellular loop (icl1) indicate that binding to nbd1 requires phosphorylation of the ri. our homology model shows that the icl1 binding site is buried when the ri is bound to the nbd core. nmr data comparing phospho and non-phospho nbd1 indicates that phosphorylation of the ri and re disrupts their interactions with the nbd core, likely exposing the site for icl1 binding. the structure of the molybdate transporter modb2c2, with the nbds in an open conformation, shows icl1 binding is cognate nbd at a different interface (hollenstein et al. nature. 2007) . icl1 in modb2c2 interacts with the nbd near y93, the analogous residue to f508 in cftr. we will show binding data of icl1 with wt and ∆f508 nbd1 in different states and also present resonance assignments of nbd1 to map the icl1 interaction sites. since the sav1866 and modb2c2 structures were solved in different states, the different icl interactions may represent structural changes during the reaction cycle. the ∆f508 mutation may thus affect intramolecular associations in certain conformations, which would account for another part of the ∆f508 defect. probing differences in interactions of wt and ∆f508 nbd1 is critical for understanding the molecular basis of normal cftr function and of the altered cftr function in cystic fibrosis. shsps target ∆f508 cftr for erad via the sumo pathway ahner, a. 1 ; brodsky, j.l. 2 ; frizzell, r.a. 1 1. cell biology and physiology, university of pittsburgh, pittsburgh, pa, usa; 2. biological sciences, university of pittsburgh, pittsburgh, pa, usa the most frequent disease-causing mutation in cystic fibrosis (cf) is a deletion of phenylalanine at position 508 (df508) in the first nucleotide binding domain of cftr. essentially all of the mutant protein, as well as 70% of wild type (wt) cftr, is retained in the er and targeted for erassociated degradation (erad) by the 26s proteasome. key mediators of cftr folding and degradation are molecular chaperones, which help protein substrates fold, but can target them for degradation if folding efficiency is compromised. to identify factors modulating erad of cftr we performed a microarray analysis, screening for transcription profile differences between yeast expressing cftr and control strains, and identified enhanced transcript levels for the small heat shock protein hsp26 in yeast expressing cftr. we then demonstrated that cftr degradation was blocked in strains lacking the genes encoding the two yeast shsps, hsp26 and hsp42 (1) . to examine whether shsps regulate cftr biogenesis in mammalian cells, we asked which of the 10 human shsp homologues are endogenously co-expressed with cftr using rt-pcr and western blot analysis. we detected 5 shsps, including alphaa-crystallin and hsp27, in calu-3 and primary hbe cells. co-expression of alphaacrystallin or hsp27 together with wt or df508-cftr in hek293 cells selectively decreased the steady state levels of df508-cftr. pulse-chase experiments showed that the rate of df508-cftr degradation was enhanced when either shsp was over-expressed, but wt cftr biogenesis was unchanged. co-immuno-precipitation experiments in hek 293 cells revealed that alphaa-crystallin and hsp27 interacted preferentially with df508-cftr. thus far, our results suggest that shsps selectively increase df508-cftr's accessibility to proteasome-mediated degradation pathways. previously, hsp27 was reported to interact with ubc9, the sumo (small ubiquitin-like modifier) conjugating enzyme (2) . sumo is covalently linked to its substrates, often at sumoylation consensus sites; similarly to ubiquitin, this occurs through a series of thiol transfer reactions. we confirmed this interaction by co-immuno-precipitation and found that ubc9 as well as the sumo specific protease, senp1, are expressed in airway epithelial cells. over-expression of ubc9 decreased, while over-expression of senp1 increased df508-cftr protein levels. pulse-chase experiments indicated that these enzymes regulate selectively the degradation of df508-cftr, similarly to the shsps. preliminary studies in vitro and in vivo indicate that cftr, and particularly purified nbd1, is sumoylated and that hsp27 facilitates this process. mutational analysis of the 3 sumoylation consensus sites within cftr suggests a cycle of sumo modification that facilitates wt cftr biosynthesis but targets df508-cftr for degradation, probably because the balance between sumo addition and removal is impaired by an excessive interaction of shsps with the mutant protein. [supported by grants from the nih and the cystic young, a. physiology and biophysics, chicago medical school, north chicago, il, usa efficient retrieval of wtcftr from the cell surface is mediated by components of the clathrin mediated endocytic pathway; however many aspects of cftr endocytosis remain to be elucidated. we have previously shown that endocytosis of wtcftr is dependent upon recognition of a tyrosinebased motif in the carboxyl tail of cftr by the ap2 endocytic adaptor complex. in contrast to wtcftr, less is known about the mechanisms whereby mutant cftr undergoes internalization. according to one model, df508 cftr undergoes accelerated endocytosis compared to wtcftr, whereas others have argued that endocytic rates of df508 cftr are comparable to those of wild-type cftr. to further define the function of proteins involved in the initial steps of wt and mutant cftr endocytosis, we investigated the role of the gtpase dynamin. dynamin has been proposed to play a key role in endocytosis by facilitating the scission of nascent clathrin coated vesicles from the cell surface. until recently, very few tools were available to modulate clathrin mediated events. we took advantage of dynasore, a newly discovered small molecule, non-competitive inhibitor of the gtpase dynamin. dynasore acts as a potent, rapid and reversible inhibitor of endocytic pathways known to depend upon dynamin, by blocking coated vesicle formation within seconds of dynasore addition. endocytosis of wtcftr was inhibited rapidly and efficiently upon addition of dynasore. furthermore, within minutes of dynasore washout, endocytosis of wtcftr resumed completely. also upon dynamin washout, normal trafficking and recycling of wtcftr was also observed. temperature correction of df508 causes expression of cftr at the cell surface, however such 'rescued' df508 cftr is not stabilized upon returning cells to 37c. application of dynasore prevented such rapid loss of mutant cftr from the cell surface. since maintenance of mutant cftr at the cell surface is critical for effective therapeutic intervention in patients with cf, our results provide insight into additional potential targets for pharmacological manipulation for the treatment of cf. richardson, j.m.; thibodeau, p.h.; watson, j.; thomas, p.j. physiology, ut southwestern medical center, dallas, tx, usa protein misfolding is the basis for a multitude of human diseases; however, the mechanisms underlying misfolding are not well understood. most cases of cystic fibrosis are associated with mutations-including the most common, deletion of phenylalanine 508 (∆f508) in nbd1-that interfere with the folding of the cystic fibrosis transmembrane conductance regulator (cftr). the resulting loss of functional cftr causes the disease. thus, elucidating how ∆f508, affects the folding of cftr is critical to understanding the pathology of the disease. cftr is composed of five domains: two integral membrane transmembrane domains (tmds), a regulatory domain (r), and two nucleotide binding domains (nbds). ∆f508 occurs in the n-terminal nbd1. both the murine wildtype and ∆f508 nbd1 can be expressed in bacteria and purified to near homogeneity. while the soluble expression yield of ∆f508 is lower than the wild type under identical conditions, ∆f508 achieves a native conformation similar to wild type as monitored by a variety of hydrodynamic and spectroscopic characteristics such as analytical size exclusion chromatography, circular dichroism, and fluorescence. recently, a non-native, but folded, species has been detected under mildly denaturing conditions. far-uv cd reveals a time and temperature dependent conversion from a mixed alpha/beta native conformation to a less helical non-native conformation, while fluorescence measurements reveal a parallel blue shift in the peak emission intensity from 343 to 330 nm. this non-native species is in a more open conformation as determined by both limited proteolysis and the change in retention time on analytical size exclusion chromatography. the conversion to this state is inhibited by the native state ligand (atp) and by the presence of the second site suppressors (g550e, r553m, and r555k). notably, the ∆f508 nbd1 protein populates the non-native state under milder conditions than the wild type nbd1. these studies reveal the properties of the native state and its conversion to a partially folded state that is affected by the ∆f508 mutation. this data highlight the proximal affect of the disease causing mutation and provide a means of assessing strategies designed to correct the defect. this work is supported by the cystic fibrosis foundation through a postdoctoral training grant and by the nih we have previously demonstrated that the stability of cftr is negatively modulated by the cftr interacting protein, cal. cal competes with nherf in binding to the pdz motif at the c-terminus of cftr and directs cftr trafficking to the lysosome. recently, silencing of cal or overexpression of nherf has been shown to restore chloride transport activity in ∆f508-cftr cell lines. to identify the molecular machineries involved in cal dependent degradation of cftr, we investigated the role of the cal interacting snare protein syntaxin 6 (stx6). stx6 protein expression and function were manipulated by sirna silencing, over-expression, and a dominant-negative mutant. we also performed a functional study of chloride transport activity in cystic fibrosis bronchial epithelial cell line cfbe41o-stably expressing either wt-cftr or ∆f508-cftr. the endogenous stx6 and cal protein expressions were knocked down by sirna techniques in multiple human cell lines including hek293, hela cell stably expressing ∆f508-cftr and cfbe41o-stably expressing either wt-cftr or ∆f508-cftr. in hek293 cells, knockdown of either stx6 or cal augments the expression of transfected gfp-tagged cftr (gfp-cftr), which is consistent with the observation that overexpression of either stx6 or cal enhances the degradation of cftr. stx6 interaction with cal was confirmed by a co-immunoprecipitation assay. because of this interaction, we hypothesize that stx6 negatively regulates cftr protein stability by interacting with cal which in turn binds to the c-ter-minal pdz motif of cftr. consistent with his hypothesis, stx6 knockdown has no effect on protein levels of a cftr mutant lacking the pdz motif (gfp-cftr∆trl). more importantly, cal knockdown eliminates the inhibitory effect of stx6 overexpression on gfp-cftr. a cell surface biotinylation assay and confocal microscopy showed significant increases in plasma membrane expression of cftr in stx6 knockdown cells without gross changes in the expression pattern. furthermore, consistent with its trans-golgi localization, stx6 knockdown has no effect on gfp-∆f508-cftr expression. however, ∆f508-cftr increases significantly in stx6 knockdown, temperature-rescued cells. to assess the effect of stx6 in bronchial epithelial cells, stx6 was knocked down in cfbe41o-stably expressing wt-cftr. consistent with the observations in hek293 and hela cells, the expression of untagged wt-cftr is also augmented. knockdown of stx6 increases cftr expression in a does-dependent manner, reciprocal to the expression level of stx6, without changing the level of expression of cal or gapdh. furthermore, stx6 knockdown lead to a does-dependent increase in cftr mediated short-circuit current that is stimulated by forskolin and inhibited by cftr chloride channel inhibitor cftrinh-172. more importantly, in temperaturerescued cfbe41o-stably expressing ∆f508-cftr, stx6 knockdown tripled the cftr mediated short-circuit chloride current. these results not only delineate the effect of stx6 on post-golgi trafficking of cftr and its dependence on cal-mediated pdz-based interaction, but also points to it as a potential therapeutic target in conjunction with the er-quality control based rescue of ∆f508-cftr. supported by the cystic fibrosis foundation and the national institutes of health. seavilleklein, g. 1 ; evagelidis, a. 2 ; amer, n. 1 ; chappe, f. 1 ; hanrahan, j. 2 ; chappe, v. 1 1. physiology & biophysics, dalhousie university, halifax, ns, canada; 2. physiology, mcgill university, montreal, qc, canada cftr channel activity is regulated by phosphorylation and de-phosphorylation of the r-domain. direct pkc phosphorylation at specific sites in the r domain and nbd1 is essential and modulates cftr activation by pka. however, the molecular mechanism by which phosphorylation of the r domain induces channel activity remains unknown. we have reported biochemical evidence for the role of phosphorylation in domain-domain association using a deltar-split construct encoding the front (tmd1-nbd1) and back (nbd2-tmd2) halves of cftr as separate polypeptides (1) . in agreement with the inhibitory role of the r domain, iodide effluxes and patch-clamp experiments confirmed that deltar-split channels were functional and constitutively active without camp stimulation. co-transfecting a plasmid that directs expression of the r domain restored the pka-dependence of re-assembled channels. co-immunoprecipitations of the 2 halves of the deltar-split with a gst-r domain fusion protein in vitro or with the cotransfected r domain in bhk cells revealed that pka phosphorylation enhances the r domain binding to the rest of the channel. we have now investigated the effect of pkc phosphorylation on the r domain binding. our data demonstrate that pkc phosphorylation also induces a strong binding of gst-r domain in vitro or co-transfected r domain in vivo when bhk cells are stimulated with pma. interestingly, the combination of pkc+pka phosphorylation induced a stronger binding than pka alone. mutation of all consensus pkc sites on the r domain to alanine (7ca) eliminated specific pkc phosphorylation but did not alter pka phosphorylation in gst-rd. western blotting and confocal microscopy experiments confirmed that expression levels of all 3 domains of the split-7ca/rd are unchanged compared to split-rd and co-localize at the plasma membrane of bhk cells. basal activity of the split-7ca/rd was similar to that of the split-rd in iodide efflux experiments. however, activation by camp+ibmx was delayed and significantly reduced (36.92 % of split-rd and 13.85% of wt-cftr activation). these results confirm the functional re-assembly of the 7ca-rd with the front and back halves of the channel and the inhibitory role of the r domain. in co-immunoprecipitation experiments, the same level of interaction was observed between the 7ca/rd and rd with the split halves suggesting that basal, inhibitory interaction of the r domain is independent of pkc phosphorylation. however, 7ca/rd binding with the split channel was not increased by pkc or pka stimulation. we conclude that pkc phosphorylation is essential for pka-dependent binding of the r domain to the rest of the channel, and that the combined effect of these kinases further strengthens the r domain interaction. these structural changes are consistent with the essential role of pkc sites for cftr activation by pka as previously reported (2) . supported by cihr, nshrf, dmrf & ccff. g.seavilleklein was supported by nserc. (1) the in vivo folding mechanism of multidomain membrane proteins, including the cystic fibrosis transmembrane conductance regulator (cftr), is poorly understood. cftr, a member of the abc transporter superfamily, contains two symmetrical halves, each consisting of a membrane spanning domain (msd1 and msd2) and a cytosolic nucleotide binding domains (nbd1 and nbd2), connected by the regulatory (r) domain. to address whether cftr follows the archetypical domain-wise folding of multidomain soluble polypeptides and/or the cooperative domain folding mechanism, first we determined domain combinations that are necessary and sufficient to escape the endoplasmic reticulum (er) quality control. one-, twoor three-domain assemblies failed to be processed efficiently in cells, measured by biochemical and morphological assays. the smallest folding unit that escaped the er quality control was composed of a four-domain assembly, containing msd1, nbd1, r or msd2 as linear or split polypeptide. as a second approach we showed that cf-causing point mutations in the msd1, nbd1 or msd2 provoked the er retention of the full-length cftr with severe conformational defect in the nbd2, measured by limited proteolysis and immunoblotting, using domain spacific antibodies. the posttranslational folding kinetics of the four-domain folding unit was comparable to that of cftr, suggesting that cooperative domain assembly is essential for the channel biogenesis. this mechanism provides an explanation for the processing defect caused by numerous cf mutations and outlines a possible paradigm for cftr folding in living cells. cftr in post-golgi compartments. our results, jointly, uncover the role of n-glycans as a critical structural determinant of cftr conformational maturation/stability in a chaperone-independent manner. karamyshev, a.l. 1 ; patrick, a.e. 1 ; johnson, a.e. 2 ; thomas, p.j. 1 1. ut southwestern medical center at dallas, dallas, tx, usa; 2. texas a&m university, college station, tx, usa cystic fibrosis (cf) is caused by mutations in cftr (cystic fibrosis transmembrane conductance regulator). cftr, a member of the abc transporter family, is a multispanning membrane protein critical to chloride and water movement in secretory epithelia. over one thousand mutations, in all parts of the cftr sequence, have been identified, many of which disrupt the function of the protein to cause the disease. some of the disease-associated mutations in the n-terminal portion of cftr were predicted to alter cotranslational interactions with factors involved in the membrane integration of cftr required for its proper function. to test this hypothesis, a photoreactive probe was incorporated into different positions of the n-terminal portion of cftr by introducing amber stop codons and translating these messages in vitro in the presence of the amber suppressor trna n ε -(5-azido-2 nitrobenzoyl)-lys-trna amb . ribosome-associated nascent chains of a specific length containing the modified amino acid at a defined position were produced using truncated mrnas. some of these cftr nascent chains photocrosslinked with proteins of approximately 50 and 100 kda molecular mass. crosslinking to the ~50 kda protein was observed only after the tm1 span had emerged from the ribosomal tunnel. moreover, truncation analysis demonstrated that tm1 is necessary for this interaction. in evaluating a range of nascent chains, the extents of photocrosslinking to the ~50 kda and 100 kda proteins often varied inversely. immunochemical and knockdown/depletion studies indicate the ~50 kda protein is srp54, a subunit of the signal recognition particle involved in targeting ribosome nascent chain complexes to the translocon in the endoplasmic reticulum membrane. highlighting the functional relevance of this interaction, in cells with reduced srp levels, nonglycosylated forms of cftr accumulate and total cftr levels decrease. notably, some cf-causing mutations in tm1 also reduce crosslinking to srp. these data reveal that mutational inhibition of nascent cftr association with srp likely contributes to or mediates some types of cf. aleksandrov, l.; aleksandrov, a.a.; riordan, j.r. the initial reversible steps of atp binding to the first (nbd1) and second (nbd2) nucleotide binding sites of cftr are divalent cation independent and dependent, respectively (aleksandrov et al, j biol chem 277, 15419-25, 2002) . subsequent to the initial binding, either mg or mn drive rapid hydrolysis at the second site while promoting trapping of the nucleotide at the first site. this occlusion at the first site of functional wildtype cftr is somewhat similar to that which occurs when the catalytic glutamates in both of the hydrolytic sites of p-glycoprotein are mutated. this occluded state has been proposed to be the result of dimerization of the two nbds and represent a transient intermediate formed during atp hydrolysis (tombline and senior, j bioenerg biomembr 37, 497-500, 2005) . to test the possible relevance of this interpretation to cftr, we have now characterized the process by which nbd1 occludes [ 32 p]-8n 3 atp and [ 32 p]-8n 3 adp. we have now found that only n 3 atp and not n 3 adp can be bound initially at nbd1 in the absence of mg. consistent with this mg requirement for the ndp binding, n 3 adp or adp is able to compete for the n 3 atp binding site only when the divalent ion is present. despite the lack of a requirement for mg for atp binding, retention of the nucleotide triphosphate at the binding site at 37°c was dependent on the cation. however, at reduced temperature (4°c), n 3 atp remains locked in the binding pocket with virtually no reduction over a 1 hour period even in the absence of mg. these apparently paradoxical temperature effects hint that different molecular events can contribute to the occlusion of nucleotide at nbd1. that the occlusion process occurs exclusively at nbd1 and does not depend on its interaction with nbd2, was shown by experiments in which identical behavior was exhibited by ∆nbd2 constructs of cftr. thus, nucleotide trapping by wild-type cftr, unlike that by mutant p-glycoprotein, is accomplished by events at a single domain (nbd1), rather than by dimerization of the two nbds. (supported by the nih and cff). cftr consists of five domains or regions, two regions that span the cell membrane (the tmds), two cytoplasmic nucleotide-binding domains (the nbds), and a regulatory (r) domain. the r domain is unique to cftr: without phosphorylation of this domain, the cftr chloride channel does not open. the tmds and the nbds of cftr are similar to those found in many atp-binding cassette proteins, the majority of which are transporters. the domains are fused in a single polypeptide chain, linked in the sequence: tmd1, nbd1, r domain, tmd2, nbd2. the first nbd is the site of the location of the mutation that is responsible for the development of cystic fibrosis in the majority of human patients (loss of phenylalanine 508). although there is a structure for nbd1, we are still lacking a structure for the entire cftr protein. we have employed electron microscopy to provide low-to medium-resolution structures for the complete cftr protein which was expressed in bhk cells and purified by affinity chromatography. using single particle analysis combined with site-specific labelling using 1.8nm diameter ni-nta nanogold, we have identified the locations of nbd2 and the region around residue 696 of the r-domain within the low resolution 3d structure. by a process of elimination, this has also allowed the identification of nbd1 and the region occupied by the two tmds. these low resolution studies have now enabled us to interpret medium resolution 3d structural data obtained for cftr. progress in obtaining two-dimensional arrays (crystals a single molecule thick) should allow higher resolution structural data to be obtained for the entire cftr protein -sufficient for the identification of the transmembrane alpha helices. these data should allow a much clearer picture of the transmembrane channel formed by cftr and will inform molecular modelling approaches aimed at the development of novel drugs for the treatment of cystic fibrosis. we explored the gating process by introducing cysteines along the entire lengths of various transmembrane domains and extra-cellular loops, and studying their ability to chemically modified. we study the changes in their reactivity in closed and open channel states to characterize the changes in transmembrane domain conformations during gating. using membrane impermeant mts-reagents, we have characterized the conformational changes in tm1-ecl1, ecl3-tm6, ecl4-tm8, and ecl6-tm12 of cftr. our results suggest that atp binding induces modest changes in the transmembrane domains and extracellular loops. channel opening involves a rearrangement of the tm helices leading to decreased hydration of residues near the extracellular end. this conformational change is either a part of, or allosterically coupled to the gating mechanism. 14 physiological role of nherf1 in the regulation of cftr raghuram, v.; yang, y.; yaemsiri, s. laboratory of kidney and electrolyte metabolism, nhlbi/nih, bethesda, md, usa in the apical membrane of epithelial cells, cftr seems to exist within a multiprotein complex, in which its activity is regulated by interactions with other proteins. the c-terminus of cftr contains a pdz-binding motif, which binds to several pdz domain-containing proteins, including members of na+/h+ exchanger 3 regulatory factor (nherf) family, nherf1/ebp50, nherf2/e3karp, cap70/nherf3, nherf4, and cal/pist/gopc. pdz domain proteins act as scaffolds to assemble signaling proteins to form signaling complexes. these signaling complexes are central to achieve specificity and efficiency of signal transduction. nherf1 and nherf2 have a c-terminal erm domain, which interacts with cytoskeleton-associated erm proteins. this interaction may anchor cftr to the actin-cytoskeleton and restrict its movement within the membrane. in addition, several functional roles have been proposed for cftr-pdz domain interactions, including as an adapter to promote efficient pka phosphorylation of cftr, apical targeting and recycling of cftr, regulation of single-channel activity, organization of β-adrenergic, and lysophosphatidic acid-2 receptor signaling complex, and trafficking of romk channels. our current understandings of the role of cftr-pdz interactions are mostly obtained from heterologous expression studies. very few studies have addressed the role of pdz domain proteins in native epithelial cells or in organ physiology. we have used rna interference techniques to produce a knockout or knockdown (kd) calu-3 cell lines using a lenti-viral vector for stable suppression. using four different shrnas, we have generated four stable nherf1-kd cell lines with varying levels of nherf1 suppression. in these cells, nherf1 expression ranged from 10-40% of calu-3nt cells (control cells). in nherf1-kd cells, steady-state cftr protein level was greatly reduced, whereas its apical localization was unaffected. we also found structural defects in the apical microvilli. in polarized nherf1-kd epithelia, membrane-permeable camp analogs were unable to stimulate cftr mediated short-circuit currents. however, forskolin induced response was nearly normal despite reduced cftr expression. examinations of βadrenergic-, and adenosine-receptor signaling pathways suggest that nherf1 plays a unique role in organizing the adenosine receptor signaling pathway. abnormal retention of the df508 cftr mutated protein in lung epithelial cells underlies the pathology in a large proportion of individuals with cystic fibrosis. a drug discovery alliance between cfft and biofocus dpi was initiated with the aim to identify novel genes which upon shrna-mediated knockdown were able to efficiently restore df508 cftr activity. a high-throughput assay based on an yfp halide reporter was developed in a human cystic fibrosis bronchial epithelial cell line (cfbe41o-), whereby the rate of halide influx directly correlated to df508 cftr activity. biofocus dpi's proprietary adenoviral shrna libraries totalling 11,334 viruses were screened in this functional assay and yielded 753 hits. these 753 hits were repropogated to generate new adenoviral stocks and rescreened at three concentrations on the yfp halide reporter assay. 39 genes were eliminated due to potential cytotoxic effects, and hit calling was performed on the remaining 714 hits. a total of 315 duplicate hits were confirmed (44%). 7 of the confirmed hits had more than one shrnas which targeted the respective gene. 36 confirmed hits were stronger in restoring df508 cftr function than the most potent positive control: syntaxin-8. in order to elucidate which of these 315 hits induce halide influx via direct rescue and activation of df508 cftr, the yfp halide reporter assay was performed in the presence or absence of the cftrinh172. these cftr dependent hits will move forward into subsequent validation experiments whereby hits will be categorised into potentiators and correctors. the final goal of this program is to prioritize targets for entry into drug discovery. restoring expression and/or function of mutated cftr provides a means to treat cystic fibrosis. biofocus dpi -a galapagos company -in collaboration with the cystic fibrosis foundation therapeutics (cfft) has established a drug target discovery project that will enable the selection of proteins that rescue mutated cftr. by screening biofocus dpi's proprietary adenoviral shrna libraries in a high-throughput primary functional assay for cftr activity in human cells, we have identified and confirmed 315 hits out of 11,334 that entered the primary screen. to further validate these proteins as potential drug targets, we have set up a series of experiments and assays focused on the rescue of cftr expression and its maturation by knock-down of specific targets. 1) in order to establish whether the knock-down of a specific target was able to promote df508-cftr plasma membrane localization, we performed immunofluorescence stainings of df508-cftr in a patient-derived human cell line carrying the df508-cftr mutation (cfbe41o-) grown in air-liquid interface culture. a transduction protocol to deliver shrna adenovirus to this polarized airway cell model system has been optimized and preliminary results showed increased levels of expression of df508-cftr at the plasma membrane level upon knock-down of syntaxin-8, our positive control. experiments performed at 27 οc also demonstrated redistribution of df508-cftr to the apical plasma membrane at low temperature. we will show the results of all the shrna hits in this assay. 2) changes in the post-translational maturation levels of df508-cftr upon knock-down of specific proteins were evaluated by determining the accumulation of band b and c by using a western blot approach. in this way, we can select targets able to promote post-translational maturation of df508-cftr and/or its escape from er in the cfbe41o-cell line. accumulation of band b and c indicates maturation of cftr and will be considered as a beneficial effect. preliminary results show increased accumulation of band b upon knock-down of specific targets and overexpression of wild-type cftr induces band c accumulation. experiments performed at 27 οc also demonstrated increased band b expression. we will show the effect of all the shrna hits in this assay on band b and c expression. we believe that the results obtained by the combination of those two approaches will guide us in obtaining very strong drug targets for entry into a drug discovery phase. because gene therapy as a corrective measure for cf is possible in the foreseeable future, it is important to understand whether the global effects of gene introduction will have significant detrimental impact on cell function. to address this question, global gene mrna expression profiles were created with the affymetrix u133a & b chip sets. wild type cftr (n=1), ∆f508 (n=1) and g551d (n=1) were transfected in separate ib3 cell cultures using lipofectamine 2000™. control expression profiles were generated from untreated ib3 cells; one from this experimental set and five from previous control experiments. differential gene expression was determined by anova using a 10% false discovery rate threshold. genespring was used for data analysis and visualization. genego was used to determine which functional categories were over represented in the list and further assisted in interpretation of the results. rt-pcr was used to confirm differential expression of selected genes and examine the response in more detail. all transfected constructs produced very similar gene expression profiles quite distinct from the untransfected cells. 256 probe sets representing 228 uniquely named genes were significantly different between transfected and untransfected cells. cftr mrna expression in all forms was increased an average of 525-fold. transfection of ib3-1 cells resulted in upregulation of a variety of genes involved in several basic processes: inflammation, protein folding and cytoskeleton framework regulation. genego canonical pathways with a significantly enriched number of genes from the data set involved glucocorticoid signaling and regulation, cytoskeleton remodeling and adhesion plus a variety of other signaling pathways including il8, chemokines, g-protein and mapk cascades. cell processes overrepresented in the list were predominately associated with protein folding, inflammatory responses and cytoskeleton regulation. geneontology categories over represented in the list were regulation of signal transduction, receptor mediated endocytosis and protein folding. the proinflammatory interleukins il6 and il 8 were upregulated 2.5 fold and 4 fold respectively. the genes most upregulated, samd9 (15-fold) and oasl (12 fold), are not well annotated. as controls, rt-pcr analysis of cells treated with either lipofectamine 2000™ alone or any form of cftr dna alone did not show upregulation of il6, il8, samd9 or oasl. in cells transfected with lipofectamine containing dna, upregulation of il6 (n=3), oasl (n=3) and samd9 (n=3) was confirmed by rt-pcr. oasl is known to interact with methyl cpg-binding protein 1, a dna transcription repressor acting at methylated promoter regions. thus it is possible that the global effects noted here leading to dramatic upregulation of two novel genes oasl and smad9 could be previously unrecognized consequences of gene therapies. cystic fibrosis (cf) is a disease which is caused by mutations of the cf gene, which codes for the cftr chloride channel. the ∆f508-cftr mutation results in a mutant protein that undergoes transcription but fails to localize to the apical membrane, where it normally functions as a camp-regulated clchannel. however, clchannel activity can be detected at the plasma membrane when ∆f508-cftr is overexpressed, synthesized at a reduced temperature, or in the presence of chemical chaperones. thus, strategies that facilitate the translocation of ∆f508-cftr to the plasma membrane by increasing cftr transcription may be beneficial for the treat-ment of cf. previous studies performed using fluorescence halide efflux measurements and short-circuit current voltage clamp have shown that treatment with pparγ (peroxisome proliferator activated receptor gamma) agonists, such as pioglitazone and fll (fmoc-l-leucine), resulted in an increased biosynthesis and trafficking of ∆f508-cftr to the cell surface. this effect was at least partially due to increased ∆f508-cftr expression. further studies were designed to investigate the effect of pparγ agonists on cftr promoter activity. two firefly luciferase-conjugated cftr promoter plasmids, containing either 0.79 kb or 3.9 kb fragments, were transfected together with a control plasmid containing a renilla luciferase in human alveolar basal epithelial (a549) cells. the ratio of the firefly to renilla luminescence was used to measure cftr promoter activity. pparγ agonists increased cftr expression acting both on 0.7 kb and 3.9 kb promoter fragments. these results were confirmed in quantitative rt-pcr studies. transcription factor binding site analysis using current online programs and matrices showed three putative ppre (peroxisome proliferator response element) consensus sequences in the cftr promoter. the interaction of these sites with pparγ was shown using emsa (electrophoretic mobility shift assay). interestingly, none of these consensus sequences appear in the 0.7 kb construct, which still maintained a significant pparγ activation response. therefore, we hypothesized that a novel pparγ agonist effector site(s) is involved in transcription activation of cftr. to characterize this novel site(s), a series of deletion constructs to the promoter region were created, using mutation-introduced restriction sites in the original promoter construct. the new constructs ranged from 0.61 kb to 24 base pairs upstream of the transcription start site. in preliminary studies we found that at least one previously uncharacterized pparγ effector site was present in the 0.61 kb promoter fragment that regulated cftr expression. additional experiments were performed using emsa to determine the proteins and cofactors which controlled the optimal conditions for cftr expression. the results of this study show that pparγ agonists, currently prescribed to treat hyperglycemia and improve peripheral insulin resistance, may also have additional benefits for the treatment of cf. our studies also show that pioglitazone and other pparγ agonists, by increasing cftr expression, are potent activators of clflux in epithelial cells expressing ∆f508-cftr. this work was supported by grant from the canadian cystic fibrosis foundation decreased by 10 µm gsno. the effect of gsno on hop expression was post-transcriptional. in cfbe41o-cells transfected with sirna duplex specific for hop (48 hr), hop expression was modestly inhibited; however, gsno dramatically augmented the inhibition. we next showed that knockdown of endogenous hop by sirna increased the steady-state levels of immature and mature forms of cftr. though overexpression of hop did not significantly affect cftr expression (immature and mature forms increased 1.3 ± 0.1 and 1.2 ± 0.3 fold respectively), in the presence of 10 µm gsno, expression of both immature and mature cftr forms significantly increased in the hop overexpressing cells (3.6 ± 0.4 and 2.7 ± 0.2 fold respectively). next, we treated cfbe41o-cells with a pulse of gsno (10 µm; 4 hr); isolated cytosol, er and golgi fractions; immunoprecipitated cftr from these fractions; and studied whether hop, co-immunoprecipitated with cftr, was s-nitrosylated (using a biotin substitution technique followed by streptavadin isolation). we found that sno-modified hop coimmunoprecipitated with ∆f508 cftr in the cytosol and er at baseline; and that gsno decreased the association between sno-modified hop and ∆f508 cftr. we speculate that the effect of gsno to decrease hop-cftr association may 1) result from increased hop s-nitrosylation; and 2) be permissive for ∆f508 cftr maturation. these data may be of relevance to the development of no donor-based therapies for cf. supported by cf foundation and the nih. mouse models for cystic fibrosis (cf) provide new possibilities to study disease pathogenesis, to correlate genotype and phenotype, and to test novel cf therapies. we use the cftr tm1eu mice homozygous for the f508del mutation in a congenic fvb background as a tool in pre-clinical testing of therapeutic strategies aimed at the correction of the f508del folding and trafficking defect. these mice show characteristic cf ion transport abnormalities such as reduced camp-and cgmp activated intestinal chloride secretion and an increase of the amiloride-sensitive sodium absorption in nasal epithelium. we developed an ex vivo assay for the rescue studies of the f508del trafficking defect in murine intestine by proteasome inhibitors based on periodical measurements of camp-activated transepithlial chloride currents in the ussing chamber. muscle-stripped ileum was incubated at 37 0 c in william's e glutamax medium supplemented with insulin (10µg/ml) and dexamethasone (20µg/ml), in the presence of vehicle or proteasome inhibitor. a partial gain of chloride secretory function was achieved by incubating the tissue for 2-4h at 37 0 c in the presence of the proteasome inhibitors (pi) epoxomicin (1µm), mg132 (10µm), nip-(leu)3-vinyl and adalys(bio)ahx3l3vs (both 2µm) and bortezomib (3nm). exposure of the tissue for 6h at 37 0 c to alln (20µm) revealed a functional recovery up to wt levels. immunohistochemical and western blot analysis of all pitreated tissues showed the appearance of cftr positive crypt cells and mature band c cftr protein. rescue by alln remained detectable following inhibition of the er to golgi transport by brefeldin a. our ex vivo experiments demonstrate that proteasome inhibitors can enhance the amount of mature, functionally active murine f508 cftr mutant protein in intact tissue by preventing proteolytic degradation and increasing its expression at the cell surface. in vivo treatment of homozygous f508del mice with bortezomib at a dose of 0.1mg/kg/day for 3 days resulted in a high morbidity. administration of a lower dose (0.04mg/kg/day ) was well tolerated and no mortality or significant weight loss could be observed. as outcome parameters for the in vivo treatment with pis we used a salivary secretion assay and bioelectric measurements of intestinal and nasal epithelium in the ussing chamber. there was no difference in isoproterenol-induced salivary gland secretion or and in cftr-mediated intestinal chloride currents between control -and bortezomib-treated animals. however we found a reduction (1.7 fold) of the amiloride-sensitive short circuit current (isc) in the nasal epithelium of bortezomib-treated mice, indicating a moderate correction of the enacmediated sodium hyper absorption, that was not paralleled by a gain of the forskolin-mediated transepithelial chloride secretion. current in vivo experiments focus on the effects of pis upon short term (2h-6h) rather than chronic treatment, to allow a direct comparison with the ex vivo treatment data. supported by grants from the cf foundation, bethesda, the sophia foundation and the dutch stomach-liver-intestine foundation. rotterdam, netherlands; 2. physiology and biophysics, university of alabama birmingham, birmingham, al, usa; 3. physiology, school of medical sciences, university of bristol, bristol, united kingdom recent progress in the development of small molecule correctors and potentiators capable of restoring cftr function have increased the need for pre-clinical test models including cultured airway epithelial cells from human cf patients as well as cf mouse models. the validity of cf mice as a surrogate for human cf patients depends on the assumption that human and mouse cftr, despite their limited sequence homology (78%) and differences in open probability (p o ) and channel subconductance state, behave similarly in their response to pharmacological correctors and potentiators. to verify this assumption, we used the 125 iodide efflux technique to compare the cftr chloride channel activity in (i) cho cells stably transfected with mouse f508del-cftr, (ii) nih-3t3 cells stably expressing human f508del-cftr, and (iii) their wt-counterparts. forskolin (10µm)-stimulated iodide efflux was very low in both mouse and human f508del cftr cells (0.5 respectively 2 % of wt values) and was stimulated to a similar extent (3.5 and 3.8fold) by the cftr potentiator genistein (100 µm). preincubation for 14 h at 26 0 c, or at 37 0 c in the presence of the vertex corrector cfcor-325 (3 µm) caused a 3-4 fold increase in forskolin-activated 125 i-efflux in both cell types that was further enhanced by 2.6-3 fold upon addition of genistein. functional correction was paralleled by the appearance of mature, complex-glycosylated mouse or human cftr on western blots. surprisingly, two other, structurally unrelated compounds known to correct the gating defect in hcftr, i.e. the vertex potentiator cfpot-532 (10 µm) and the uncharged nppb analogue nppb-am (20 µm; cf. wang w et al. 2005 ; j biol chem 280: 23622) mimicked the effect of genistein on hf508del-cftr but failed to potentiate mf508del-cftr in the 3t3 cells. both cfpot-532 and nppb-am likewise failed to potentiate forskolin-stimulated, cftr-mediated transepithelial chloride currents ex vivo in muscle-stripped ileal mucosa from homozygous f508del-cftr tm1eu mice (congenic fvb) mounted in ussing chambers under conditions in which genistein (100µm) enhanced the current response by 2-3 fold. in contrast, preincubation of the tissue in william's e glutamax medium for 14h at 26 0 c, or for 5 h at 37 0 c in the presence of cfcor-325 (10µm) enhanced the forskolin+genisteininduced transepithelial clsecretion up to 70-100% of the response in cftr+/+ littermates, and was associated with the appearance of band c on western blots. these results indicate that mouse and human f508del-cftr (1) respond to low temperature incubation or to a small molecule corrector with a similar gain in surface expression and function, supporting the use of cf mouse models for in vivo tests of cftr correctors; (2) respond differently to various classes of cftr potentiators, in line with their pronounced differences in gating behaviour, and emphasizing the specificity of cftr-potentiator interaction. further studies focussing on human/mouse cftr chimera and aimed to identify the domain(s) accounting for the differential response to the cftr potentiators are in progress. cell apoptosis is a highly regulated process which is central to the maintenance of pulmonary vascular homeostasis. recent investigations implicated cftr in the regulation of intracellular sphingolipid levels. our laboratory demonstrated that an imbalance in endothelial sphingolipid levels triggers apoptotic cell death. we therefore hypothesized that a dysregulated cftr function in human lung endothelial cells inhibits programmed cell death in response to stress. methods: patch clamp analysis was used to verify the expression of cftr on human lung endothelial cells and the effect of specific inhibitors. we inhibited cftr with dpc, cftr inh -172, and nppb or specific sirna followed by treatment with prototypical stressors that induce endothelial cell apoptosis, staurosporine, h 2 o 2 , or tnf-α. apoptosis was quantified by caspase-3 activity or annexin/ propidium iodide staining. lipid extraction was performed by a modified bligh and dyer method and intracellular sphingolipids were measured by tandem mass spectrometry. results: patch clamp analysis confirmed functional cftr expression on endothelial cells and a complete inhibitory effect of dpc and nppb but no effect of dids. as expected, treatment with staurosporine, a general protein kinase inhibitor and h 2 o 2 , as an oxidative stress stimulus resulted in significant endothelial cell apoptosis. similarly, tnf-α treatment (20 ng/ml) in combination with cycloheximide caused activation of caspase-3 associated with a significant upregulation of the pro-apoptotic sphingolipid ceramide. knock-down with specific cftr sirna was validated with western blot and whole cell patch clamp analysis. dpc, nppb, and cftr inh -172 consistently and significantly inhibited apoptosis in human lung and primary mouse lung endothelial cells. caspase-3 activity was decreased by 38-95% (% decrease in caspase units/µg/min) in hlmvec treated with h 2 o 2 after pretreatment with dpc (90%), cftr inh -172 (95%), and nppb (38%). however, we observed that dids, a non-specific chloride channel inhibitor also inhibited staurosporine induced caspase-3 activation (93%), suggesting the possible involvement of other chloride channels. in conclusion, our data suggest a role of cftr in modulating endothelial cell apoptosis in response to various stresses. the absence of functional cftr impaired stress-induced apoptosis, a process which may be mediated via alterations in sphingolipid trafficking. this abnormal cftr function in cf leading to aberrant cellular responses to stress may perpetuate an activated, pro-inflammatory phenotype of the lung vasculature. in turn, aberrant vascular activation may have a significant impact to the abnormal airway remodeling and persistence of a chronic inflammatory state characteristic of cf. supported by: cystic fibrosis foundation clinical fellowship training grant hegedus, t. 1, 2 ; serohijos, a.w. 1, 3 ; dokholyan, n.v. 1 ; he, l. 1,2 ; riordan, j.r. 1, 2 1. department of biochemistry and biophysics, unc at chapel hill, chapel hill, nc, usa; 2. cystic fibrosis treatment and research center, unc at chapel hill, chapel hill, nc, usa; 3. department of physics and astronomy, unc at chapel hill, chapel hill, nc, usa cftr (cystic fibrosis transmembrane conductance regulator) is a camp dependent chloride channel. the phosphorylation of its 200 amino acid r domain by protein kinase a (pka) is obligatory for channel gating under normal conditions. r domain contains multiple pka phosphorylation sites, which participate in activation, but no specific site is essential for channel function. in spite of numerous studies of the role of r domain in cftr regulation, the mechanism is largely unknown. one reason for this is the lack of information on r domain structure and its interactions with other parts of cftr. it has been shown that r domain lacks well-defined secondary structural elements and is intrinsically disordered. in this study, we used computational methods to explore the structure and function of the disordered r domain. our results show a specific disorder-order pattern in r domain, which is conserved among species even though the primary structure is not conserved. this result implies a role of the disorder-order pattern in r domain function and provides a basis of a novel working model. to gain some insight into the three dimensional structure of r domain, we per-formed ab initio folding of r-domain using discrete molecular dynamics with an all-atom force field. as disordered domains can not be characterized with a single structure, an ensemble was generated by selection of structures with the lowest energy from a large number of folding simulations. these structures were clustered to determine the dominant r domain conformations. they reveal features of secondary and tertiary structure, the positions of phosphorylation sites in 3d, and residue contacts within the r region that might be important for structure and function. to computationally confirm the mechanism proposed in our working model, an ensemble of phosphorylated r domain structures was also produced and characterized. supported by nih (r01dk051619), cff (dokhol07i0), and novartis (06123815). physiology system that utilises the planar patch clamp technique (finkel et al 2006) . this format utilises perforated patch clamp recording of 384 wells in parallel, permitting the generation of up to 4000 data points per day. we have employed this system to develop assays to detect modulators of mutant cftr. human cftr-g551d and df508, over expressed in bhk and cho cells respectively, were cultured to approximately 80% confluence before harvesting. df508 cells were temperature corrected at 26°c for 72 hours prior to experimentation to facilitate channel trafficking to the plasma membrane. cells were placed on the quattro system and whole cell chloride currents measured using asymmetric chloride selective solutions such that the chloride reversal potential was significantly different from that of leak currents (0mv). currents were assessed using a voltage ramp from -100 to +100mv applied prior and 10 min subsequent to compound addition. to validate the assays we used the prototypical cftr potentiator, genistein, which was added simultaneously to cells under study with 10 µm forskolin. after assessing the effects of genistein, the specific cftr inhibitor glyh101, was added at 30 µm to confirm the currents were mediated via activation of cftr. the ec 50 values determined for genistein were 3.8 ± 0.7 µm (mean ± s.e.m, n=7) for df508 and 16.3 ± 2.6 µm (mean ± s.e.m, n=4) for g551d. these values agree with literature values (bulteau-pignoux et al 2002 ) . two alternative potentiators, phenylglycine-01 (pg-01) and vrt-532 were also tested in the presence of 10 µm forskolin and were active against both df508 and g551d. the ec 50 values for pg-01 were 1.1 ± 0.1µm (mean ± s.e.m, n=10) for df508 and 3.2 ± 0.5µm (mean ± s.e.m, n=8) for g551d. the ec 50 values for vrt-532 were 2.8 ± 1.2µm (mean ± s.d, n=3) for df508 and 20.8 µm (mean, n=2) for g551d. these values agree with published data from ion transport studies (pedemonte et al 2005; van goor et al 2006) . in summary the ionworks quattro can be used to identify compounds which modulate the activity of cftr. this assay has a number of advantages over other formats in that it is direct, sensitive and employs voltage control of the cells under study. these assays can be used for primary screening of small to medium compound sets or as a secondary screen to triage the output from higher throughput indirect formats such as membrane potential. references it is very well agreed that ∆f508 cftr is recognized as a misfolded protein by the er quality control and targeted for degradation by the proteasome. during the course of experiments for aav gene therapy for cf we created an aav2/5 vector expressing a truncated cftr insert (∆264) driven by a chicken beta actin promoter (raav-cb-∆264-cftr). ∆264-cftr contains the normal coding sequence of wt cftr except that it is missing the first 264 amino acids. we observed that cos7 cells transiently transfected with raav-cb-∆264 cftr express cftr at significantly lower levels as compared to cells similarly transfected with a wt-cftr construct. to examine why, cells were transfected with raav-cb-∆264-cftr and treated for 16 hours with 5 µm of proteasome inhibitor, mg132. in the cells treated with mg132, ∆264-cftr protein expression was significantly increased. because lysosomal inhibitors did not have a significant effect, the data suggest that a large frac-tion of ∆264-cftr protein is degraded in the proteasome. to evaluate the rate of degradation, cells were treated with cycloheximide. time course experiments showed that ∆264 is more rapidly degraded than ∆f508-cftr. importantly, in cos7 cells cotransfected with ∆f508 and ∆264-cftr, there was a significant increase in the mature c-band of ∆f508 cftr compared to cells transfected with ∆f508 alone. this suggests that ∆264-cftr enhanced the maturation of ∆f508 from b to c bands. enhanced maturation of cftr from b band to c band also occurred when ∆264-cftr was transfected into hela cells stably expressing ∆508 cftr (hela ∆508) and cfbe 41o-cells stably expressing ∆508 (cfbe 41o-∆508). a number of proteins participate in the er to determine the fate of cftr. ∆f508-cftr is known to associate with retrograde translocation protein complex on the er membrane containing vcp (valosin-containing protein) and derlin 1. vcp is also found in aggresomes containing ∆f508-cftr in association with cytosolic histone deacetylase, hdac6. we hypothesized that since ∆264 is more efficiently degraded as compared to ∆f508-cftr it may have higher affinity with vcp protein complex. we verified that ∆264-cftr interacts more readily with vcp and also has a higher degree of association with hdac6 as compared to ∆f508-cftr. our data suggest that ∆264 is more readily degraded by the proteasome as compared to ∆f508-cftr and lacks biosynthetic arrest in the er. ∆264-cftr binds more readily to vcp and associates avidly with hdac6, two outcomes that are expected to enhance proteasomal degradation. by engaging key quality control proteins, ∆264-cftr allows more ∆f508-cftr to mature. our data suggest that truncated forms of cftr (∆264) may be useful for cf gene therapy by affecting the maturation of endogenous cftr. ∆f508, the most common disease causing cftr mutation, has impaired conformational maturation during its biosynthesis, and thus is unable to exit the endoplasmic reticulum (er). a well-defined di-acidic er exit code has been identified within the first nucleotide binding domain (nbd1) of wildtype cftr (jcb 167: 65-74, 2004) , the disruption of which prevents cftr from efficiently exiting the er. this is consistent with the role of the diacidic code in the copii-mediated cargo concentration process. however, it is not known whether other types of er-to-golgi trafficking signals play a role in cftr trafficking in the early secretory pathway. even less is known about the trafficking signal(s) that are responsible for the rescue of ∆f508 cftr by a number of means. to achieve a better understanding of the trafficking signals dictating the export of cftr from the er, we constructed a series of cftr mutants to probe the potential functional targeting signals within both wild-type and ∆f508 cftr. it has been shown that nbd2 is not required for the export of wild-type cftr from the er. we further found that an internal deletion mutant lacking membrane spanning domain 1 (msd1) and nbd1-r but retaining the n-terminal cytoplasmic region, msd2 and nbd2 does not have sufficient signal to allow er export, suggesting that the signal(s) that are responsible for cftr export largely reside within the nbd1-r. unlike ∆f508 cftr, the di-acidic code mutants are not temperature-sensitive. dissecting the di-acidic code revealed that both acidic residues contribute to er export, and that simultaneous substitution of both leads to a much decreased export of cftr. we found that the temperature-dependent export of ∆f508 relies on the presence of the di-acidic code within the nbd1, suggesting that this code has a functional role in the temperature-dependent rescue of ∆f508 cftr. in our effort to further examine the role of the di-acidic code in the rescue of ∆f508 cftr by second site mutations, we found that ∆f508 cftr rescue by the simultaneous disruption of two of the arginine-framed tripeptides (afts) is also dependent upon the di-acidic code. surprisingly, we found that while nbd2 is not necessary for the export of wild-type cftr, it is required for the aft-mediated rescue of ∆f508 cftr, suggesting a role for cytoplasmic inter-domain interactions in the aft-mediated rescue of ∆f508 cftr. a detailed biochemical analysis of these mutants provides valuable insights into the roles of different cftr "trafficking" motifs in the er-to-golgi transport of cftr, the specific conformational defects of ∆f508 cftr and the potential molecular mechanism underlying the rescue of ∆f508 cftr. supported by cff. sonawane, n.d. 1 ; zhao, d. 1 ; galietta, l. 2 ; zegarra-moran, o. 2 ; verkman, a. 1 1. medicine, university of california san francisco, san francisco, ca, usa; 2. genetica molecolare, istituto gianina gaslini, genova, italy cftr inhibitors are predicted to prevent intestinal fluid secretion in enterotoxin-mediated secretory diarrheas such as cholera. cftr inhibitors that are not absorbed across the intestinal wall are attractive for diarrhea therapy because they may provide safe oral therapy. we previously discovered low affinity glycine hydrazide (glyh, ic50 ~5 µm) cftr inhibitors that block cftr at its external pore (muanprasat et al., j. gen. physiol. 2004; 124:125-137) . in order to increase inhibitor potency and prevent washout during severe secretory diarrhea, we synthesized a series of glycocalyx interactive cftr inhibitors containing a malonic acid hydrazide (malh) cftr pore blocking moiety linked to a lectin (sonawane et al., gastroenterology 2007; 132:1234 -1244 . lectin conjugation improved cftr inhibitory potency by ~100-fold (ic50 to 50 nm). high-affinity cftr inhibition was abolished by malh-lectin heat denaturation, protease digestion, or competition by mannose or unconjugated lectin. fluorescently labeled malh-lectin remained membrane-bound for >6 hours after washout, whereas washout occurred in a few minutes without the lectin. malh-lectins blocked cholera toxin-induced intestinal fluid secretion in closed intestinal loops in mice with ec50 50-100 pmol, and greatly reduced mortality in a suckling mouse model of cholera. we recently synthesized mono-and divalent cftr inhibitors consisting of malh coupled via a disulfonic stilbene linker to flexible mono and bifunctional polyethyleneglycols (pegs) of molecular size 0. 2, 0.75, 2, 3, 5, 10, 20, 40 and 100 kda, with calculated solution lengths of 1-23 nm, with the larger size pegs potentially spanning cftr dimers or inducing their formation. ic50 for inhibition of cftr chloride current was 10-15 µm for monovalent malh-pegs, but substantially lower and size-dependent for divalent malh-pegs, decreasing from 1.5 µm to 300 nm with increasing peg size. the mechanisms responsible for the improved and size-dependent potency of divalent malh-pegs were studied by whole cell, single-channel patch-clamp and by functional analysis of multivalent malh-conjugated dextrans and asymmetric divalent pegs. whole-cell experiments revealed reversible voltage-dependent block of cftr currents, with outward (positive) currents being more strongly blocked. in outside-out patch-clamp experiments, inhibitors caused a reduction of the mean open time. for malh-peg20kda-malh (2 µm) , the mean open time decreased from 189 ± 44 to 97 ± 29 ms. the effect on currentvoltage relationship and channel kinetics are consistent with a mechanism involving occlusion of the cftr pore from the extracellular side. luminally added divalent malh-pegs blocked by > 90% cholera toxin-induced fluid secretion in mouse intestinal loops with ic50s of <10 pmol/loop, and greatly reduced mortality in a suckling mouse model of cholera. nonabsorbable, multivalent cftr inhibitor-macromolecule conjugates may be useful as anti-secretory agents in the treatment of enterotoxin-mediated diarrheas. supported by cff and nih. activation of the cystic fibrosis transmembrane conductance regulator (cftr) clchannel is primarily controlled by pka-dependent phosphory-lation of the r domain. once the r domain is phosphorylated, atp binding and enzymatic activity at two nucleotide-binding domains (nbds) open and close the cftr clchannel. we hypothesized that r domain phosphorylation regulates cftr activity by modulating atp interactions within two nbds. to test this hypothesis, we studied the activity of wild-type cftr and five variants with deletions of portions of the r domain between residues 708 and 835. to alter atp-dependent channel gating, we tested three cftr stimulators, pyrophosphate (pp i , 2 mm), 2'deoxy-atp (2'datp, 1mm) and caatp (1mm) . consistent with previous studies, pp i , 2'datp and caatp all increased current of wild-type cftr about two-fold. each stimulator produced a similar increase in the r domain variants. the similar effects of stimulators on the channel activities of wild-type cftr and r domain variants suggest that the r domain does not have a major role in regulating atp-dependent channel gating. the r domain deletions did not alter the single-channel current amplitude of the r domain variants. however, r domain variants missing the sequence between residues 784 and 835 markedly reduced the open state probability, suggesting that this region is required for normal gating. these data suggest that the r domain does not control cftr activity by modulating atp interactions with nbd binding sites. instead, we speculate that the c-terminal part of r domain might participate in the channel-gating machinery downstream of atp regulation. supported by the cystic fibrosis foundation and howard hughes medical institute. haggie, p.m.; verkman, a. medicine and physiology, cvri, u.c.s.f., san francisco, ca, usa it was recently reported that phagolysosomes of alveolar macrophages from cf mice acidify in a cftr-dependent manner and that defective phagolysosomal acidification impairs bactericidal activity (di et al., nat. cell biol. 2006, 8:933-944) . these findings suggested a unifying hypothesis for cf disease progression: defective phagolysosomal acidification in cf macrophages permits the initiation and promotes progression of bacterial infection in the lungs. to reassess the central finding of that study we measured phagolysosomal ph using a fluorescent ph indicator containing oregon green® 488 (pka ~4.7) and tetramethylrhodamine covalently bound to zymosan. phagolysosomal ph was insensitive to cftr inhibition (10 µm cftrinh-172) in j774 macrophages (ph 5.08 ± 0.08 vs. 5.10 ± 0.03), alveolar macrophages from mouse lung (ph 5.34 ± 0.05 vs. 5.37 ± 0.07), and alveolar macrophages from human lung (ph 5.30 ± 0.08 vs. 5.35 ± 0.07). phagolysosomal ph in alveolar macrophages from ∆f508 cf mice was not significantly different from that in alveolar macrophages from wild-type mice (ph 5.34 ± 0.05 vs. 5.40 ± 0.06). to account for their finding of defective phagolysosomal acidification in alveolar macrophages, di et al. reported that lysosomal acidification was cftr-dependent and that fusion of lysosomes to phagosomes (at ~20 min after phagocytic uptake) was responsible for phagosomal acidification. we measured lysosomal acidification in j774 macrophages using a dextran-conjugate containing oregon green® 488 and tetramethylrhodamine and found that acidification was not impaired by cftr inhibition (ph 4.48 ± 0.03 vs. 4.42 ± 0.03) . we also measured the kinetics of phagosomal acidification using a zymosan-conjugate containing 5-(and-6)-carboxyfluorescein (pka ~ 6.4) and tetramethylrhodamine. phagosomal acidification in j774 macrophages and murine alveolar macrophages began within 3 min of phagocytosis and reached steady-state by 10-15 min, in agreement with prior data in murine peritoneal and bone marrow-derived macrophages. acidification kinetics in j774 macrophages was not altered by cftr inhibition, nor was acidification kinetics different in wild-type vs. cf alveolar macrophages. our findings do not support the conclusion that phagolysosomal acidification in alveolar macrophages is cftr-dependent, nor that it is impaired in cf. the mechanism of phagosomal acidification proposed by di et al. is not in accord with our data or precedents in the literature. because phagolysosomal acidification is central to the proposed mechanism linking defective cftr chloride channel function with cf lung disease, our results do not support the involvement of cftr in defective macrophage function in the pathogenesis of cf lung disease. supported by cff and nih. pared to that of wild-type cftr. the hsp70 family of molecular chaperones play important roles in the protein quality control process within the er. hsp70 atpase activity is regulated by multiple co-chaperones such as hsp40, bag-1 and hspbp1. hsp105, a high-molecular-weight member of the hsp70 family was recently shown to display nucleotide exchange activity for hsp70s in vitro. furthermore, hsp105 was identified as a component of a cftr-associated multiple protein complex using a global proteomic approach (cell 127: 803-815, 2006) . in an attempt to explore the role of hsp105 in cftr conformational maturation in the er, we over-expressed the co-chaperone in hek293 cells and quantitatively analyzed its effect on the maturation and degradation of cftr. consistent with its role as a nucleotide exchange factor for hsp70s, over-expression of hsp105 inhibits the er export of wild-type cftr and promotes its degradation. however, in striking contrast, over-expressing hsp105 stabilizes ∆f508 cftr and promotes its er export at reduced temperature. this effect is less pronounced at physiological temperature. the apparently opposite effects of hsp105 on wild-type and ∆f508 cftr maturation and quality control, suggests distinct conformational maturation pathways for the two cftr molecules, and reveals a specific role for hsp105 in regulating ∆f508 cftr refolding. such conclusion is reinforced by rnai experiments and further supported by quantitative co-immunoprecipitation. rnai-mediated down-regulation of hsp105 expression destabilizes ∆f508 cftr and reduces its export at reduced temperature, and hsp105 shows more extensive association with the er form of ∆f508 cftr than that of wild-type cftr. further analysis revealed that hsp105 not only modulates the chaperone activities of hsp70s but also alters their steady state levels within the cell, creating secondary effects on cftr maturation and quality control. further studies are necessary to achieve a better understanding of the machinery, pathway and mechanism of ∆f508 cftr conformational maturation at reduced as well as physiological temperatures, and this in turn will provide critical insights and key factors that are of potential value to the rescue of the trafficking defect of ∆f508 cftr. farinha, c.m. 1, 2 ; pissarra, l.s. 1 ; amaral, m.d. 1, 2 1. department of chemistry and biochemistry, faculty of sciences, university of lisboa, lisboa, portugal; 2. ctr hum genet, nat inst health, lisboa, portugal the most frequent mutation in the cystic fibrosis (cf) gene, f508del, causes retention of its protein product, f508del-cf transmembrane conductance regulator (cftr) in the endoplasmic reticulum (er) as a core-glycosylated intermediate that is rapidly degraded. therefore, most f508del-cftr fails to traffic to the plasma membrane, where wild-type (wt) cftr normally functions as a chloride (cl-) channel. retention, however, is not due to lack of function, since the mutant still retains some function if it reaches the membrane. instead, it results from misfolding which is recognized by the er quality control (erqc) in which many intervenients, including molecular chaperones, participate. identification of molecular partners involved in the disposal of f508del-cftr to the proteasome is therefore crutial. it was recently shown that casein kinase ii (ck2), a pleiotropic constitutively active protein kinase involved in several processes, such as neoplasia, cell survival and viral infections, binds wt-cftr near the f508 residue, phosphorylating its first nucleotide binding domain (nbd1) at s511 [1] . interestingly, deletion of f508 abrogates this interaction, which is the first described f508del-dependent protein-protein interaction. our aim here was to identify whether ck2 interaction affects the early steps of cftr biogenesis, turnover and processing. to this end, novel bhk cells were produced which stably express wt-or f508del-cftr in which the consensus residue s511 was substituted by either a neutral (alanine -s511a) or an acidic residue (aspartate -s511d). initial biochemical analyses of these cell lines revealed that: 1) cftr proteins bearing d or a at position 511 are processed; 2) despite producing equivalent levels of cftr transcripts, s511a expressing lines consistently show lower levels of cftr protein. metabolic labelling and pulse-chase experiments followed by cftr immunoprecipitation were also performed in these lines. after quantification of bands b (immature form) and c (mature form) of cftr, these preliminary results show that substitution of s511 (by a or d) does not affect the turnover or processing of either wt-or f508del-cftr. the effect of ck2 inhibition on the turnover and processing of cftr was also studied, by incubating cells with 20 µm of the ck2 inhibitor tetrabromobenzotriazole (tbb) for 90 min. pulse-chase experiments under these conditions show that: 1) the steady-state levels of both wt-and f508del-cftr are reduced; 2) the turnover of wt-cftr (but not of f508del-cftr) is increased; and 3) processing of wt-cftr is decreased. such data are consistent with a putative stabilizing role for ck2 upon wt-cftr [1] . however, in our preliminary results this effect does not appear to be dependent on residue 511 (nor on the putative charge added by the kinase on this residue), thus suggesting that this effect may be indirect. further studies are underway to identify the mechanism by which ck2 affects the turnover and processing of cftr. 1. treharne et al (2007) pissarra, l.s. 1 ; xu, z. 2 ; farinha, c.m. 1, 3 ; sheppard, d.n. 2 ; amaral, m.d. 1, 3 1. department of chemistry and biochemistry, faculty of sciences, university of lisboa, lisboa, portugal; 2. dep physiology, univ bristol, school med sciences, bristol, united kingdom; 3. ctr hum genet, nat inst health, lisboa, portugal g550e and 4rk (the simultaneous mutation of four arginine-framed tripeptides (afts): r29k, r516k, r555k and r766k) are second site mutations that rescue the processing and function of f508del-cftr [1, 2] . these revertant mutations rescue f508del-cftr from retention within the endoplasmic reticulum by distinct mechanisms: g550e likely alters the conformation of the first nucleotide-binding domain (nbd1), whereas 4rk plausibly allows f508del-cftr to escape er retention/retrieval mediated by afts [3] . both g550e and one of the afts (r555k) lie close to the residue g551, where a common cf-causing mutation g551d occurs, generating a correctly localized clchannel with a severe gating defect. our aim here was to assess whether the revertants g550e and 4rk influence the folding, processing and gating behaviour of the g551d mutation, by employing biochemical and functional approaches. to test this idea, we introduced the g551d mutation into cftr cdnas containing either g550e or 4rk in the pnut vector and stably expressed these constructs in bhk cells. preliminary results from biochemical studies indicate that the mature fully-glycosylated form of cftr protein (band c) of both g550e-g551d-cftr and 4rk-g551d-cftr were present at similar levels to those of g551d-cftr. analysis of cftr-mediated iodide efflux from these cells revealed that g550e is unable to rescue the functional defect of g551d. however, 4rk generated an efflux of iodide larger than that elicited by cells expressing g551d, albeit smaller than that of cells expressing wild-type (wt) cftr. consistent with these data neither 4rk nor g550e rescued the defective channel gating of g551d-cftr. however, both revertants caused a small increase in g551d-cftr activity by attenuating the prolonged interburst interval of g551d. we conclude that f508del and g551d disrupt cftr channel gating by distinct mechanisms. altogether, our data suggest that at least when in cis with the g551d mutation, the afts (together or individually) might have a direct effect on cftr channel gating. this raises the possibility that 4rk, in addition to its well-described effect on trafficking, may act on cftr structure and/or folding, as previously suggested for r555k [4] . work supported by grant pocti/sau/mmo/58425/2004 and pluriannual funding of cigmh (fct, portugal) and the uk cf trust. l. pissarra was a recipient of bd/9095/2002 doctoral fellowship (fct, portugal) . [1] carvalho ac, gansheroff lj, teem jl (2002) j biol chem 277, 35896-35905. [2] chang xb, cui l, hou yx, jensen tj, aleksandrov aa, mengos a, riordan jr (1999) mol cell 4, [137] [138] [139] [140] [141] [142] [3] roxo-rosa m, xu z, schmidt a, neto m, cai z, soares cm, sheppard dn, amaral md (2006) proc natl acad sci usa 103, 17891-17896. [4] teem jl, carson mr, welsh mj (1996) improves ∆f508-cftr intracellular trafficking in cf epithelial cells, although the mechanism by which this occurs is not clear. to identify gene products with atlered abundance in response to 4pba, we performed a differential display rt-pcr screen on rna isolated from ib3-1 cf epithelial cells treated with 4pba for 0-24 hours. we isolated a cdna encoding stip-1, a putative human stat-3 (signal transducer and activator of transcription-3) interacting protein and confirmed that 4pba causes transiently increased stip-1 mrna and protein abundance after 8 hours of exposure. stip-1 was originally described as a scaffold protein that is required for ligand-dependent activation of stat-3. stip-1 is also identical to elongator protein 2 (elp-2), a subunit of the multicomponent elongator complex that stimulates rna polymerase ii activity. interestingly, recent data suggests that elongator may also regulate polarized secretion (rahl, et al. (2005) , mol. cell, 17:841-853). we therefore hypothesized that stip-1/elp-2 would regulate ∆f508-cftr intracellular trafficking. in ib3-1 cells, stip-1/elp-2 associates with ∆f508-cftr in coimmunoprecipitation experiments after 4pba treatment. in ib3-1 cells overexpressing stip-1/elp-2, immunofluorescence experiments suggested that ∆f508-cftr trafficked to the plasma membrane even in the absence of 4pba treatment. stip-1/elp2 co-localized with markers of the golgi (58 kda golgi protein) and exocytic vesicles (snap-25) when overexpressed in these cells. in contrast, overexpression of a deletion mutant of stip-1/elp-2 lacking the wd domain, or stat-3 binding region, blocks the improvement of ∆f508-cftr trafficking in response to 4pba in immunofluorescence and surface biotinylation experiments. this stip-1/elp-2 deletion mutant did not co-localize with golgi or exocytic vesicle markers. furthermore, immunofluorescence experiments also suggested that 4pba causes colocalization of stip-1/elp-2 and elp-1, another member of the elongator complex that is often defective in familial dysautonomia. these data are consistent with stip-1/elp-2 regulating ∆f508-cftr intracellular trafficking in response to 4pba. this regulation may occur in the context of 4pba stimulating assembly of elongator, which in turn may promote trafficking of exocytic vesicles carrying ∆f508-cftr to the plasma membrane. supported by grants from niddk. fold [1, 2] . however, functional studies demonstrate significant differences in the gating behaviour of hcftr and mcftr [3] . a powerful approach to investigate cftr structure and function is to examine interspecies differences and identify regions of conservation and divergence. to understand the structural basis for the functional differences between hcftr and mcftr, we generated hmcftr chimeras containing mcftr domains on an hcftr backbone. for this purpose, we replaced all or part of nbd1, nbd2 or the rdomain of hcftr with the equivalent regions of mcftr and investigated their biochemical and functional properties. the in vivo folding of these hmcftr chimeric proteins was indirectly evaluated from their maturation status, after their stable expression in novel bhk cell lines. like wt-hcftr, most chimeric proteins were processed within the cell. however, two chimeras failed to mature: clone 12b (mnbd1, amino acid (aa) residues 518-585) and clone 114c (mnbd2, aa 1260-1412). we compared the murine sequence of these two chimeras with that of hcftr to determine the respective physico-chemical distances (pcds) of their aa changes. changes with higher pcd values were selected and in vitro mutagenesis performed to introduce these aa alterations into hcftr cdna. for clone 12b the selected changes were: e527q, e528q (pcd = 29); s531t (pcd = 58); k536q (pcd = 53), i539t (pcd = 89) and k584e (pcd = 56). for clone 114c the changes were: p1290t (pcd = 38), k1302q (pcd = 53), y1307n (pcd = 143), s1311k (pcd = 121), c1344y (pcd = 194), d1394g (pcd = 94) and e1409d (pcd = 45). biochemical analyses of these mutants stably expressed in bhk cells revealed that for clone 12b, e527q, s531t, k536q and i539t were processed, whereas k584e was not. for clone 114c, p1290t, k1302q, y1307n, d1394g and e1409d were processed. iodide efflux showed that s531t, k536q, i539t, e1409d and d1394g were functional, but not k584e. we consider that with additional functional analyses, our approach will identify critical residues responsible for conformational changes and hence, functional differences between hcftr and mcftr. work supported by the bbsrc and grant pocti/mgi/47382/2002 (fct, portugal) and pluriannual funding of cigmh (fct, portugal) . ac dapaula is a recipient of phd fellowship sfrh/ bd/17475/2004 (fct, portugal we recently reported that the upr decreases endogenous wild-type (wt) cftr expression. as a follow up of these studies, we investigated the role of the folding deficient, ∆f508 cftr on upr induction and identified a mechanism by which the upr decreases cftr expression. for these studies, we developed a cell line expressing recombinant ∆f508 cftr on the endogenous wt background (calu-3 ∆f) and established individual clones with different ratios of endogenous (wt) to recombinant (∆f508 cftr) expression. two clones which express 1:1 (calu-3 ∆fc3) and 1:8 (calu-3 ∆fc5) ratios of wt to ∆f508 cftr and cfpac-1 cells expressing endogenous ∆f508 cftr were tested as models. the upr was constitutively active in calu-3 ∆fc5 cells only. in calu-3 ∆fc3 induction of ∆f508 cftr expression resulted in upr activation, indicating that high expression of ∆f508 cftr is required for upr induction. furthermore, following pharmacological induction of the upr, endogenous cftr mrna decreased to undetectable amounts both in calu-3 ∆f and cfpac-1 cells. the decrease in cftr mrna levels was not the result of shortened mrna half-life. in contrast, using a human cftr promoter driven reporter (pcftr-pluc), we demonstrate suppression of the cftr promoter when the upr is activated. considering that correction of ∆f508 cftr is the main therapeutic approach for cf, it is important that ∆f508 cftr expressed at low levels, such as in vivo, does not activate the upr. however, our results also reveal that ∆f508 cftr correctors have to be tested for upr activation, since transcriptional inhibition in this setting may contribute to inefficient rescue in native cells or in vivo. genistein, a naturally occurring tryrosine kinase inhibitor, at low concentrations (50µm) stimulates wt -cftr-mediated chloride secretion in a variety of cell and tissue types by an adenosine 3',5'-cyclic monophosphate (camp)-independent pathway. genistein has been shown to potentiate the channel function of ∆f508-cftr and g551d-cftr channels by prolonging channel open time (hwanget al., am j physiol cell physiol 273: c988-c998, 1997; illeck et al. am j physiol cell physiol 277: c833-c839, 1999) patch clamp studies have provided indirect evidence that genistein may bind directly to cftr and potentiate channel open probability by modifying the function of the nucleotide binding domains (weinreich et al., pflügers arch 434: 484-491, 1997) . the goal of the present study was to determine whether genistein activated purified and reconstituted cftr protein as an initial step toward defining its molecular mechanism of action. the effect of genistein on purified and reconstituted wt-cftr was first studied following its reconstitution into planar lipid bilayers. in the presence of 100µm atp, phosphorylated cftr exhibited a low open probability (po= 0.12). the addition of 50µm genistein, caused a significant increase to the open probability of cftr (po=0.76). using an electrogenic flux assay on a population of purified and phosphorylated cftr molecules, we determined that 50 µm genistein caused a potentiation (70% increase) of chloride channel activity in the presence of 100 µm atp. these results suggest that this macroscopic assay of purified cftr function is sufficiently sensitive to detect the effect of this potentiator. currently, the consequences of genistein on the channel activity and atpase activity of purified reconstituted ∆f508-cftr and g551d-cftr are being assessed. preliminary results suggest that in the presence of low atp concentrations (10-100 µm),50µm genistein inhibits the atpase activity of ∆f508-cftr, suggesting that it interacts directly with the nucleotide binding domains to alter their function. previous studies indicated an acute coordination between the activities of cystic fibrosis transmembrane conductance regulator (cftr) chloride channel and the amiloride sensitive epithelial sodium channel (enac) so that both these channels are either activated or deactivated in a synchronous fashion in the human sweat duct (reddy et.al, 1999) . however, the mechanisms responsible for such cooperativity between these ion channels are unknown. previous studies indicated that: cytoplasmic ph controls cftr activity through effects on phosphorylation (reddy et.al. 1998) . at ph 6.0, cftr chloride conductance was reduced to 10±5 ms/cm 2 , but incresed to 42±7 ms/cm 2 at ph 8.0 (n=number of ducts=5). enac channel activity is also a function of cytosolic ph in heterologous expression systems (chalfant et.al, 1999) . the objective of this study was to test the hypothesis that the cytosolic ph may mediate the cooperative effects that occur between cftr and enac. we used basolaterally a-toxin permeabilized apical membrane preparations of native human sweat duct which expresses enac and cftr robustly as an experimental system. we showed that while luminal ph had no effect, changes in cytosolic ph acutely affected enac activity. that is, acidic ph inhibited, while basic ph activated enac activity. alkalinizing cytosolic ph from 6.8 to 8.5 increased enac conductance (genac) by 35±1.8 ms/cm 2 (n=6). ph regulation of enac activity appears to be independent of cftr and endogenous kinase activities because basic ph stimulated enac: a.) after deactivating cftr by removing camp and atp in normal ducts, b.) in the absence of cftr in cf ducts, and c.) after blocking endogenous kinase activity with the non-specific kinase inhibitor, staurosporine. na + /h + exchanger (nhe) may mediate changes in cytosolic ph as a function of intracellular na + activity. nhe mrna is expressed in the sweat duct and cytosolic ph responds to changes in na + gradients across the basolateral membrane. conceivably, when transport conditions are favorable and intracellular na + is low, alkaline ph would allow enac and cftr to cooperatively admit na + and clthrough the apical membrane. when conditions are unfavorable and intracellular na + is excessive, acid ph would limit na + and clentry to protect the cell from disruptive changes in cell volume. thus, changes in cytosolic ph may play a crucial role in coordinating the activities of enac and cftr during transepithelial salt transport. acknowledgements: kirk taylor and sucharitha madireddi for expert technical assistance. funded by nih-ro1 de14352, nih-ro1hl08042 and the nancy olmsted trust. the cystic fibrosis transmembrane conductance regulator (cftr) is an anion channel that is normally expressed in the plasma membrane but is mislocalized in cf. to monitor trafficking we inserted super ecliptic phluorin, a ph-sensitive variant of green fluorescent protein (gfp) (miesenbock g, de angelis da, rothman je (1998) nature 394, 192-5) into the fourth extracellular loop of cftr. this construct, which we call cftr-phluor, has weak fluorescence intracellularly that increases ~10-fold when it is inserted into the plasma membrane and the phlour becomes exposed to more alkaline extracellular ph . thus cftr-phluor should be less fluorescent than cftr with the normal gfp fused to the n or c termini when situated in intracellular vesicles. despite the insertion of an entire gfp in the middle of the channel protein, cftr-phluor trafficked to the membrane and patch clamp studies revealed little change in unitary conductance or open probability compared to wild-type cftr. cftr-phluor enabled visualization of vesicle insertion events at the plasma membrane by total internal reflection fluorescence (tirf) microscopy. faintly fluorescent vesicles containing cftr were observed under the plasma membrane and eventually fusing with it to generate a burst of fluorescence. phluorin inserted at the same position in ∆f508-cftr, the predominant mutant in cf, was retained in the er where the phluor remained partially quenched. when the folding defect was corrected by culturing cells at 29°c or treating them with known correctors, ∆f508-phluor trafficking was partially restored and total cell fluorescence increased. this enabled a quantitative analysis of ∆f508-cftr correction based on the increase in fluorescence in individual cells by flow cytometry. cftr-phluor may be most useful for microscope-based high-content screening studies of cftr and ∆f508-cftr trafficking. support: the breathe program, canadian cf foundation, canadian institutes of health research and cf foundation therapeutics (usa). tukaye, d.n.; guggino, w.b. physiology, johns hopkins university, baltimore, md, usa type iii secretion system (t3ss) toxins of p. aeruginosa are important virulence factors in p. aeruginosa infections. one of the t3ss toxins, exos, has been shown to facilitate uptake and invasion of p. aeruginosa at airway surface epithelium. in mouse models of pseudomonas pneumonia, infection with p. aeruginosa exos+ strains caused increased levels of fluid in lungs as determined at autopsy in contrast to infection with p. aeruginosa exosstrains. the exact molecular mechanism underlying these observations is not known. exos is a bifunctional protein with gtpase activating protein (gap) activity at the n terminal domain and adp ribosyl transferase (adprt) activity at the c terminal domain. we found that exos-gap activity increases total cellular levels of mature (c band) wild type (wt) cftr in cfbe41o-cells as measured by western blot analysis. this increase was mediated by decreased delivery of wt cftr for lysosomal degradation. in contrast, exos-gap failed to increase total levels of ∆508 cftr (b band). interestingly, exos-gap increased total levels of ∆508 cftr, bands c and b, following rescue with 4-phenylbutyrate. this indicates that targets of exos-gap exist in cftr trafficking beyond the er degradation pathway. exos-gap also brought about a corresponding increase in surface levels of mature wt cftr as measured by surface biotinylation. in conclusion, we have shown that p. aeruginosa t3ss exos-gap, upregulates total and surface levels of mature wt cftr by modulating cftr trafficking beyond er, most likely by decreasing lysosomal degradation. the decrease in lysosomal degradation could be occurring in part by inactivation of small molecular weight g-proteins involved in delivery of wt cftr to lysosomes. increases in total and hence surface levels of cftr could in part explain increased amounts of fluids seen in p. aeruginosa pneumonia mouse model treated with exos+ strains. the post-maturational trafficking and localization of cftr is regulated by a wide variety of proteins. among the most prominent are several pdz domain proteins that bind to the c-terminal residues of cftr. in particular, two of these proteins, nherf1 and cal, have been shown to mediate opposing effects on the apical membrane levels of the disease-associated ∆f508-cftr mutant, by controlling the balance between endocytic recycling and lysosomal degradation. in particular, whereas overexpression of nherf1 rescues ∆f508-cftr at the cell surface, overexpression of cal has the opposite effect on cftr. cal-mediated downregulation of cftr requires a functional pdz binding site. we have now shown that suppression of endogenous cal cooperates with temperature rescue to stabilize functional ∆f508-cftr channels at the apical membrane in polarized bronchial epithelial cells. this work supports the hypothesis that the cal pdz binding site is a therapeutic target for treatment of cystic fibrosis. a more direct test of this hypothesis will require competitive inhibitors that can efficiently block the cal:cftr interaction. in addition, given the large-number of protein-protein interactions formed by both the cal pdz domain and the cftr c-terminus, these inhibitors should ideally exhibit bidirectional selectivity, neither disrupting the favorable nherf1:cftr interaction, nor strongly displacing other membrane proteins that interact with cal. using a fluorescence polarization assay and peptide-array technology, we have now identified a high-affinity, cal-selective peptide inhibitor. compared to cftr, this sequence binds cal more tightly, but nherf1 more weakly. furthermore, we have shown that the cal:cftr interaction is the weakest among the known cal-binding proteins, and that cftr should thus be susceptible to selective displacement from the cal binding site. finally, we have designed cell-permeable peptides that allow us to test the hypotheses (a) that such compounds will specifically disrupt the cal:cftr interaction and (b) that such targeted stereochemical inhibition will stabilize the functional cell-surface expression of ∆f508-cftr in airway epithelial cells. the pathways for the endoplasmic reticulum associated protein degradation (erad) of misfolded proteins in the mammalian cells are incompletely understood. we investigated the role of molecular chaperones in erad for the cystic fibrosis transmembrane conductance regulator (cftr) and its common folding mutant, ∆f508cftr. we found that hsp70 and its cochaperone hdj-2 interacted significantly with ∆f508cftr and wt-cftr in airway epithelial cells. hsp70 interacted with the immature form of cftr while hdj-2 associated with both immature form and ubiquitylated cftr. structure-function studies showed that hdj-2 recognized ubiquitylated cftr via its zn-binding domain. immunoprecipitation and gst-fusion protein pulldown experiments revealed that hdj-2 interacted with ubiquitin. in steady-state, over-expression of hdj-2 elicited increasing both immature and mature forms of wt-cftr, but it resulted in decreasing immature form of ∆f508cftr. pulse-chase studies showed that co-expression of hdj-2 promoted ∆f508cftr degradation, reducing its half-life from 85 to 50 min. in contrast, hdj-2 expression did not significantly affect wt-cftr biogenesis. these data are consistent with our finding that hdj-2 shows a selective physical interaction with ∆f508cftr, which translates into a functional discrimination between the mutant and its wt counterpart. the hpd motif within the j-domain of hdj-2, which is necessary for the co-chaperone to bind hsp70 and stimulate its atpase activity, was required for hdj-2 mediated ∆f508cftr degradation. mutation of the hpd motif retarded the degradation of ∆f508cftr and increased the steady-state levels of ∆f508cftr 3-fold. a role for hsp70 in hdj-2 mediated ∆f508cftr degradation was tested in which hsp70 knockdown increased ∆f508cftr expression 3-4 fold. hjd-2 regulates ∆f508cftr maturation and knockdown of endogenous hdj-2 promoted ∆f508cftr and its camp-dependent anion conductance in airway epithelial cells. in contrast, we were unable to detect maturation of ∆f508cftr in hsp70 knockdown experiments, indicating that the maturation of ∆f508cftr mediated by reduced hdj-2 expression may be independent of hsp70 function. taken together, our data demonstrate that hdj-2 is a molecular sensor that can detect differences in the folding related to the ∆f508cftr maturation. these data also highlight a novel pathway for hdj-2 linked, ubiquitin-dependent degradation of ∆f508cftr. reduction of hdj-2 expression or its association with ∆f508cftr promotes maturation of this mutant and therefore represents a potential therapeutic approach for cystic fibrosis. [supported by nih and cf foundation] how the loss of cftr function results in cholesterol accumulation within the cell is currently unknown. cftr activation is driven by the camp and we propose that cf cells respond to the loss of cftr activity by increasing the camp pathway in order to increase cftr expression. to test this hypothesis, epithelial cells were treated with the phosphodiesterase-3 (pde3) and pde4 inhibitors milrinone and rolipram, respectively. inhibition of pde4 function with rolipram in wildtype cells leads to peri-nuclear free cholesterol accumulation identical to what is observed in cf epithelial cells as viewed by filipin staining, strongly implicating a camp-dependent pathway in the regulation of cholesterol trafficking. the pde3-selective inhibitor milrinone had no effect on cholesterol trafficking suggesting specificity for the pde4 pathway. our preliminary data support this hypothesis in that both cf-model 9hteo-pcepr cells and mouse nasal epithelium (mne) from cftr -/-mice exhibit reduced protein expression of the campspecific phosphodiesterases pde4 compared to wt controls. the proposed consequence of a chronic amplification in camp signaling is altered cholesterol transport regulation. to address this potential role of pde4 in cholesterol trafficking, free cholesterol was visualized using filipin staining in wild type cells that were treated with rolipram, a pde4-specific inhibitor. conversely, treatment of cf-model pcepr cells with the pka inhibitor rp-camps significantly improves cholesterol processing, further pointing to the involvement of the camp pathway. we propose that the camp pathway influences cholesterol processing through the regulation of β-arrestin-2 (βarr2) according to the premise that chronic increase in the camp pathway would initiate an elevation in βarr2 expression. βarr2 is an important regulator of adrenergic receptor recycling and organelle trafficking. because βarr2 is pivotal in regulating endocytotic recycling pathways, it could also impact cholesterol processing. this predicted increase in βarr2 protein expression is observed in both cf-model pcepr cells and cftr -/-mne compared to respective controls. over expression of βarr2 in wt epithelial cells leads to cf-like peri-nuclear cholesterol accumulation further implicating a role for βarr2 in the development of this phenotype. altered cholesterol trafficking in cftr would lead one to expect different βarr2 localization throughout the cell. further understanding of the implications of altered camp signaling and its relationship to aberrant cholesterol accumu-rationale: we recently reported that vcp (valosin containing protein) physically interacts with gp78/amfr (autocrine motility factor receptor) to couple retrograde translocation of ∆f508-cftr to proteasomal degradation (vij et al. jbc 2006) . recent studies have revealed an alternative system to the proteasome for degradation of polyubiquitinated misfolded proteins termed the aggresome. histone deacetylase-6 (hdac6) is a unique cytoplasmic deacetylase capable of interacting with ubiquitin and mediating the accumulation of ∆f508-cftr in aggresome bodies. hypothesis: vcp competes with hdac6 to dissociate ∆f508-cftr perinuclear aggregates. methods: ib3-1 (∆f508/w1282x) cf bronchial epithelial cells were transiently transfected with ∆f508-cftr and vcp-gfp constructs and treated with a hdac6 inhibitor (tubacin), proteasome inhibitor (ps-341 or mg-132), lysosome inhibitor (baflomycin a1) or nil-tubacin (control) for 4, 8 or 12 hrs. the effect of these inhibitors on ∆f508-cftr and vcp:∆f508-cftr interactions were quantified by immunoprecipitating these protein complexes followed by immunoblotting with vcp, hdac6 or clathrin antibody. ∆f508-cftr levels were measured by metabolic labeling using tran-35s-label (250 µci/ml) for a 30 min pulse and 2 hrs chase. the subsequent effects of these inhibitors on vcp localization was detected by fluorescence microscopy of gfp moiety. results: the co-immunoprecipitation experiments showed that hdac6inhibition by 10µm tubacin (12 hrs) promotes vcp:∆f508-cftr and prevents hdac6:∆f508-cftr interaction. the proteasome inhibition (mg132 10µm) resulted in maximal hdac6:∆f508-cftr at 4hrs with minimal at 12 hrs while vcp:∆f508-cftr increased overtime (4 to 12hrs). adding tubacin (10µm; 4 hrs) reversed the proteasomal inhibitor effects. in pulse-chase experiments, mg-132 increased the accumulation of cftr bform and poly-ubiquitinated-∆f508-cftr as compared to the untreated control. tubacin suppressed the levels of mg-132 induced poly-ubiquinated-∆f508-cftr as well as ∆f508-cftr b-form. the vcp localization by microscopy showed the accumulation of vcp-gfp in perinuclear aggregates in the presence of proteasome inhibitor (ps-341 10µm). the 10µm tubacin blocked these ps-341 induced perinuclear aggregates. we observed that vcp is associated with endocytic protein-complexes containing clathrin in the presence of ps-341 indicating cytosolic re-localization of vcp. we further confirmed the cytosolic re-localization of vcp-gfp in the presence of lysosome inhibitor (bafilomycin a1 10µm) by fluorescent microscopy. conclusion: we found that a small molecule inhibitor of aggresome function prevents hdac6:∆f508-cftr interaction, promotes vcp:∆f508-cftr interaction, and suppresses the accumulation of poly-ubiquinated-∆f508-cftr. wang, y. 1 ; jiang, y. 1 ; zhu, n. 1 ; feng, x. 1 ; yang, h. 1, 2 ; ma, t. 1, 2 1. membrane channel research laboratory, northeast normal university, changchun, china; 2. biopharmaceutical center, liaoning normal university, dalian, china deletion of the codon encoding the phenylalanine residue at position 508 (∆f508) in the cystic fibrosis transmembrane conductance regulator (cftr) is the most common mutation causing cystic fibrosis (cf). the ∆f508 mutation results in a cftr protein with impaired folding, trafficking and gating in human and rodents. the consequences of ∆f508-cftr mutation in other species have not been studied. the purpose of the present study was to characterize the ∆f508 mutant of porcine cftr in cell culture model. cdna sequence encoding full-length porcine cftr (pcftr) was cloned from the lung by rt-pcr using primers designed according to porcine genomic sequences in two bac clones (genbank accession no. ac092497 and ac092478) containing all exons of the porcine cftr. pcftr encodes a 1482-amino-acid protein that shows 92.3% identity to human cftr. the phenylalanine residue at position 508 is conserved in pcftr. functional analysis of pcftr stably expressed in fisher rat thyroid (frt) epithelial cells by short-circuit current assays indicated a campactivated cl-channel similar to human cftr except that the sensitivity of pcftr to the specific cftr inhibitor cftrinh-172 is 25-fold lower than that of human cftr. a ∆f508 mutation of porcine cftr was generated by site-directed mutagenesis. surprisingly, ∆f508-pcftr expressed as a func-tional chloride channel activated by forskolin in stably transfected frt cells. western blot analysis of cos7 and bhk cells transiently transfected with cmyc-tagged ∆f508-pcftr indicated a protein pattern identical to that of wildtype pcftr. immunofluorescence analysis of the transiently transfected cells demonstrated similar plasma membrane expression pattern of ∆f508-pcftr and wildtype pcftr. these results clearly demonstrated unimpaired cellular processing of ∆f508-pcftr. however, the sensitivity of ∆f508-pcftr to forskolin activation (ic50~2.5 µm) was dramatically lower than that of wildtype pcftr (ic50~0.5 µm), indicating a defect of channel gating by pka phosphorylation. the present study provided the first evidence that the ∆f508 mutation in mammalian cftr does not result in impaired cellular processing that is closely associated with cf phenotype. we have optimized airway epithelial cell spheroid cultures for functional measurements of cftr chloride conductance in order to screen smallmolecule activators and inhibitors. spheroids, which are sealed spheroidal monolayers of airway surface epithelial cells with diameter 75-250 micron, were generated within 2 to 4 days after nasal brushing and suspension culture. yields of up to 2000 spheroids were obtained from nasal brushing of a non-cf or cf subject. approximately 3,000 spheroids could be prepared from one million freshly isolated human bronchial epithelial cells. we compared several electrophysiological and fluorescence methods to assay cftr function in spheroids. whole-cell current was measured in single spheroids following pipette immobilization and micropipette puncture of the solutionexposed apical cell membrane. large, forskolin-stimulated whole-cell chloride currents were measured in non-cf spheroids. as an independent approach, transepithelial potential difference was measured by micropipette insertion into the spheroid lumen. for fluorescence detection, cell cytoplasm was stained with the halide indicators mqae or lzq. fluorescently stained spheroids were immobilized on a coated coverglass or in a custom microfluidic chamber. halide flux across the apical surface was measured in response to chloride-nitrate or iodide-chloride solution exchange, in the absence and presence of camp activators. the fluorescence assay is suitable for medium-throughput screening of ∆f508 correctors and potentiators, with definitive verification of cftr chloride channel function by whole-cell current analysis. an airway spheroid cultures provide a powerful approach to prioritize the efficacy of ∆f508 correctors because of their rapid generation without prolonged culture, and their suitability for quantitative analysis of cftr function. supported by cff and nih. background and aims: cftr has been shown to be expressed and functional in ventricular cardiomyocytes. however, to date, no physiological role for cftr in the heart has been established. the aim of this study was to determine if cftr plays a role in the regulation of cardiomyocyte contraction rate. methods: cardiomyocyte contraction rate was assessed using the neonatal mouse beating assay. ventricular myocytes were isolated from neonatal mice and cultured for 2-3 days until they formed a functional in sitium. contraction rates were captured by video imaging and analyzed by metamorph in the presence and absence of beta-adrenergic receptor stimulation by isoproteronol (10 µm). assessment of cftr activity was determined by pharmacological inhibition by glibenclamide (100 µm), dpc (500 µm), and cftr inh-172 (10 and 20 µm). the role of k atp channels was assessed by 5-hd (100 µm). results: baseline contraction rate of vehicletreated (dmso or h 2 o) cardiomyocytes was unchanged. addition of isoproteronol caused a significant increase in contraction rate, which was sustained for at least 30 minutes. in contrast, application of glibenclamide to beating ventricular myocytes significantly, but transiently, inhibited cardiomyocyte contraction rate, with recovery occurring within 20 minutes. glibenclamide did not affect the initial increase in contraction rate caused by isoproteronol stimulation, but inhibited sustained increases in isoproteronol-stimulated contraction rate. to determine if this was an effect of k atp channel inhibition we examined the effect of 5-hd on contraction rate. in contrast to glibenclamide, 5-hd had no inhibitory affect on basal or isoproteronol-stimulated contraction rate, indicating glibenclamide's inhibitory effect was due to its inhibition of cftr. to further verify this, we examined the effect of two other cftr inhibitors, dpc and cftr inh-172 on ventricular cardiomyocyte contraction rate. both dpc and cftr inh-172 elicited similar responses as glibenclamide, with a quick, but transient inhibition of baseline contraction rate and inhibition of sustained isoproteronol-stimulated increases in cardiomyocyte beating. summary and conclusions: using neonatal murine ventricular myocyte cultures inhibition of cftr leads to a significant, but transient inhibition of baseline ventricular cardiomyocyte contraction rate. additionally, while inhibition of cftr did not affect initial increases in beta-adrenergic receptor-stimulated contraction rate, cftr inhibition did prevent sustained increases in contraction rate. the data suggest that cftr plays a role in modulating ventricular cardiomyocyte contraction rate during resting and adrenergic-stimulated conditions. drevillon, l. 1,2 ; tanguy, g. 1, 2 ; hinzpeter, a. 1,2 ; arous, n. 1,2 ; goossens, m. 1,2 ; fanen, p. 1, 2 1. génétique, inserm u.841, créteil, france; 2. faculté de médecine, université paris 12, créteil, france cystic fibrosis is mainly caused by mutations that interfere with the biosynthetic folding of the cftr protein. the aim of this study was to find cellular proteins capable of interacting with cftr and modifying its processing or its trafficking. we have used a genetic screen in yeast to identify such proteins and identified commd1 that preferentially interacted with the third cytoplasmic loop of cftr. commd1 is a regulator of copper transporter atp7b in hepatocyte and sodium epithelial channel enac, but its exact biochemical function and physiological relevance remain elusive. here, we report that first, commd1 interacted with wild-type and f508del-cftr in epithelial cells and second, commd1 subcellular distribution was not only both nuclear and cytoplasmic, but also in cytoplasmic vesicular compartments. we wondered if commd1 was involved in the processing and/or the trafficking of cftr protein in epithelial cells. we demonstrated that commd1 was not involved in cftr processing but that cell surface expression of wild-type cftr at the plasma membrane was cut by half when commd1 expression was 90% reduced by rna interference. we drew the conclusion that commd1 is a major component of cftr trafficking and/or recycling at the plasma membrane, precise characterization of commd1 in these vesicular compartments is under process to decipher its function in cftr trafficking. commd1 has been labelled as the prototype of a newly described protein family that plays a role in inhibiting nf-kappab signalling. moreover, subcellular distribution of commd1 was different in cf and non cf cells, nuclear form of commd1 being increased in f508del cells compared to wild-type cells. thus, this opens up a new area of investigation about commd1 nuclear function. finally, these data indicate that commd1 is involved in multiple cellular processes of outstanding interest in cf pathophysiology. understanding how its modulation modifies transepithelial transport and inflammation in cf versus non cf cells should give new therapeutic clues to reduce exaggerated inflammation and improve fluid secretion in cf patients. supported 1 ; fanen, p. 2 ; galietta, l.j. 1 1. lab. of molecular genetics, istituto giannina gaslini, genova, italy; 2. inserm u.841, creteil, france class iii cystic fibrosis mutations cause reduced cltransport by impairing the process of cftr channel opening. typical class iii mutations include g551d, g1349d, and f508del, all of them affecting nucleotide binding domains. it has been reported that mutations residing in other regions of the cftr protein may also reduce channel activity. we investigated the extent of loss of function caused by such mutations, in particular by missense mutations localized in cftr intracellular loops, and the sensitivity to pharmacological stimulation with cftr potentiators. to determine cftr activity, plasmids carrying the coding sequence for wild type or mutant cftr were co-transfected in cos-7 cells together with the halide-sensitive yellow fluorescent protein yfp-h148q/i152l. the rate of cftr-dependent anion transport was measured after short-term stimulation with forskolin alone or in combination with potentiators (felodipine, phenylglycine pg-01, or sulfonamide sf-01). processing of cftr protein was assessed by examining glycosylation status of cftr by immunoprecipitation experiments. when stimulated with forskolin alone, cftr mutants showed the following anion transport compared to the wild type protein: i148t (45 %), i175v (153 %), q179k (54 %), e193k (9 %), g970r (6 %), d1152h (29 %). interestingly, all mutants having a reduced anion transport responded to stimulation with potentiators pg-01, sf-01, and felodipine (5 µm) with an increase in anion transport that approached the level of normal cftr protein. the correction of defective cftr activity by potentiators was confirmed in frt cells expressing e193k and d1152h mutants using short-circuit current measurements. our results indicate that some missense cf mutations, like e193k and g970r, really cause a severe reduction in cftr activity. others, like d1152h, cause only a partial loss of function which may explain their association with a milder form of the disease. conversely, some other amino acid changes (with the extreme example of i175v that seems to cause a gain of function) do not impair cftr activity and therefore may represent polymorphisms and not cf-causing mutations. interestingly, all mutants having a mild or more severe anion transport defect were sensitive to potentiators. this finding indicates that cftr potentiators have a wide efficacy on many class iii mutants and therefore may represent a promising therapeutic strategy for a significant number of cf patients. supported by cfft and telethon-italy. cftr, a cl-channel membrane protein, is a member of the atp-binding cassette (abc) super-family. mutations in cftr result in a misfolded protein which fails to mature, leading to impaired flow of cl-ions across the membrane of the epithelial cells lining the airway, and to cf. the most common mutation leading to cf is df508. currently available structural information relevant to cftr modeling includes low resolution structures of cftr (20å) and p-gp (8å) and high resolution structures of other abc transporters as well as nucleotide binding domains (nbd) . of particular relevance is the newly published structure of the bacterial multidrug abc transporter, sav1866, whose topology can serve as a starting point for cftr modeling. as a first step towards the development of cf therapeutics, epix has undertaken the modeling of the 3d structure of cftr (excluding the r domain) in its conducting (i.e., open-channel, nbd-dimer) state, using our proprietary membrane-protein modeling technology (predicttm) in combination with other modeling tools. to date, we have modeled the cytosolic part of the receptor, namely the nbds:icls construct and work is ongoing on modeling the transmembrane (msd1:msd2) region. the now available partial model has been scanned for putative binding sites for df508-cftr correctors/potentiators. several potential sites have been located, and epix has initiated its in silico screening technology in order to identify potential drug candidates. this work is supported by the cfft. the deletion of a phenylalanine residue at position 508 (f508del) in the first nucleotide-binding domain (nbd1) of cftr is the principal cause of cystic fibrosis (cf). the altered interaction of f508del cftr with endoplasmic reticulum (er) quality control proteins, primarily chaperones, promotes its proteasomal degradation. however, it is believed that crucial cftr-interacting proteins (cips) remain unknown [1] . moreover, there is little information currently available on the strength of cip-cftr interactions. our goals here are two-fold: 1) to quantify cftr-cip interactions; and 2) to isolate and characterise unidentified cips. to address our first goal, we used surface plasmon resonance (spr; biacore tm ) to quantify real-time binding of hsc70 (a well-known cip) to bacterially expressed wt-and f508del-nbd1. hsc70/hsp70 was covalently immobilised onto the surface of carboxymethyl dextran (cm5) sensor chips (500 µm) and the real-time binding of purified nbd1 and control proteins quantified. in control experiments, anti-hsc70 antibody 1b5 (20 nm; against residues 373-650) bound specifically to immobilised hsc70 with high affinity (k d , 0.46 ± 0.07 nm; n = 3) whereas bovine serum albumin (15 µm) did not interact (n = 10). given the difficulties associated with expression of hnbd1 carrying the f508del mutation [2] it was not used in our spr analyses. instead, we used purified murine (m) nbd1 to quantify the impact of f508del on the strength of the hsc70-nbd1 binding. in the presence of low concentrations of atp (≤5 um), the affinity of nbd1 binding to immobilised hsc70 was strengthened when f508 was deleted (wt, k d app , 1.20 ± 0.16 µm; f508del, k d app , 0.43 ± 0.11 µm; n = 3; p < 0.05). interestingly, raising the concentration of mgatp weakened the binding of nbd1 (0.5 µm) to hsc70 (wt, k i , 29 µm atp; n = 2). furthermore, deletion of f508 dramatically increased the concentration of mgatp required to destabilise the nbd1-hsc70 interaction (f508del, k i , 221 µm atp; n = 2). in summary, we used spr to demonstrate that: (i) nbd1 of cftr binds specifically to hsc70, and (ii) the f508del mutation enhances the association of nbd1 with hsc70. we are presently investigating the effect of co-chaperones and small molecules on the interaction of hsc/hsp70 with wt-and f508del-nbd1. to identify novel cips, purified nbd1 was immobilised onto metalaffinity resin and used to capture cips from epithelial cell lysates. by this approach, cips were captured from human respiratory cell (calu-3) lysates and analysed by 2d-electrophoresis. relevant protein spots so far identified by mass spectrometry include: 1) raichu404x (thr/ser kinase); 2) profilin 2 isoform b; 3) annexin a5; 4) ifapsoriasin (intermediate filament-associated ca 2+ regulatory protein); 5) mgc35308 (member of the er reticulon family). current analyses are underway to determine their functional roles in cftr trafficking and function. [1] amaral ( billet, a. 1 ; melin, p. 1 ; norez, c. 1 ; bilan, f. 2 ; vandebrouck, c. 1 ; mettey, y. 3 ; becq, f. 1 1. physiologie, cnrs umr 6187, poitiers, france; 2. gmas umr 6187, poitiers, france; 3. faculté medecine et pharmacie, poitiers, france in order to gain a better insight into the structure-activity relationship (sar) of cftr protein, we have functionally characterized a cystic fibrosis mutation: the cftr-g622d, identified in patients with oligospermia (vankeerberghen et al., 1998) . we examined cftr chloride channel activity by patch-clamp analysis, in whole cell configuration, using cos-7 cells transiently transfected with gfp-cftr-wt or with the mutant gfp-cftr-g622d. cftr channels were activated by 10 µm forskolin (fsk), and, after stable activation 10 µm cftrinh-172 was added. as gfp-cftr-wt, g622d chloride channels elicit a time and voltage independent current in presence of 10 µm fsk. gfp-cftr-g622d current density at +40mv (52.08 ± 5.8 pa/pf) is 1.5 fold less than gfp-cftr-wt current density at +40mv (76.67 ± 9.3 pa/pf). g622d mediated currents are blocked by 10 µm of the specific cftr-inhibitor, cftrinh-172 (ma et al., 2002) . as expected, gfp-cftr-wt channels are stimulated by 100 µm of the potent activator 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (mpb-91, derand et al., 2001 ) with a current density at +40 mv of 127.44 ± 25.49 pa/pf. interestingly, no activation of g622d channels is recorded in presence of mpb-91: difference between currents recorded in basal condition or in presence of mpb-91 is not significant. using iodide efflux experiments, we showed that cftr-g622d mutant does not respond to several benzo[c]quinolizinium derivatives. however, the mutant channel can be activated by forskolin, ibmx and genistein. thus, the mechanisms of activation by xanthine and isoflavone are not affected by the mutation. these results show that: (1) cftr-g622d channels are functional: activation by camp pathway, inhibition by cftrinh-172. (2) camp chloride currents elicited by cftr-g622d protein are weaker than cftr-wt. (3) cftr-g622d channels are refractory to benzo[c]quinolizinium activation. the replacement of the glycine by a negative charged amino acid in position 622 affects cftr chloride channel activity and prevents activation by benzo [c] quinolizinium. this amino acid g622, localized in the interface of nbd1 and r domain between two β sheets (callebaut et al., 2004) seems to have a crucial role in pharmacological activation of cftr by mpbs. further experiments will be performed to evaluate the role of the charge and of the nature of the amino acid at position 622 in cftr protein. supported by vaincre la mucoviscidose and cnrs. cysteine string protein (csp) is a j-domain containing protein that regulates the extent of wild type cftr maturation. over-expression of csp blocks the formation of mature, glycosylated (band c) cftr and increases the level of immature (band b) cftr, whereas knockdown of csp elicits a marked increase in cftr maturation (zhang et al., j. biol. chem. 281: 11312, 2006) . thus, csp appears to block the exit of cftr from the er. when the er exit of cftr is blocked by a different mechanism, i.e. the expression of mutant sar1-gtp (sar1 h79g interferes with the formation of copii vesicles), the steady-state level of immature cftr is two-fold greater than that resulting from csp over-expression. this finding suggests that csp, in addition to blocking er exit of cftr, may facilitate the degradation of the immature protein. in agreement with this concept, csp expression increased cftr ubiquitylation. a mutation within the conserved hpd motif of the j-domain (h43q) disrupts the ability of csp to interact with and stimulate the atpase activity of hsp70. csp h43q does not block cftr maturation (above ref); in addition, the level of immature cftr was ~60% higher when h43q csp was over-expressed than the level observed with mutant sar1. this finding suggests that the pro-degradative effect of csp on immature cftr requires hsp70 binding/activation. chip (carboxy terminus of hsp70-interacting protein) is an e3 ubiquitin protein ligase that can target wt and ∆f508 cftr for proteasome-mediated degradation (meacham et al., nature cell biol. 3: 100, 2001) . we sought to determine weather chip was involved in the csp-mediated degradation of cftr. indeed, co-immunoprecipitation (co-ip) experiments showed that chip associated with csp. also, csp h43q, which has reduced interaction with hsp70, showed a similar level of chip interaction by co-ip, suggesting that their association did not depend on hsp70 as a linker between the two proteins. a csp mutant that is truncated after the cysteine string domain, which is its site of membrane attachment, blocked cftr maturation as did wt csp; however, this mutant did not decrease cftr band b levels like wt csp did. in addition, the csp mutant missing its cterminus did not associate with chip. these findings further implicate chip in the csp-mediated degradation of immature cftr, and they suggest that the c-terminus of csp is a chip binding domain. when a dominant-negative mutant of chip, which lacks the u-box needed for chip-mediated ubiquitylation (chip∆ub), was co-expressed with cftr, csp was no longer able to facilitate the degradation of cftr in the er. these data show that it is possible to separate the effect of csp on cftr maturation from its ability to mediate the degradation of immature cftr. thus, csp blocks the exit of cftr from the er, and this requires a j-domain mediated activation of hsp70 but not the csp c-terminus. the action of csp on cftr degradation requires both a functional j-domain and the csp c-terminus, and this may require a trimeric complex involving csp, hsp70 and chip. [ the cystic fibrosis transmembrane conductance regulator (cftr) represents the main clchannel in the apical membrane of epithelial cells for camp-dependent clsecretion. mutations of this channel causes cystic fibrosis disease ; thus discovery of pharmacological activators of cftr is crucial to design future medicament for protein therapy. recently, we reported on the synthesis and screening of a small library of 6-phenylpyrrolo [2,3b] pyrazines (named rp derivatives) evaluated as activators of wild-type cftr, g551d-cftr and f508del-cftr clchannels (noël et al, 2006) . this preliminary structure-activity relationship study identified 4-hydroxyphenyl and 7-n-butyl as determinants required for activation of cftr (rp-107 and rp-108). here we studied structure-function relationship of more than 190 compounds prepared by chemical synthesis, and the subsequent activation of cftr channels. within the 6-phenylpyrrolo [2,3-b] pyrazines family, we observed by iodide efflux technique that rp-173 bearing a 2hydroxyphenyl substituant is more potent (ec 50 = 16 nm) than rp-107 having 4-hydroxyphenyl substituant (ec 50 = 150 nm). by whole-cell patch clamp recording analysis, we confirmed that nanomolar concentrations of rp-173 activate linear chloride current in cho cells stably transfected with human wild-type cftr. this current was inhibited by 10 µm of cftr inh -172. we also found significant stimulation of short circuit current (i sc ) by rp-173 (ec 50 = 9 nm) on colon of cftr +/+ but not of cftr -/mice mounted in ussing chamber. stimulation of i sc by rp derivatives was inhibited by glibenclamide. the structural analogue , was less potent (ec 50 = 347 nm). as for rp-107 compound, we found that the 7-nbutyl chain is crucial for rp-146 and rp-173 activity, and that the 2-hydroxyphenyl compounds without 7-n-butyl chain are unactive on cftr. in this study, we showed that the presence of an hydroxyl group at position 2, 3 or 4 of the pyrrolopyrazine cycle determined the highest activity on cftr. the most potent compound is the 7-n-butyl-6-(2-hydroxyphenyl)5hpyrrolo [2,3b] pyrazine . the potency of these agents indicates that compounds in this class may be of therapeutic benefit in cftr-related diseases, including cystic fibrosis. the most common mutation causing cystic fibrosis (cf) is the deletion of f508 in the cftr gene, which results in inefficient trafficking of the cftr protein from the endoplasmic reticulum to the plasma membrane. the surface expression of the cftr is usually quantified biochemically with western blot or functionally with electrophysiological assays. however, these methods are labor intensive. we explore using fluorescence-based techniques for monitoring cftr surface expression. wild-type cftr was tagged at the amino terminus with green fluorescent polypeptides (gfp-cftr). the gfp-cftr was either transiently or stably expressed in chinese hamster ovary (cho) cells. the cells were then treated with a red fluorophore that specifically labels plasma membranes. the fluorescent images were acquired with an olympus laser scanning confocal microscope. two image channels were taken simultaneously. the green channel was excited with a 488nm laser, and signals within 500-545nm were collected. the red channel was excited with a 559nm laser, and signals within 570-670nm were collected. for a single focal plane, each image was composed of 1024*1024 pixels. threshold values were given to both green and red channels. pixels with an intensity value above the threshold were marked as green/red pixels. the green pixels in the first channel represent the signal of gfp-cftr, and the red pixels in the second channel represent the location of the plasma membrane. thus, pixels that are both green in the first channel and red in the second channel are the cftr surface expression, and denoted as yellow pixels. the percentage of successfully trafficked cftr is thus quantitatively estimated by the ratio of yellow to green pixels in the single plane. alternatively, a stack of images from the same cell at different depth was collected and analyzed. the total amount and surface portion of cftr expression are then determined by summing the correspondent pixels. the percentage of cftr surface expression was then calculated and will be verified both biochemically and functionally. the approach is modified to study deltaf508 cftr trafficking. we anticipate that the method can be used to screen cftr correctors from a small or intermediate sized chemical database. supported by cfft and nih. the most common mutation of the cystic fibrosis transmembrane conductance regulator (cftr) gene (deletion of phe-508 (∆f508) in the first nucleotide binding domain (nbd1)) causes retention of the ∆f508 protein in the endoplasmic reticulum (er). ∆f508 mutation prevents conformational maturation of cftr protein without altering profoundly the local structure of nbd1, but possibly by disrupting the interaction between nbd1 and nbd2. however, the individual role of nbd1 and nbd2 in biogenesis, folding, maturation and membrane stability of a full length cftr is still under debate. using splicing by overlap extension method (soe by pcr), we generated six recombinant cftr proteins with inverted or suppressed nbds. the native boundary domains of nbd1 and nbd2 were respected for all mutants (riordan j.r. et al. 1989 ) and their c-terminal tails preserved. the constructs were transiently or stably expressed in cos-7 and bhk cells respectively. immmunodetection and immunolocalization assays confirmed that deletion of nbd2 domain (n1-cftr) did not alter the trafficking or the plasma membrane expression of cftr. both processes were abolished in the presence of ∆f508 mutation. however, using whole cell patch-clamp configuration, we did not detect any camp-stimulated clcurrent. pulse-chase experiments established that the turnover of n1-cftr is 3-4 fold faster (t1/2~ 5h) than its wild type counterpart (t1/2~ 18h). when we replaced nbd2 with nbd1 (2n1-cftr), we could detect a faint plasma membrane expression associated with a weak cl-channel activation. more surprisingly, the t1/2 of 2n1-cftr coreglycosylated form was around 12h. when nbd1 was deleted (n2-cftr) or substituted with nbd2 domain (2n2-cftr), the expression and functional pattern were similar to cftr ∆f508. furthermore, we showed that the n1-cftr folding was not attenuated as demonstrated by protease susceptibility and sdsresistant thermoaggregation tendency, supporting the notion that n1-cftr membrane stability requires nbd1-nbd2 interaction. altogether, our results suggest that nbd1 domain is essential to form a foldable cftr that satisfies er quality control (erqc) but correct inter-domain assembly is mandatory for its stability at plasma membrane. the results are also in agreement with previous suggestion that ∆f508 mutation alters the interactions between nbds and transmembrane domains of cftr. supported 1 1. inserm u845, inserm, paris, france; 2. inserm u845, inserm, paris, france; 3. inserm u845, inserm, paris, france; 4. inserm u845, inserm, paris, france; 5. inserm u845, inserm, paris, france; 6. inserm u845, inserm, paris, france; 7. inserm 845, inserm, paris, france; 8. inserm u845, inserm, paris, france the physiological role of cftr in renal epithelium is not known. in the proximal tubule, cftr co-localizes with the sodium-phosphate cotransporter npt2a, a protein involved in phosphate (pi) reabsorption. the aim of our work is to determine if there is a functional interaction between cftr and npt2a. to this end, the arnm coding for cftr and the arn coding for npt2a (bearing a myc tag in c-ter) were injected in xenopus laevis oocytes. electrophysiological or biochemical experiments were performed 3 days after arns injection. currents induced by 1mm pi (ipi) were measured in voltage-clamped (-50 mv) oocytes that expressed npt2a alone, or in oocytes co-expressing cftr + npt2a. for immunoprecipitation experiments, 24-1 cftrab and myc ab were used. results ipi was significantly reduced in oocytes expressing cftr + npt2a compared to that measured in oocytes expressing npt2a alone. using a two-bath electrodes configuration during voltage-clamping did not change this observation. this result suggests that when expressed in oocytes, cftr is in functional interaction with npt2a. kinetic analysis (ipi as a function of pi concentration) of npt2a showed a reduction in vmax but not in phosphate km. this suggests that npt2a membrane expression and/or activity is altered when cftr is co-expressed. co-immunoprecipitation experiments performed on protein lysate derived from oocytes co-expressing cftr and npt2a suggested an interaction between the 2 transporters. using a pka activating cocktail as a pkc activator induced cftr-mediated currents in cftr+npt2a expressing oocytes. in these oocytes, ipi is decreased by pkc activation (as expected from npt2a regulation) but is increased shortly after exposure to the pka activating cocktail. the increase of ipi observed under this condition is at variance to that expected from classical npt2a regulation. in oocytes expressing npt2a alone, both pka activation and pkc activation reduced ipi. these observations suggest that in oocytes, cftr expression modifies npt2a regulation. our study suggests that, after expression in xenopus laevis oocytes, cftr interacts functionally with npt2a, and modifies its regulation. an interaction is also suggested by the co-immunoprecipitation of cftr and npt2a. these results cannot discriminate between a direct or an indirect (via a third protein) interaction. to determine if an intracellular chloride concentration change may participate to the functional interaction, we are now measuring in cftr+npt2a expressing oocytes intracellular chloride activity (using chloride-sensitive microelectrodes) during the use of the pka activating cocktail, and measuring ipi when pka is stimulated in the presence of a specific cftr inhibitor. cystic fibrosis (cf), the most commonly inherited lethal pulmonary disorder in caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (cftr). to date, more than 1000 mutations were identified in cftr and were associated with clinical disease. a phenylalanine deletion at position 508 accounts for 70% of cf genotypes in caucasian populations, and determines cftr misfolding and degradation by proteasome. consequently, limited cftr abundance leads to multiorgan disease, affecting the lung, pancreas, gut, liver, sweat glands and the reproductive organs. the past several years have witnessed an explosion of information regarding the identification and elucidation of molecules and pathways that are regulated by cftr or that regulate cftr activity. genomics and proteomics technologies now offer the opportunity to examine global alterations in the mrna and protein expression patterns of cf cells and tissues to elucidate the pathways linking defective cftr to clinical disease. here we describe systems biology methods that integrate heterogeneous datasets, including protein-protein interaction networks, gene expression and mass-spectrometry profiles, and mutation and genetic variation information, in order to identify the regulatory circuits and active subnetworks that are responsible for the progression and the severity of cf disease. the study will focus on correlations that define the key events in the cftr folding and trafficking pathways that lead to pathogenesis and dysfunctions, and could provide insights and targets for intervention and therapy. the number of cftr cl-channels in the plasma membrane, and thus the transepithelial cl-secretion is controlled, in part, by the endocytic trafficking of cftr. rab gtpases regulate the endocytic trafficking by acting as molecular switches that cycle between the gdp-bound (i.e. inactive) and the gtp-bound (i.e. active) state, associate with target membranes, and recruit downstream effectors. rab5 facilitates cftr endocytosis and rab11a facilitates the recycling of internalized cftr to the plasma membrane. furthermore, rab7 and rab9 facilitate cftr trafficking to the lysosome. the mechanism of sorting internalized cftr for either recycling to the plasma membrane or degradation in the lysosome is not well understood. recent evidence demonstrates that protein sorting occurs in rab4 specific sorting endosomes. presence of cftr in rab4 compartment has been previously demonstrated in human airway epithelial cells by electron microscopy. yet, the role of rab4 in cftr trafficking has not been determined in these cells. rab4 did not control cftr trafficking in fibroblasts (bhk cells). however, it negatively regulated the plasma membrane expression of cftr in the colorectal cells . the goal of this study was to elucidate the role of rab4 in the endocytic trafficking of cftr in polarized human airway epithelial cells (calu-3 cells and cfbe41o-cells stably expressing wt-cftr or deltaf508-cftr). endogenous rab4 coimmunoprecipitated with cftr in calu-3 cell and cfbe41o-cells. to determine if rab4 plays a role in cftr trafficking, cfbe41o-cells were transfected with either the flag-tagged, dominant negative (dn) rab4 mutant deficient in gtp binding (gdp-locked; flag-rab4-n121i) or with a non-specific cdna control (gfp). if rab4 sorts internalized cftr to the recycling pathway, the dn rab4 should inhibit recycling, and, thus, should decrease the plasma membrane expression of cftr. if, on the other hand, rab4 sorts internalized cftr for degradation the dn rab4 should inhibit cftr degradation, increase cftr recycling and thus, should increase the expression of cftr in the plasma membrane. the dn rab4 increased cftr expression in the plasma membrane as determined by cell surface biotinylation. these data are consistent with the role of rab4 in sorting internalized cftr for degradation. similar to overexpression of the dn rab4, silencing endogenous rab4 with double stranded small interfering rna specific for the human rab4 sequence increased the plasma membrane expression of cftr and increased the cftr mediated cl-secretion across polarized cfbe41o-cells in ussing chamber experiments. the plasma membrane half-life of deltaf508-cftr is decreased compared to wt-cftr. however, after silencing rab4, the plasma membrane half-life of deltaf508-cftr was similar to that of wt-cftr, as determined by cftr disappearance from the plasma membrane following incubation with cycloheximide. our data are consistent with the role of rab4 in sorting the internalized cftr for degradation and are consistent with the changing paradigm for the role of rab4 in protein trafficking (supported by the nih grant 1 p20 rr018787 and the cff swiate03qo). the conformational changes underlying activation of phosphorylated cftr by atp remain unclear. in the present study we assessed the utility of labeling endogenous cysteine(s) in cftr using an environmentally-sensitive fluorescent probe alexa-488 in monitoring such changes in structure. these studies were performed using purified and functionally reconstituted wild-type cftr. we labeled pka-phosphorylated cftr with 138 µm alexa-488 maleimide prior to reconstitution in phospholipids liposomes. changes in fluorescence intensity were monitored in a suspension of proteoliposomes following addition of 1 and 5 mm mg-atp or 5 mm mg-amp-pnp in order to evaluate nucleotide dependent changes in conformation. in order to identify the labeled cysteines, the protein was subjected trypsin-mediated proteolysis and analysis by mass spectrometry. we have shown that cftr is labeled using alexa-488 maleimide. a significant increase in fluorescence emission occurred in alexa-488 labeled cftr of 17 ± 5 (au) relative to empty liposomes one minute after the addition of activating nucleotide, 1 mm mg-atp (n = 3). the non-hydrolyzable analogue, 1mm mg-amp-pnp, failed to cause an increase in fluorescence of alexa-488-labeled cftr. however, the higher concentration of 5 mm mg-amp-pnp evoked an increase in fluorescence similar in magnitude to that of 5 mm mg-atp suggesting that the fluorescence response reflects structural changes relating to atp binding to cftr. analysis by mass spectrometry identified the labeled residue as c647. this cysteine resides at the junction between nbd1 and the "r" domain in a region which has been suggested to be highly dynamic on the basis of multiple crystal structures. our studies suggest that this flexible region around c647 of cftr undergoes conformational change upon mg-atp binding such that it moves the alexa 488 fluorophore into a relatively more polar environment. this finding presents a first case where conformational changes in the cftr nbds induced by atp binding could lead to changes in the conformation of the r domain. further, these studies suggest that fluorescence methods can be used to probe dynamic conformational changes in purified reconstituted cftr. acknowledgements cystic fibrosis is caused by mutations in the apical chloride channel cftr with 90% of patients carrying at least one allele of the ∆f508 mutation. this mutant form of cftr is characterized by a trafficking defect in which it fails to exit the endoplasmic reticulum (er). we have previously reported that ∆f508 cftr is found in complex with hsp90 and its co-chaperones in cell extracts suggesting that either this mutant form of the protein accumulates in on-pathway folding intermediates which result in its accumulation in the er or that these intermediates include inhibitory factors leading to retention of ∆f508 cftr in this compartment. we have examined the role of one of these hsp90 co-chaperones, fk506 binding protein 8 (fkbp8), in the life cycle of cftr. fkbp8 is a unique member of the immunophilin family of peptidyl-prolyl isomerases (ppiases) which mediate the cis/trans interconversion of peptidyl-prolyl bonds, which is thought to represent a rate limiting step in protein folding. in order to establish a functional role for fkbp8 in cftr stability and trafficking, we modulated the expression level of this protein in human bronchial epithelial cells endogenously expressing either wild type or ∆f508 cftr. a knockdown of fkbp8 by sirna resulted in a destabilization of both wild type and ∆f508 cftr as well as a concomitant loss in channel activity of wild type and temperature corrected ∆f508 cftr. conversely, over expression of fkbp8 resulted in a stabilization of the er glycoform of ∆f508 cftr in both mammalian and yeast systems. these data suggest that fkbp8 is an essential component of the hsp90-mediated folding machinery for both wild type and ∆f508 cftr, which supports the hypothesis that ∆f508 cftr accumulates in the er in an on-pathway folding intermediate. dmh cftr folding involves a series of sequential, although potentially overlapping steps that involve i) formation, orientation and integration of (tms), ii) helical packing, iii) folding of cytosolic and extracytosolic domains, and iv) establishing proper domain-domain contacts. this process begins cotranslationally and is facilitated by the sec61 er translocation machinery and a diverse group of lumenal and cytosolic chaperones. the most common inherited cftr mutation, deltaf508, disrupts one or more early steps along this folding pathway. a major obstacle in understanding how deltaf508 causes cf is that the native folding environment, comprised of lipids, cytosolic and er proteins, is not amenable to traditional biochemical and biophysical approaches. to overcome this problem we are developing a flu-orescence based analytical approach that enables one to directly access nascent ribosome-attached folding intermediates at virtually any stage of synthesis. the strategy requires incorporation of fluorescent probes into the substrate protein, which provide a highly selective means to monitor structural features at sub-nanomolar concentrations in highly complex biological mixtures. polypeptides are synthesized in vitro from truncated mrna templates to generate uniform cohorts of ribosome-bound nascent chains. progressive stages of folding can then be evaluated using mrna templates of different length. as proof-of-principle we have shown that fully synthesized gfp variants can be readily trapped in an unfolded state and complete their folding only after the entire nascent peptide has exited the large ribosomal subunit. this approach enabled us to measure real-time folding kinetics of extended peptide domains following synchronous ribosome release and compare fluorophore maturation of four fluorescent protein variants with different spectral properties cfp, gfp, venus and mcherry. to extend this approach to cftr and other general protein substrates, we are employing fluorescence resonance energy transfer (fret) using "donor" and "acceptor" probes incorporated simultaneously into the substrate protein. an in frame n-terminal fusion to cfp provides the "donor" fluorophore, while the "acceptor" fluorophore is incorporated at an engineered stop codon using a synthetic modified aminoacyl-suppressor trna. we are currently developing a 'molecular ruler' to correlate the efficiency of energy transfer with changes in the relative distance between probes in ribosome-bound and ribosome-released cftr folding intermediates. fret-based experiments are underway to evaluate folding of both n-terminal (1-80aa) and nbd1 (387-674aa) domains. these studies will provide new insight into the mechanism of cotranslational cftr domain folding and provide a potential means to identify agents that correct the deltaf508 folding defect. relationships between hypoxia, ado release, and prostenoid production in ariway cells are not defined. in the present studies, we examined mechanisms governing hypoxia-induced production of prostenoids in cfbe41o-and calu-3 monolayers. using elisa-based detection of prostenoid and leukotriene production, both calu-3 and cfbe41omonolayers treated with ado (10 µm) or arachidonic acid (aa, 10 µm, the parent molecule of prostenoids and leukotrienes) for 16 hrs led to increases in pge 2 levels (1198-1283 ± 64-145 pg/mg of total proteins vs controls 732 ± 89 pg/mg in calu-3 cells, 756-839 ± 8-12 pg/mg vs controls 408 ± 42 pg/mg in cfbe41o-cells, p<0.05 in both airway cell types compared with control). in contrast, ltb 4 and ltc 4 release were not stimulated by both maneuvers. we next used sirna techniques to knockdown a2b adenosine receptor expression, and demonstrated durable knockdown of a2b ar expression to~25% of scrambled sirna-treated controls following lipid-based transfection with sirna directed against the a2b ar in cfbe41o-cells. following sirna knockdown of a2b ar expression, cells were exposed to hypoxic conditions (mucosal volume = 2 ml in 12-well plate), normoxic conditions (mucosal volume = 0.2 ml, fio 2 = 0.21), and ado (10 µm). hypoxic stress led to high levels of mucosal pge 2 production (1613 ± 102.8 pg/mg vs controls 495 ± 19.6 pg/mg, p<0.01) that was sensitive to a2b ar knockdown (743 ± 70.6 pg/mg, p<0.01). exogenous ado also stimulated pge 2 production, but this effect was small relative to hypoxia. we next examined basolateral effects of ado and aa on transmucosal clsecretion in cfbe41o-and calu-3 cells grown as monolayers on permeable supports and conducted ussing chamber analysis under voltage clamp conditions. addition of ado and aa to the basolateral membrane (10 µm) activated transmucosal clsecretion [18.94 ± 4.07 µa/cm 2 (ado) and 14.30 ± 2.6 µa/cm 2 (aa) for calu-3 cells; and 12.65 ± 2.31 µa/cm 2 (ado) and 18.96 ± 1.24 µa/cm 2 (aa) for cfbe41o-cells expressing wtcftr] that was sensitive to basolateral blockade with barium cl -(10 mm). isc by either agonist was stimulat-ed in the presence and absence of apical glybenclamide (200 µm), adenosine deaminase (ada -2u/ml) and hexokinase (hexo -2u/ml), confirming that the effects were specific for the basolateral membrane and could be accomplished with or without cftr activity. these results demonstrate that hypoxia is sufficient to stimulate pge 2 production through a2b ars, and that both ado and aa activate basolateral k + channels to promote transepithelial clsecretion. the findings further support a model in which ado (and prostenoids) regulate cltransport through effects on both the apical and the basolateral membrane, and elucidate molecules that couple the two membranes as part of the normal clsecretory response. hypoxia would be predicted to increase local prostenoid production through ado release and stimulation of a2b ars, and further highlight a unifying model by which local hypoxia promotes inflammation in airway epithelia. supported by the nih and cff. low levels of tissue oxygen -observed in lung diseases such as cystic fibrosis -initiate a signaling cascade resulting in altered transcription of genes possessing a hypoxia response element (hre). we recently used gene chip mrna array, quantitative rt-pcr, protein analysis, and functional (short circuit current) assays to show that cftr expression is strongly repressed by hypoxia in vitro and in vivo. among several thousand human mrnas suppressed by low ambient oxygen, cftr was among the most strongly inhibited (by 10-20 fold) in a cell model system. however, cftr and the vast majority of other repressed genes lack a traditional hre. because hypoxia inducible factor (hif) acts primarily as a transcriptional activator, the mechanisms underlying hypoxia mediated suppression of mammalian genes are not well understood. we therefore tested the hypothesis that micrornas play a central role during transcriptional regulation of genes such as cftr. micrornas are evolutionarily conserved, short noncoding rna sequences believed important for repression of gene expression. some reports have estimated that at least 30% of protein-coding genes are regulated by micrornas. thousands of micrornas have been predicted in the human genome by available algorithms (e.g. palgrade, mirscan, and promir) and several hundred experimentally validated. the cftr gene contains 16 predicted target sites for 13 micrornas in the 3'utr, including hsa-mir-383 (nucleotides 1309-1331 of the cftr 3'utr), hsa-mir-449 (nt 1443-1463) and hsa-mir-509 (nt 1041-1063). however, no study to date has examined the effect of micrornas on cftr message levels, or the influence of micrornas during the cftr response to environmental perturbations such as hypoxia. we therefore performed a global, genome-wide analyses of both mrnas and micrornas expressed during hypoxia (using air/liquid or liquid/liquid interface culture systems) in ht29 (colonic epithelial) cells that robustly express cftr. we found that approximately 9% of micrornas were significantly altered by hypoxia, as judged by mirna profiling (ambion mirvana™ bioarray 1566v2, asuragen microrna expression profiling service). comparison of these hypoxia-related micrornas with expression profiles of their predicted targets indicated a much lower level of correlation than has been previously hypothesized by others. we show that none of the micrornas predicted to regulate cftr are altered in ht29 (in response to hypoxia). these findings are therefore consistent with the notion that highly expressed genes in a particular tissue type are not co-expressed with their predicted regulatory micrornas. our data also indicate that micrornas do not mediate the hypoxic repression of cftr via the 3' utr. supported by nih and cff. 1 1. physiology, mcgill university, montreal, qc, canada; 2. biochemistry, mcgill university, montreal, qc, canada the ∆f508 mutation impairs cftr maturation and trafficking to the plasma membrane and results in a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. using our highthroughput trafficking assay, we previously showed that the quinoline km60, a structural analogue of sildenafil, corrects the ∆f508-cftr processing defect and leads to a significant increase in cftr surface expression after 2 h treatment with 10 µm and after 24 h treatment with 10 nm according to detection of mature cftr in western-blots. additional studies have now been carried out with other preparations to assess the functionality of ∆f508-cftr following rescue by km60 treatment. different time and concentration exposures were tested on the cftr function using iodide efflux assays with both bhk and human bronchial epithelial cells over-expressing ∆f508-cftr. exposure to 10 µm km60 for 2 h partially restored iodide efflux responses to 10 µm forskolin + 50 µm genistein in both cell line. after 24 h incubation, rescue of the mutant protein in bhk cells still required 10 µm, whereas 100 nm was sufficient in the human cell line cfbe. iodide efflux assays were performed at different times following km60 wash out to functionally assess the stability of the rescued mutant protein. camp-stimulated iodide efflux was still detectable 6 h after removing km60, but responsiveness was lost after 24 h in both cell types. electrophysiological studies were performed to examine the channel activity of ∆f508-cftr rescued by km60 treatment. incubation with 10 µm km60 for 48 h increased camp-responsive ∆f508-cftr current density in hek293 whole-cell patches ~6-fold. the conductance was time-and voltage-independent, anion selective, and strongly inhibited by glibenclamide. incubating polarized cfbe monolayers with 20 µm km60 increased the forskolin + genistein-stimulated short-circuit current after 48 h, and this response was inhibited by cftrinh172 (10 µm). permeabilization of the basolateral membrane with nystatin confirmed the apical location of the forskolin + genistein-stimulation. ussing chamber experiments also revealed trafficking correction in ileum that had been excised from cftrtm1eu mice homozygous for ∆f508. when the ileum was pre-incubated with 20 µm km60 for 4-6 h, the forskolin + genistein-stimulation of short-circuit current was significantly increased when compared to untreated intestine. these results confirm partial correction of ∆f508-cftr by km60 in unpolarized cells and in human polarized epithelial cell monolayers and ex-vivo intestine isolated from ∆f508-cftr mice. this work was supported by the breathe program funded by the ccff and cihr, cihr, cfft, and génome québec. primary sequence and overall structure of human cftr overlaps significantly with murine, rabbit and other homologues, a feature that has limited generation of conformation specific reagents to address cftr folding. because regions of ∆f508 cftr exposed extracellularly may exhibit aberrant topology (jbc 279:39620-7), probes that identify cftr from the extracellular surface could be useful to 1) verify plasma membrane localization of ∆f508 cftr, 2) discriminate wild-type from mutant protein, and 3) provide a means of determining whether small molecule "correctors" of the ∆f508 processing lead to a native (wildtype) cftr configuration. our laboratories are investigating phage-display techniques suitable for detecting properly folded extracellular domains of cftr. two large phage libraries encoding 7 or 12 amino acid sequences (approximately 10 9 phage per library) were panned against extracellular regions of cftr in hela cells. following incubation in blocking solution containing pbs and 1% bsa at 4°c, unbound fraction was incubated with recombinant helas expressing wild-type or temperature corrected ∆f508 cftr (protein expression verified biochemically and functionally). bound bacteriophage was dislodged after multiple washings by exposure to an acidic elution buffer and amplified to increase copy number (five rounds of antigen selection for each library, eur j biochem 268:2004-12). supernatants from microtiter wells expressing phage were analyzed for binding to parental (no cftr), wt or ∆f508 cftr expressing hela cells. using this method we obtained enrichment of phage at the surface of cells expressing wt or ∆f508 cftr. an example using a 7-amino acid sequence (10 10 phage) with specificity for ∆f508 cftr (representative of four repeat experiments) is shown in a binding assay with 10 6 hela cells/condition. reagents such as these can be further optimized by constructing second generation libraries using core recognition sequences (derived from the initial screen) and surrounding the core epitopes with random amino acid diversity. these experiments are intended to identify peptides with high binding affinity (nm binding constants) that recognize native cftr epitopes exposed extracellularly. because there is evidence that low temperature corrected ∆f508 cftr in the plasma membrane is misfolded, these probes may ultimately distinguish wild-type cftr from the surface targeted ∆f508 mutant and be useful as drug discovery reagents. supported by cff and nih. bridges, r.; nagubadi, s.; thakerar, a.; jia, y.; bradbury, n.a. physiology and biophysics, rosalind franklin university of medicine and science, north chicago, il, usa the goal of this project was to compare several reported correctors of ∆f508-cftr biosynthesis for their efficacy on chloride channel activity under a standard set of experimental conditions. fisher rat thyroid (frt) cells were transfected with ∆f508-cftr and a stable cell line selected with g418. cells were grown on transwell filters and studied under short circuit current (i sc ) conditions in ussing chambers. i sc measurements were performed at 27°c. the basolateral membrane was permeabilized with α toxin and chloride currents were measured with a serosal to mucosal chloride gradient. atp (1 mm) and gtp (100 µm) were added to the serosal bath. cftr channel activity was stimulated with camp (100 µm), ibmx (100 µm) and genistein (30 µm). cells were treated with correctors or vehicle (dmso) at 24 and 48 hours before the i sc measurements. a subgroup of cells were temperature corrected at 27°c for 48 hours for comparison. because only modest currents were observed even in the temperature corrected cells (3 -6 µa/cm 2 ) cells were treated for 48 hours with 5 mm sodium butyrate and 30 nm dexamethasone to enhance the observed currents. under these conditions temperature corrected cells displayed stimulated chloride currents of ~100 µa/cm 2 that were 6-8-fold greater than cells maintained at 37°c. correctors c4 (n5' ]bithiazolyl-2'-yl]-benzamide) and c1 (6-(1h-benzomidazol-2-ylsulfanylmethyl)-2-(6-methoxy-4-methyl-quinazolin-2-ylamino)pyrimidin-4-ol) caused a modest rescue of ∆f508-cftr chloride currents compared to cells maintained at 37°c (c4, 1.7-fold; c1, 1.3-fold) but far below the effect of temperature correction. rescue by c4 was concentration dependent with a significant observable effect at 300 nm and a maximum effect at 1 µm. rescue by c1 was observed at only 3 µm, lower concentrations had no effect and higher concentrations caused an inhibition of the stimulated chloride currents. compounds c2 -piperazin-1-yl]-ethyl}-4-piperidin-1-yl-quinazoline) (1 to 10 µm), c3 (4-cyclohexyloxy-2-{1[4-(4methoxy-benzensulfonyl)-piperazin-1yl]-ethyl}-quinazoline) (1 to 10 µm) and 4 phenylbutyrate (3 mm) did not significantly increase the stimulated chloride currents. all four compounds c1 -c4 caused significant decreases in the transepithelial resistances at the higher concentrations (3 -10 µm) suggesting some degree of cytotoxicity. our results indicate only c1 and c4 demonstrate efficacy in frt cells and that these effects are modest compared to temperature correction. supported by the cystic fibrosis foundation. cftr contains two membrane-spanning domains (msd), two nucleotide-binding domains (nbd), and a regulatory domain that require proper assembly for chloride channel activity. the most prevalent disease causing mutation, cftr∆f508, arises from deletion of phenylalanine 508 in the nbd1 domain. the cftr∆f508 mutant is translated and inserted into the endoplasmic reticulum (er) membrane, but is unable to attain its native state and accumulates in a kinetically trapped conformation that is degraded by the ubiquitin proteasome system (1) . the nature of the misfolded cftr∆f508 intermediate that is selected for premature degradation is not clear and is a topic of great interest (2) . recent work in the cyr lab indicate that defects in cftr and cftr∆f508 folding are sensed by two different e3 ubiquitin ligase complexes that are located in the er membrane and cytosol (3) . the membrane-associated derlin-1/rma1 e3 complex appears to recognize early folding defects of cftr that may involve assembly of msd2 into a complex with the amino-terminal regions of cftr. in contrast, the cytosolic hsc70/chip e3 complex appears to sense folding defects that occur after synthesis of the nbd2 domain. misfolded cftrs recognized at either checkpoint are retained and degraded via the proteasomal pathway. the presence of dual protein quality control (qc) checkpoints suggests a mechanism by which the folding status of cftrs membrane and cytosolic domains are sequentially monitored prior to its escape from the er. currently little is known about how these erqc complexes sense misfolded substrates. small-molecule chemical correctors have recently been identified which rescue the folding defect of cftr∆f508 in model cell culture systems. in the presence of these chemical correctors a subset of cftr∆f508 is able to localize to the cell surface and retain channel function. however, how the chemical correctors rescue cftr∆f508 folding is not clear. we have examined the extent to which the molecules correct folding defects that are recognized by the derlin-1/rma1 or hsc70/chip qc checkpoints. in addition, because it is unknown at what point of cftr biogenesis that the chemical correctors act to facilitate folding, we have determined how the chemical correctors affect the folding of different biogenic intermediates. our results indicate that the correctors act in a post-translational manner to influence assembly of cftr sub-domains. interactions with members of the qc pathway have also been used to monitor the folding and assembly process of cftr in the presence of chemical correctors. ultimately the results from these studies will enhance our knowledge of how chemical correctors permit passage of the cftr mutants past different erqc checkpoints. supported by -cystic fibrosis foundation combination therapy has proven successful in treating a wide variety of human diseases, including cancer, infectious disease, and diabetes. historically, combination treatments have been discovered through trial and error using drugs with proven disease modifying effects. we have developed a combination high-throughput screening (chts™) technology to systematically and efficiently find synergistic combinations that target multiple pathways underlying disease biology, and that may be developed into new therapeutics. we are applying chts technology to discover and develop combination therapeutics for the treatment of cystic fibrosis. we have optimized two high throughput screening assays for identifying correctors and potentiators of the cftr (cystic fibrosis transmembrane protein regulator) ∆f508 mutation. one assay uses the flipr membrane potential dye (fmp) and the other uses genetically modified yellow fluorescent protein yfp h148q/i152l to measure changes in halide flux through ∆f508 cftr. both assays were optimized and validated on the flipr tetra using the panel of cf correctors, potentiators and inhibitors provided by the cf foundation. using our automated chts, we systematically evaluate pairwise combinations of a library of ~2,000 approved drugs and other bioactive molecules to identify novel disease-relevant biological interactions. candidate single agents and combinations are tested in secondary assays and prioritized for testing in animal efficacy models. combination therapies can act on multiple pathways in a coordinated way. chts may prove to be a useful tool for identifying combination therapeutic candidates with novel multi-target mechanisms and a way to enhance co-therapy regimens for the treatment of cystic fibrosis. supported the highly variable clinical phenotypes of cf airway disease suggest that a number of genetic factors, other than cftr, play a role in its pathophysiology. some of these modifier genes are expected to play a role in the endoplasmic reticulum quality control (erqc), since the major defect caused by f508del is misfolding, retention and degradation of the mutant protein by this cellular surveillance system. the nematode caenorhabditis elegans is an excellent multicellular genetic model, which has been successfully used in studies of human diseases. its ~20,000-genes genome has been fully sequenced, it has short life-span (2-3 weeks) and life cycle (~ 3.5 days) and it is easily cultured and amenable for gene disruption, both by knockout or rnai. our goal is to generate a c. elegans model for the cftr folding defect, so as to take advantage of this model for the identification of genes involved in cftr folding and/or degradation. to this end, and because no cftr orthologue has been described in c. elegans, we constructed two previously described human p-gp-/cftr chimeras (p-gp/wt-cftr and p-gp/f508del-cftr) [1, 2] to be used as an in vivo folding substrate in this organism. the two chimeric cdnas and intact human p-gp cdna were cloned into the c. elegans ubiquitous expression vector ps235 and injected into mutant nematode strains for multidrug resistance genes [3] . the effect on the nematodes phenotype was evaluated by an assay of heavy metal sensitivity (2.0 mm arsenite), as described [3] . our quantitative results show that the p-gp/wt-cftr chimera increases the resistance to arsenite when injected into the pgp-1/pgp-3 c. elegans double mutant, whereas p-gp/f508del-cftr causes no effect. these preliminary results indicate it is possible to generate two distinct nematode phenotypes caused by each of the transgenic chimeras (p-gp/wt-cftr and pgp/f508del-cftr). they also suggest that the same folding defect impairing f508del-cftr function in cf may be responsible for the loss of heavy metal resistance function by the p-gp/f508del-cftr chimera. these chimeras are currently under test in pgp-1/pgp-3 double and in pgp-1/pgp-3/mrp-1 triple c. elegans mutants. analysis of these strains by rt-pcr showed that the mrnas from the two p-gp/cftr chimeras have low expression levels. this led us to produce more robust constructs, including: 1) to test chimera expression under the c. elegans endogenous pgp-1 or intestinal-specific (potent) promoters; and 2) to develop p-gp chimeras with a full cftr-nbd1. these c. elegans models will be used in genome-wide rnai screens to identify genes involved in the erqc of the p-gp/cftr chimeras. work channel gating of cftr is regulated by atp binding and hydrolysis at the nbds and by pka phosphorylation at multiple sites, primarily within the intrinsically disordered regulatory (r) region. phosphorylation at multiple sites is required for full activation of channel function, but no one specific phosphorylation site is required. the phospho-regulation of r region interactions with nbd1 likely contributes to the regulation of cftr channel function by modulating r region interactions with nbd1 at the nbd1/nbd2 dimerization interface, consistent with nbd1 crystal structures that include the regulatory extension (re) comprising the first 20 residues of the r region (1). this regulation likely occurs via a dynamic complex where multiple segments of nonphosphorylated r region with αhelical propensity transiently engage nbd1 and are released. pka phosphorylation disrupts r region α-helical propensity and decreases binding of each interacting segment (2) . regulation of the ∆f508 cftr by pka is altered, with the rate of channel activation by pka 7-fold slower than for wild-type (3). we hypothesize that the dynamic interactions between the r region and nbd1 are affected by the ∆f508 mutation. to characterize the differences in r region binding between wild-type and ∆f508 nbd1 at residue-level resolution, we use nuclear magnetic resonance (nmr) techniques. experiments are performed using either r region or nbd1 samples labeled with nmr-active nuclei and monitoring their structural changes upon the addition of unlabeled binding partner, allowing us to confirm r region binding to nbd1 at the nbd1/nbd2 dimerization interface and to identify other potential interaction surfaces on both the r region and nbd1. together, these experiments will allow us to characterize the dynamic associations between the r region and nbd1 in order to better understand the molecular basis for the pathogenesis associated with ∆f508 cftr. funded by the canadian institutes for health research, the canadian cystic fibrosis foundation, and the us cystic fibrosis foundation 1. lewis, h.a., et al, embo j. 23, 1-12 (2004) . 2. baker, j.m.r., hudson, r.p., kanelis, v., choy, w-y, thibodeau, p.h., thomas, p.j., and forman-kay, j.d. submitted. the ∆f508-cftr mutation, the most common gene mutation in cystic fibrosis (cf), results in diminished plasma membrane expression of cftr, leading to loss of functional cftr and altered mucociliary clearance. this impairment promotes chronic infection of cf patients by the opportunistic pathogen, pseudomonas aeruginosa. we previously reported that a secreted protein from p. aeruginosa (cftr inhibitory factor (cif)) reduces the wt-cftr and ∆f508-cftr-mediated chloride secretion and the plasma membrane expression of cftr by decreasing endocytic recycling of cftr. the aim of the current study was to investigate the mechanism by which cif is secreted by p. aeruginosa and delivered to human airway epithelial cells (cfbe41o-). in this study, we show that cif is packaged in outer membrane vesicles (omv) for secretion from p. aeruginosa. interestingly, cif secretion via omv was increased when the bacteria were exposed to airway epithelial cells. changes in cif protein level or bacterial growth did not account for the increase in cif secretion. purification and application of p. aeruginosa omv to airway epithelial cells caused a decrease in plasma membrane expression of cftr. the decreased plasma membrane cftr expression was followed by increased ubiquitination and lysosomal degradation of cftr. cif delivery to the airway epithelial cells via omv showed a more robust decrease in plasma membrane cftr expression, in comparison with cif delivery as purified protein. to investigate the mechanisms whereby cif enters epithelial cells, optiprep gradient fractionation experiments were conducted in polarized airway epithelial cells. in airway epithelial cells treated with p. aeruginosa omv, cif was associated with airway cell lipid rafts. pharmacological inhibition of lipid raft formation with filipin iii blocked the decrease in cftr plasma membrane expression upon treatment with omv. these studies demonstrate that cif is released by p. aeruginosa via omv and enters the airway epithelial cells through lipid rafts. upon entry into the epithelial cell, cif increases the ubiquitination and degradation of cftr and redirects cftr trafficking from the recycling pathway to the lysosomal-degradative pathway. these data suggest that chronic infection of p. aeruginosa in the cf lung may reduce the efficacy of therapeutics developed to increase plasma membrane expression of the mutant ∆f508-cftr (supported by the nih 5 ro1hl074175-06 and 5 t32-dk007301-29). 1 1. physiology, dartmouth medical school, hanover, nh, usa; 2. microbiology and immunology, dartmouth medical school, hanover, nh, usa we have identified and cloned a secreted protein from pseudomonas aeruginosa (p. aeruginosa pa14 and clinical isolates), designated cftr inhibitory factor (cif). cif reduces the apical membrane expression of cftr and inhibits the cftr-mediated chloride ion secretion by human airway epithelial cells expressing wt-cftr and df508-cftr. cif does not have general effects on protein trafficking, as the localization and expression of gp114, na + -k + -atpase, and the transferrin receptor were not affected. cftr is a member of the atp-binding cassette (abc) transporter superfamily. abc proteins are atp-dependent transporters involved in exporting a wide variety of cytotoxic agents across the plasma membrane. p-glycoprotein (pgp; abcb1, mdr1 gene product), also an abc transporter, is one of the major drug-efflux pumps expressed in normal tissues, as well as in many human cancers. over-expression of pgp results in reduced intracellular drug concentration and reduced cytotoxicity, conferring multi-drug resistance (mdr) to cancer cells. the aim of the current study was to examine whether cif also affects pgp expression in the plasma membrane, which could be exploited as a potential therapeutic strategy for restoring the sensitivity to pgp-transported drugs in cancer chemotherapy. cif significantly reduced the apical expression of pgp in mdck cells stably transfected with gfp-tagged pgp (mdck-gfp-mdr1 cells) and in caco-2 and calu-3 cells, which express endogenous pgp in the apical plasma membrane. the drug sensitivity of mdck-gfp-mdr1 cells to doxorubicin, a pgp substrate, was evaluated in the absence and presence of cif by measuring the ec 50 . cif reduced the cytotoxicity of doxorubicin by 100-fold. neither cif nor vehicle alone had a cytotoxic effect. the drug sensitivity of the parental cell line, mdck-c7, that expresses 4-fold less pgp than the transfected mdck cells, to doxorubicin was also increased by 20-fold. by contrast, cif had no effect on the ec 50 values of cisplatin (a substrate of mrp2) and etoposide (a substrate of mrp1), suggesting that the alteration of the sensitivity to doxorubicin was due to decreased apical expression of pgp, but not other abc transporters. these results suggest that although cif may be an obstacle to therapeutic attempts to restore the apical expression of cftr in the cf lung, it could be useful for the development of a novel class of inhibitors of pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs (supported by the nih (5 ro1 hl074175)). hamai, h. 1 ; keyserman, f. 1 ; worgall, t.s. 1, 2 1. pathology, columbia university, new york, ny, usa; 2. pediatrics, columbia university, new york, ny, usa cystic fibrosis (cf) is characterized by an inflammatory state and susceptibility to chronic lung infections. a common clinical finding is dyslipidemia characterized by altered plasma free fatty acid patterns, low plasma hdl levels and increased phospholipase a2 (pla2) activity. it is not clear how cftr mutations relate to lipid abnormalities. it was shown that defective cftr is associated with decreased uptake of sphingolipids (sl). sl homeostasis is tightly regulated and metabolites are relevant to lipid metabolism and cf. sl synthesis (sls) correlates with activity of srebp, a key transcription factor of lipid metabolism; ceramide-1-phosphate, a sl metabolite, is a regulator of pla2 that is highly activated in cf. we investigated the hypothesis that sls is increased in cf. experiments were carried out in airway epithelial cell lines that express defective cftr (c38 / ib3), no cftr (16hbe sense / antisense) and overexpress cftr (a549 cells infected with adcftr or adcontrol). sls was assessed using radioactive tracers 3h-serine (de-novo sls) and 3h-sphinganine (recycling pathways). ceramide and sphingomyelin mass were determined enzymatically. neutral and alkaline sphingomyelinase activity was assessed using 3h-sphingomyelin as substrate. srebp mediated gene transcription was assessed using sre-promoter assays. cholesterol synthesis was evaluated by incorporation of 3h-acetic and 3h-mevalonic acid. cholesterol mass was determined by gas-chromatography. abca-1 expression was determined by western blot analysis. pla2 activity was measured using fluorescent phospholipid substrates. sls was post-transcriptionally increased in cells expressing defective or no cftr through the de-novo pathway (133 % ± 15% for ib3; 78%± 12% for 16hbe-antisense; p<0.05 ) and the recycling pathways (38% ± 11% or ib3; 27% ± 12 % for 16hbe-antisense; p<0.05). ceramide mass was increased by 50 % (± 18 %) in 16hbe antisense, 36 % ± 9% in ib3 cells. sphingomyelin mass was increased 2-fold in ib3, up to 3-fold in 16hbe antisense cells. neutral and alkaline sphingomyelinase activity was increased up to 3-fold in both cell lines. sre-mediated gene expression was increased up to 2-fold in ib3 and by 50 % in 16hbe-antisense cells. overexpression of cftr in a549 decreased sls and srebp activity up to 40%. free cholesterol synthesis was increased by 60 % in ib3 and by 30 % in 16hbe antisense. expression of the cholesterol efflux receptor abca-1 was decreased in ib3 and 16hbe antisense compared to controls. sl mass, sre-mediated gene transcription and pla2 activity were decreased following incubation with inhibitors of sls in ib3 and 16hbe antisense cells. conclusion: defective cftr or lack of cftr expression correlates with 1) increased sls synthesis and sl mass; 2) increased sremediated gene transcription and free cholesterol synthesis; 3) decreased expression of the abca-1 cholesterol efflux receptor. inhibition of sls decreases sre-mediated gene transcription and pla2 activity. dysfunctional sls is a newly recognized pathway associated with defective cftr and a possible therapeutic target in cf. the ability to make accurate, reproducible measures of mucociliary and cough clearance (mcc/cc) in cf patients is critical to assessment of new therapies designed to improve mcc/cc function, and thus decrease pulmonary infections and decline in lung function. for example, using mcc/cc methods developed in our laboratory, we showed that 2-week treatment with aerosolized hypertonic (7%) saline (hs) led to a sustained increase in mcc and improved lung function in cf patients (donaldson et al, n eng j med 2006; 354:241) . the mcc/cc technique involves patient inhalation of radiolabeled particles followed by gamma camera scanning to determine the rate of particle movement from the lungs. while a few cf centers have shown the capability to measure mcc/cc, the techniques across these centers are not standardized, making comparison of results difficult. it is vital to develop standard, simplified techniques for these measures to make larger cf patient populations available for such studies. to standardize a radiolabeled particle inhalation technique that can be easily used by other cf centers, we have compared three protocols in healthy and cf subjects. protocol #1 was identical to that used in recent studies, and incorporates a devilbiss 646 jet nebulizer to generate 5 µm mmad (gsd = 2.0), aqueous droplets containing suspended tc99m-sulfur colloid particles. tidal volume and inhaled/exhaled flow rates were guided by a volume signal displayed on an oscilloscope. protocol #2 was further standardized and simplified to allow use at other centers. this protocol utilized the same nebulizer, but triggered by a commercial dosimeter (spira electro 2, finland) during inhalation as the subject breathed in time to a metronome (30 breaths/min) at flow rate of 0.5l/sec. subjects controlled inspiratory flow rate by feedback from a digital flow readout on the dosimeter. repeat measurements of mcc/cc were made in 8 healthy subjects and 5 mild cf patients to compare mcc/cc variables following radioaerosol inhalation using protocols #1 and #2. there were no differences in particle deposition (i.e. central to peripheral ratio, or "c/p"), or clearance of particles through 90 minutes and 24 hours in either subject group. these data suggest that the new methodology (protocol #2) will be useful and easily employed by other cf centers to test effectiveness of new therapies for cf. the third protocol (#3) was then tested in five of the healthy subjects. this protocol used a nebulizer that generated very large droplets (9.5 um mmad, gsd =2.0) and subjects inhaled at very slow flow rates (~80ml/sec). it was designed to provide greater bronchial airway deposition and thus an "improved signal" for mcc/cc evaluation. indeed, this methodology produced a larger average c/p and significantly greater particle clearance from the lung compared to the other standardized methodology (protocol #2) (c/p = 1.77 vs. 1.60; 90min clearance = 25% vs. 18%; 24hr clearance = 54% vs. 35%, respectively). interestingly, the greater fraction of clearance between 90min and 24hr may also reflect heightened ability to assess clearance from smaller airways, which could be particularly important in cf. protocol #3 will be tested further in cf patients. supported by cf foundation. previous clinical trials in cystic fibrosis (cf) indicate that anti-inflammatory therapy probably will not result in improvement in lung function, but will slow the rate of decline. this imposes constraints on study design for new anti-inflammatory agents, requiring that they use many patients over long periods. it is highly desirable to design a strategy for evaluation of antiinflammatory agents that will allow for the selection of only the most promising agents for phase iii trials. sputum induction (si) samples lower respiratory tract secretions and permits measurement of inflammatory markers. the purpose of this study was to assess the measurement of inflammatory markers in induced sputum as one such strategy by using ibuprofen as the test agent because it has demonstrated clinical benefit in cf. if changes in inflammatory markers are detectable in ibuprofen treated patients, then si might serve as a quick, noninvasive method by which to select the most promising anti-inflammatory agents for further study. methods: in this twoarm (ibuprofen and no treatment), open-label, parallel group study, 114 cf patients >/= 10 years of age with mild to moderate lung disease were screened at 16 sites. si was performed on days 0, 14, 42 and 56. patients in the ibuprofen group received drug on days 14-42. 82 subjects met eligibility criteria. 40 were randomized to ibuprofen and 40 to no treatment. 63 subjects (32 ibuprofen and 31 no treatment) completed the study through day 42 and comprise the intent-to-treat efficacy population. a maximum of 60 subjects (32 ibuprofen and 28 no treatment) and a minimum of 47 subjects (23 ibuprofen and 24 no treatment) across all markers had acceptable inflammatory marker values at both days 14 and 42. within group and between group comparisons were made. results: with respect to the within group comparisons (before and after ibuprofen), most inflammatory markers slightly decreased in the ibuprofen group and increased in the no treatment group. for ibuprofen patients, mean changes were most apparent for il-6 (-0.13 log pg/ml, p=0.04) and % neutrophils (pmns) (-4%, p=0.073). with respect to the between group comparisons (ibuprofen vs. no treatment), differences were strongest for il-6 (0.03 log pg/ml, p=0.0237), percent pmns (7%, p=0.085), and absolute pmn count (0.14 cells/ml, p=0.166). fourteen days after discontinuing ibuprofen, inflammatory mediators tended to increase. there was no difference in aes between the two groups, and si was generally safe. conclusions: 1. overall, si is well-tolerated in patients >/= 10 years, 2. a 4-week treatment period may not be long enough to study anti-inflammatory drugs, 3. with respect to changes in inflammatory markers associated with anti-inflammatory therapy, the effect size is small and the variance of the effect is small in clinically stable patients. 4. measuring inflammatory markers in induced sputum has potential as a screening tool for studying anti-inflammatory drugs in cf, 5. further studies of si in cf are warranted. supported by cystic fibrosis foundation therapeutics, inc. in preparation for our gene therapy clinical trial programme we are currently assessing a number of sputum biomarkers including viscosity and elasticity, total solids and dna content and 24 hr sputum weight. we tracked and correlated these biomarkers in cf patients (12 years and over) during a course of iv antibiotics (ab) by collecting samples on several occasions [visit (v)1: at the start of ab treatment, v2: at the end of ab treatment (generally after 2 weeks) and interim periods]. to ensure adequate reproducibility of the results viscosity/elasticity measurements were carried out in triplicate using a csl 100 rheometer which required a comparatively large volume (5 mls) of spontaneously expectorated sputum. because of this requirement paired samples could only be obtained from approximately 50% of the patients. there was no change in viscosity/elasticity (n=13), solid or dna (n=15) content when comparing samples at the beginning and end of iv ab. in contrast, 24 hr sputum weight was significantly (p<0.05) lower at the end of the ab treatment (visit 1: 57.74±14.58 g, visit 2: 49.41±24.01 g, n=16). there was a strong and significant correlation between solid content and viscosity/elasticity (r=0.8, p<0.0001) and a modest correlation between 24 hr sputum weight and % predicted fev1 (r=-0.42, p<0.05) and 24 hr sputum weight and patient scored symptom severity (r=0.48, p<0.001). surprisingly, sputum dna content did not correlate with viscosity/elasticity, despite being generally thought of as a contributor to viscosity, which may in part be related to the assay not being able to discriminate between free and cell-enclosed genomic dna.in summary, after a course of iv antibiotics which lead to significant subjective and objective (fev1) improvement the overall quantity of expectorated sputum was significantly reduced but, based on analysis available to date, none of the other parameters (viscosity, elasticity, solid and dna content) changed significantly. considering the difficulties we encountered in collecting sufficient sputum during this period of an exacerbation, sputum viscosity/elasticity measurements may not be feasible parameters to measure in a gene therapy trial to which stable patients re likely to be recruited. this study was funded by the cf trust. chronic neutrophilic inflammation is a feature of cystic fibrotic (cf)related inflammation and serves as a significant contributor to morbidity and mortality associated with the condition. recently, our group has described a novel collagen-derived neutrophil chemoattractant, proline-glycine-proline (pgp), in a murine model of lipopolysaccharide-induced inflammation. the purpose of this study was to explore both the presence of this peptide in sputum from cf patients and the determination of a proteolytic system involved in pgp generation. we show that lower airway secretions from cf subjects have 30-fold increase in pgp levels compared to normal controls and that cf sputum (p<0.05) is able to generate pgp from intact type i and ii collagen 100-fold above that seen from normal control sputum ex vivo (p<0.01). we further demonstrate that production of pgp is dependent on two specific matrix metalloproteases (mmps), mmp-8 and mmp-9. finally, we show that the generation of pgp can be significantly inhibited (80-90% inhibition) by the use of either mmp-8 or mmp-9 specific inhibitors (p<0.01 versus no inhibitor); a nonspecific mmp inhibitor (doxycycline) also demonstrates significant attenuation of pgp production. the determination of the requirements of these proteases in pgp generation allows for the identification of logical targets for disease-modifying therapeutics in cf. 1 1. cfrc, uab, birmingham, al, usa; 2. cfrc, university of north carolina, chapel hill, nc, usa cystic fibrosis (cf) lung disease is characterized by chronic neutrophilic inflammation. the discrete collagen breakdown product prolineglycine-proline (pgp) is a potent neutrophil chemoattractant thought to be generated by a specific proteolytic cascade present during cf pulmonary exacerbation (nat med 12(3):317, 2006) . high-mobility group box 1 (hmgb1) is a potent inflammatory mediator found in sepsis, rheumatoid arthritis, and other inflammatory diseases characterized by neutrophilic inflammation. the role of pgp and hmgb1 as pmn attractants in cf is unknown. in this study we utilized specimens derived both from humans and scnn1b-transgenic mice (βenac mice) in which overexpression of the βenac subunit in the airways leads to sodium hyperabsorption and airway surface liquid depletion, mimicking the cf muco-obstructive phenotype. pgp was quantified using tandem ms. neutrophil chemotaxis was measured in vitro following preincuabtion of sputum or balf with anti-hmgb1 neutralizing antibody. murine balf was assayed for hmgb1 by western blot and elisa. human sputum was similarly evaluated after normalizing for total protein concentration. in vivo hmgb1 activity was measured after intratracheal instillation in wt mice. βenac mice had 60% higher cell counts and a greater percentage of pmns (37.3% vs. 0.6%) in bal samples than wild-type (wt) littermates (p<0.005, n=15/genotype). pgp was elevated in balf of scnn1b animals (48.0 vs. below limits of detection in wt controls, p<0.001, n=11), increased in serum from cf subjects (834 vs. 340 pg/ml normal controls, p<0.005, n=5), and detected at high levels in cf sputum (54,700 pg/ml). balf screened for hmgb1 by western blot revealed elevated levels in βenac mice (p<0.05 by densitometry, n=10/genotype); elisa confirmed 52% greater hmgb1 concentration (61.8 vs. 40.6 ng/ml, p<0.02). hmgb1 was also detected by western blot in human cf sputum (19 of 23) and at higher levels than secretions from normal subjects (p<0.05 by densitometry; n=23 cf, 4 control). purified hmgb1 induced dose-dependent pmn chemotaxis in vitro (peak 50-250 ng/ml, p<0.05, n=5) and cf sputum caused potent chemotaxis (7-fold over control, p<0.005, n=12) that was inhibited by preincubation with hmgb1 antibody (p<0.005). balf from βenac mice (but not wt controls) was also chemotactic (2-fold greater than media control, p<0.01, n=5) and inhibited by anti-hmgb1 antibody (p<0.05). exogenous hmgb1 (25 ng) administered intratracheally to lps resistant mice (c3hej, toll-4 receptor mutant) and balb/c mice caused pmn influx in balf at 24 hours (c3hej: 9.0 x 10 5 pmns after hmgb1 vs. 2.0 x 10 5 after vehicle, p<0.02; balb/c: 3.5 x 10 5 vs. 0.8 x 10 5 , p<0.01), and generation of pgp (189.7 vs. 80.7 pg/ml in c3hej, p<0.05, n=3) . in summary, pgp and hmgb1 are elevated in balf of βenac mice that present with cf-like lung pathology; in cf airway secretions, hmgb1 is present in vivo at concentrations that induce neutrophil chemotaxis in vitro and pgp production in vivo, and is inhibited by blocking antibody. the role of pgp and hmgb1 in cf deserve further evaluation as they may be potential therapeutic targets of dysregulated inflammation. esther, c.r. 1 ; jasin, h.m. 2 ; collins, l.p. 3 ; boysen, g. 3 ; boucher, r.c. 2 1. pediatric pulmonology, university of north carolina at chapel hill, chapel hill, nc, usa; 2. cf research center, univeristy of north carolina at chapel hill, chapel hill, nc, usa; 3. center for environmental health and susecptibility, univeristy of north carolina at chapel hill, chapel hill, nc, usa biomarkers of airway inflammation are needed in cystic fibrosis (cf) to aid clinical management and assess the efficacy of novel therapies. we describe a simple and non-invasive method to measure airway purines, signaling molecules previously shown to be biomarkers of neutrophilic airway inflammation. airway secretions were obtained using exhaled breath condensate (ebc) collection, a technique that was easily performed in the outpatient setting on children as young as three years. the purines adenosine and amp in ebc were measured using ultra-sensitive liquid chromatography/tandem mass spectrometry (lc/ms/ms). in addition, we used lc/ms/ms to simultaneously measure urea as a dilution marker to control for the known dilutional variability of airway secretions within ebc. detection of adenonosine, amp, and urea was optimized using positive mode ms with selected reaction monitoring, and stable isotope dilution was utilized to improve quantification. detection was linear with concentration over four orders of magnitude, with coefficients of variation <15%. the method was sensitive, with limits of detection for adenosine and amp (~0.1 nm) and urea (~0.5 µm), below expected ebc concentrations. applying the methods to ebc demonstrated that detection of purines and urea was reliable, with intraclass correlation coefficients greater than 0.9 between duplicate measures (n=7). we also measured purines and urea in ebc samples collected prospectively from children with cf (n=17) and healthy controls (n=6) during regular clinic visits. all samples were lyophilized and reconstituted to increase concentration 10-fold. adenosine, amp, and urea were detected in all samples. neither adenosine nor amp levels differed between groups, but urea was significantly lower in cf (6.6 ± 2.3 µm) versus healthy control (15.1 ± 4.9 µm, p=0.02), suggesting that airway secretions were more dilute in cf samples. when dilutional variability was controlled using purine to urea ratios, the amp to urea ratio was found to be elevated in subjects with cf (0.50 ± 0.14) compared to healthy controls (0.09 ± 0.03, p=0.015), consistent with previous findings. the amp to adenosine ratio was also elevated in cf (4.3 ± 1.9) compared to control (0.87 ± 0.30), although the difference did not reach statistical significance in this small sample set (p=0.19). experiments are ongoing to increase sample size and assess ebc purine levels before and after treatment of a cf exacerbation. these results demonstrate that lc/ms/ms analysis of ebc provides a non-invasive method to measure purine biomarkers of cf airway inflammation. given the flexibility of ms, this methodology could also prove applicable for study of other small molecule biomarkers. importantly, we also show that dilution of airway secretions in ebc may be altered in cf, and lc/ms/ms can be used to control for this variability through measurement of urea as a dilution marker. introduction: mucociliary clearance and antimicrobial peptide activity may be affected by airway surface liquid (asl) ph, which is regulated by epithelial ion transport. asl ph is abnormal in disease states in which il-17a is elevated, including cystic fibrosis (cf), and the il-17a receptor is expressed at the basolateral surface of bronchial epithelium. therefore, we investigated the effects of il-17a on vectorial ion transport in well-differentiated hbe cells. methods: hbe cells were grown with an apical air interface and incubated with il-17a at 0 or 50 ng/ml in the basolateral medium for 48 hours prior to being studied with standard short-circuit current (isc) techniques. results: il-17a treated cells had a minimal increase in resting and amiloride-sensitive isc compared to control cells, and had a dosedependent, statistically significant increase in forskolin-stimulated isc. at a dose of 50 ng/ml, il-17a doubled forskolin-stimulated isc (10.5 ± 1.72 µa/cm2 for control vs. 20.3 µa/cm2 for il-17a). the increased isc was not bumetanide-sensitive, but was sensitive to acetazolamide and dnds, and was not present in hco 3 --free solutions, suggesting it was due to hco 3 secretion. to investigate whether this hco 3 secretion was cl --dependent, we studied hbe cells in cl --free solutions, and there was no difference between untreated and il-17a treated hbe cells. to test the hypothesis that il-17a promoted cl -/hco 3 exchange, we performed experiments in hbe cells mounted in cl --free solutions, and after addition of amiloride and forskolin, 30 mm nacl was added to the mucosal bath and 30 mm nagluconate was added to the serosal bath. under these conditions, one would predict a decrease in isc due to mucosal to serosal clmovement down its electrochemical gradient. in untreated hbe cells, we saw a decrease in isc followed by a recovery. in il-17a treated cells there was a sustained increase in isc, suggesting that movement of cldown its electrochemical gradient resulted in exchange of clfor an anion at the apical membrane. in the absence of hco 3 -, both untreated and il-17a treated cells responded to claddition to the mucosal bath with decreases in isc. to assess the cftr dependence of the il-17a-induced isc, we stimulated primary cf hbe cells treated with il-17a. in cf cells, there was a minimal increase in amiloride-sensitive isc (30.6 ± 3.35 µa/cm2 for control vs. 37 ± 8.24 µa/cm2 for il-17a ) and in forskolin-stimulated isc (1.2 ± 0.56 µa/cm2 for control vs. 3.4 ± 1.16 µa/cm2 for il-17a ). conclusion: the proinflammatory cytokine il-17a induces cl -/hco 3 exchange in hbe cells. notably, the observed cl -/hco 3 exchange appears to be cftr-independent. however, cftr activation is required for maximum cl -/hco 3 exchange as demonstrated by the smaller isc generated in cf cells. these data suggest that the cytokine milieu of the airway epithelium can alter ion transport and potentially asl physiology. furthermore, they suggest that cftr may interact with or regulate novel proteins in the presence of inflammation. supported by cff and nih periciliary fluid balance is maintained by the coordination of sodium and chloride (cl-) channels in the apical membranes of the respiratory epithelia. in the absence of the cystic fibrosis transmembrane conductance regulator (cftr), cl-secretion is diminished and sodium reabsorption becomes exaggerated. activation of non-cftr-dependent cl-channels can provide an alternate pathway for cl-secretion in the airways and gastrointestinal tract. the ph and voltage-dependent type-2 cl-channel (clc-2) is expressed in airway epithelial cell luminal membranes. we hypothesize that topically applied clc-2 agonists may restore cl-secretion in cystic fibrosis (cf) murine airways. using in vivo nasal potential difference measurements, we quantified clc-2-mediated cl-transport in both cf and wild-type mice during nasal perfusion with lubiprostone (a prostone compound and specific clc-2 agonist; sucampo, pharmaceuticals, inc., bethesda md) or vehicle control. wild-type (c57bl6, n = 9) and cf knock-out (cfko; n=10(jax stock# 002364)(jackson laboratories, bar harbor, me) mice were sedated and intubated to protect the lower airways from aspiration of perfusate. nasal and subcutaneous bridges were connected to fluid-filled silver chloride electrodes. baseline (in ringer's), amiloride-inhibited, low cl-(cl-free, gluconate-substituted ringer's with amiloride), and low cl-plus lubiprostone (with increasing concentrations of lubiprostone or vehicle) mice were perfused and potential differences were measured with a high impedance voltmeter (world precision instruments, sarasota fl) and continuously recorded. a clear dose-response relationship was detected in both wild-type and cf mice. at 20 µm lubiprostone, wild-type mice showed hyperpolarization of -7.8 ± 3.4 mv and cf mice responded with -4.7 ± 1.5 mv hyperpolarization. a paired t-test of low cl-perfusion and lubiprostone perfusions revealed significant (p<0.001) differences in both genotypes. five clc-2 cfko mice were similarly tested and showed no response to lubiprostone (+1.1 ± 1/5 mv). cftr inhibitor-172(50 µmol) (calbiochem, san diego ca) added to the low cl-perfusion in 6 additional wild-type mice eliminated the low cl-response but did not abolish the lubiprostone response, confirming that clc-2 is present and independent of cftr regulation. we conclude that direct application of a clc-2 agonist in the cf murine upper airways restores near normal levels of cl-secretion. cftr is an apical membrane chloride channel whose activity is required to maintain airway surface liquid (asl) volume and efficient mucociliary clearance. cftr is activated by camp dependent stimulation of protein kinase a. however, studies have suggested that the actin cytoskeleton may also be required for regulation of cftr, by acting as a structural element in second messenger compartmentalization (1) . to determine whether an intact cytoskeleton is required for asl volume homeostasis, adenosine (ado), atp, or isoproterenol (iso) (all at 200 µm) were added apically to human bronchial epithelial cultures (hbec's) before or after the cytoskeleton had been disrupted by cytochalasin d exposure (5µm for 1h). in control cultures ado, atp and iso all resulted in a significant increase in asl height (∆asl) within 10 min (∆asl; ado, 6.2±1.2µm; atp, 7.2±2.3µm; iso, 8.3±1.3µm). however, in cytochalasin d exposed cultures ado was without effect while atp and iso were still able to evoke an increase in height (∆asl; ado, -0.7±1.2µm; atp, 7.4±3.1µm; iso, 10.2±3.1µm). however, we showed that a2b receptor function was not compromised by cytoskeleton disruption as camp levels were seen to increase both after addition of adenosine and adenosine in the presence of cytochalasin d. thus, the effect of cytochalasin d on asl height appeared specific for adenosine-mediated cl-secretion. to further test the effect of cytochalasin d on the relationship between adenosine and cftr, we designed a fluorescent resonance energy transfer (fret) pair of cfp-cftr (labeled at the n-terminus) and yfp-a2b receptor (labeled at the c-terminus). addition of adenosine (100µm) increased fret by 2.3 times (% efficiency; control, 15.2±2.3%, ado, 35.3±3.5%) compared to the vehicle control (n=13). cytochalasin d exposure (5 µm for 1 h) completely inhibited the increase in fret that was observed after adenosine addition (p<0.01). in contrast, cytochalasin d had no effect on fret between cftr and the β2adrenoceptor. to determine whether cytochalasin d treatment (5 µm for 1 h) had any direct effect on cftr protein levels and localization we used a cell line stably expressed with an exotope-tagged cftr (hb8 cells) (2) . cftr protein levels were found to be significantly reduced as compared to nontreated controls by both western blot analysis and chemiluminescence detection (n=3). cells were also stained with an antibody against the external cftr exotope and imaged by confocal microscopy and a significant decrease in staining at the plasma membrane was observed after cytochalasin d treatment (n=3). we conclude that cytochalasin d exposure decreases cftr protein levels and specifically inhibits adenosine-mediated regulation of cftr activity in the airways, leading to disruption of asl homeostasis and inhibition of mucociliary clearance. numerous studies have reported evidence that cftr is expressed in the apical membrane of serous cells in the airway submucosal glands. thus, it has been reasoned that (1) cftr plays some role in normal anion and fluid secretion by airway glands and (2) loss of this channel's function in cystic fibrosis (cf) airways plays some role in the etiology of cf lung disease. a recent study by joo et al. (jbc 277:50710-50715, 2002) indicates that cf airway glands lose the ability to secrete fluid when treated with forskolin or vip but maintain the ability to secrete when treated with muscarinic agonists. this result suggests that vip and muscarinic agonists mediate secretion by cftr-dependent and cftr-independent pathways, respectively. the present study was undertaken to explore the dynamics of fluid secretion by individual submucosal glands to determine if further differences in their responses to these two agonists could be distinguished. pig tracheas were obtained from a local slaughterhouse. after removal of the cartilage, tissues were mounted in a warm (37°c) observation chamber that permitted exposure of the submucosa to krebs solution. the mucosal surfaces of the airways were dried with a stream of warm dry air and then layered with mineral oil. liquid secreted from the gland duct openings formed spherical aqueous droplets within the oil layer, and their volumes were estimated from spatial dimensions taken from sequential digital images. liquid secretion rates were estimated for individual glands present within a 28.9 mm 2 region of interest. rates were determined for two experimental protocols. for one group of tissues, secretion rates were determined for a control period, followed by a period of exposure to 1 µm vip, and then a period of exposure to both vip and 10 µm acetylcholine (ach). in the second group, the order of vip and ach treatment was reversed. these agonist concentrations were expected to produce near maximum rates of secretion. when the agonists were applied first, secretion rates were significantly (p<0.05) increased from the control period by both ach (2.01±0.44 nl/min to 4.52±0.38 nl/min) and vip (2.26±0.63 nl/min to 4.64±0.78 nl/min). the secretion rates for individual glands were highly variable with both agonists (ach: 0.11-19.20 nl/min; vip: 0.03-32.21 nl/min). when ach application followed vip treatment, the mean secretion rate was significantly increased further to 6.13±0.87 nl/min; however, when vip application followed ach treatment, the secretion rate was only marginally and insignificantly increased to 4.97±0.48 nl/min. we conclude that both ach and vip are efficacious secretagogues for porcine airway glands and that the rate of secretion from individual glands to either agonist is very heterogenous. because vip has no effect following stimulation by ach, we speculate that ach induces secretion by activating all transporters relevant to vip-induced secretion including cftr. however, ach must also activate channels and/or transporters not induced by vip since its effects are additive to vip. this most likely includes activation of a non-cftr anion channel. ( serous cells are thought to serve as the principal anion and fluidsecreting cells of airway submucosal glands. mucous cells primarily secrete gel-forming mucins. these respective physiological roles for these two cell types imply that the mucous cells, which secrete the thick mucus gel, should lie downstream of the serous cells so that their watery secretions can flush the mucins out of the ducts and onto the airway surface. early studies indicated that such arrangements existed within glands but reports of cell distributions have been descriptive rather than quantitative. because of resurgent interest in serous cell function, we revisited this issue to formally document the distribution of these cell types within the secretory tubules of the submucosal glands as well as at the surfaces of the glands, where serous cells are most accessible for study. a piece of porcine lung, containing a 5mm diameter bronchus, was treated with formalin fixative, and 250 consecutive 5µm sections were taken. the apices of virtually all gland epithelial cells, except the cells lining the collecting and ciliated ducts, stain positive for pas indicating that these cells are either serous or mucous cells. however, pas staining does not allow distinctions to be made between these two cell types. consequently, all slide sections were stained with hematoxylin and eosin. with these stains, mucous cells were identified as cells containing lucent granules in their apices whereas serous cells were considered to be cells where the apical cytoplasm stained a uniform dark reddish color. first, the distribution of serous and mucous cells in the secretory tubules were determined. the acini of individual tubules were located in the slide sections, and cells were identified and counted in consecutive sections that approached the collecting ducts until the tubules were no longer distinguishable as discrete tubes. secretory tubules from 6 individual glands were studied. in the acini, serous cells accounted for 95.0±3.2% of total cells whereas mucous cells accounted for 3.8±2.2% of total cells (remaining cells were not distinguishable as either cell type). there was a significant (p<.05) negative correlation of serous cell numbers with distance from the acini so that at 35µm from the acinus serous cells accounted for only 73.4±13.5% of total cells. there was a coincident significant positive correlation with mucous cell numbers so that at 35µm from the acini mucous cells accounted for 20.1±10.5% of total cells. next, we examined the cell distribution at the adventitial and mucosal surfaces and at the lateral margins of the glands. serous cells accounted for 82.7%, 85.4%, and 96.9% of the total cells at these respective locales. mucous cells represented 15.7%, 10.1%, and 2.4% of total cells in these same respective regions. we conclude that serous cells are by far the dominant cell type in secretory tubules of porcine submucosal glands. mucous cells are rare in acini but increase in frequency with distance from the acini. the outer surfaces of the glands are dominated by serous cells as well. at the lateral margin of the glands, serous cells outnumber mucous cells by 20-to-1. (supported by nih hl63302) . many of the membrane transporters that participate in this process have been identified; however, the identity of the class of kchannels that maintains resting membrane potential and/or cell membrane polarization during the secretion process remains poorly characterized. liquid secretion by porcine submucosal glands is insensitive to numerous k + channel blockers including ba 2+ , tetraethylammonium, apamin, charybdotoxin, iberiotoxin, clotrimazole, penitrem a, 4-aminopyridine, and quinidine. recently, a new class of k + channels, the "tandem-pore" or k 2p channels, have been described. potassium channels of this class are insensitive to many traditional k + channel inhibitors but are blocked by local anesthetics, such as lidocaine and bupivacaine. additionally, many k 2p channels are sensitive to changes in extracellular ph. in the present study, we considered whether k 2p channels might participate in the liquid secretion response to acetylcholine (ach). intrapulmonary bronchi, excised intact from the lungs of pigs, were cleared of accessible luminal liquid, cannulated, and treated with 10 µm ach to induce secretion. paired airways were pretreated for 1 h with 3 mm bupivacaine. bupivacaine blocked 90.4±1.8% of the liquid secretion response to ach. the ic 50 for the bupivacaine inhibition was approximately 220 µm. the inhibitory effect of bupivacaine was not due to nonspecific toxicity since tissues exposed to bupivacaine for 1 h and then washed with fresh inhibitor-free buffer recovered to 80% the control achinduced secretory rate. the bupivacaine effect was not due to inhibition of voltage-gated na + channels in intrinsic neurons since 1 µm tetrodotoxin did not inhibit ach-induced secretion. no significant effect was seen in the secretion rate when the extracellular solution ph was lowered from 7.43±0.01 (normal hco 3 --buffered krebs gassed with 5% co 2 ) to 6.60±0.01 by gassing with 40% co 2 gas (balance o 2 ). while we recognize the relative nonspecificity of this agent, the inhibitory effects of bupivacaine on liquid secretion by porcine bronchi are consistent with k 2p channel participation in this secretory response. the failure to demonstrate inhibition with acidification of extracellular solution could signify a k 2p channel subtype that is insensitive to low extracellular ph. ( 4 1. novartis, horsham, united kingdom; 2. rosalind franklin university, chicago, il, usa; 3. mount sinai medical center, miami, fl, usa; 4 . genomics institute of the novartis foundation, la jolla, ca, usa enac activity in the human airway epithelium has been reported to be partially-sensitive to the broad spectrum trypsin-like protease inhibitors aprotinin and placental bikunin (bridges et al., 2001 am j physiol 281:l16-23; donaldson et al., 2002 jbc 277:8338-45) . a low molecular weight inhibitor of the airway channel activating protease (cap) would represent a potential therapeutic approach to attenuating enac function in cystic fibrosis (cf) lung disease. the aims of the current study were to: (1) further characterise the in vitro efficacy of cap inhibitors in human bronchial epithelial cells (hbes), (2) to evaluate the relevance of a cap mechanism to the regulation of enac function in vivo, and (3) to establish whether the inhibition of the airway cap can modulate mucociliary clearance in vivo. primary cultures of hbes (normal and cf), cultured under air-liquid interface conditions, demonstrated an amiloride-sensitive short circuit current (isc), that was sensitive to aprotinin but that was insensitive to inhibition by sbti, α1-anti-trypsin and α1-pdx. the amiloride sensitive isc was also attenuated by the low molecular weight cap inhibitor nvp-qau145 (hbc276) with an ic50 value of approximately 30nm. nvp-qau145 caused a time dependent inhibition of the isc with a t 1 ⁄ 2 of approximately 20 min and this effect could be reversed by the addition of excess trypsin. in vivo, aprotinin attenuated the guinea pig tracheal potential difference (tpd) from -10.0±0.8mv to -4.7±0.6mv (n=4-11) with an ed50 value of 40pmoles/kg, measured at 2 hours following intra-tracheal instillation. the tpd was unaffected by either sbti or α1-anti-trypsin at doses up to 1500pmoles/kg. the combination dosing of an enac blocker with aprotinin did not attenuate the tpd beyond the effect observed with either agent alone, consistent with aprotinin attenuating enac function in this model system. intra-tracheal instillation of nvp-qau145 attenuated the tpd from -10.3±1.0mv to -4.7mv with an ed50 value of 3µg/kg (n=6-8). in the sheep, the administration of nvp-qau145 into the airways by aerosolisation of a 1mg/ml solution resulted in a 3-4 fold enhancement of the rate of clearance of 99mtc sulfur colloid from the lungs compared with the vehicle control. these studies indicate that the cap mechanism of enac regulation can translate through in vitro human assays, into in vivo models. cap inhibitors can attenuate tpd in the guinea pig in vivo and enhance the rate of mucociliary clearance in the sheep, thereby representing an approach to the therapeutic regulation of enac function in cf lung disease. cotton, c. pediatrics, physiology and biophysics, case western reserve university, cleveland, oh, usa cystic fibrosis is caused by mutations in the gene that encodes cftr, a camp-activated apical membrane anion channel. loss of cftr-mediated anion conductance is a primary defect in cf but secondary defects such a sodium hyperabsorption are also implicated in cf pathophysiology. it is generally accepted that mucociliary clearance is compromised in cf airways due to reduced fluid secretion and/or increased fluid absorption. the recent generation of a transgenic mouse model with enac overexpression that exhibits cf-like lung disease highlights the importance of sodium hyperabsorption. therapies designed to reduce mucus production and viscosity, increase lumenal water content, stimulate fluid secretion, and inhibit fluid absorption are under development for treatment of cf airways. although controversial, several recent studies have demonstrated that serca pump inhibitors partially correct the trafficking defect associated with delta f508 mutant cftr. the goal of this work is to determine if serca pump inhibitors affect enac-mediated sodium absorption. a renal collecting duct epithelial cell line and primary cultures of well-differentiated human tracheal epithelial cells (air/liquid interface culture) were used for these studies. amiloride-sensitive short circuit current was determined as a measure of enac-mediated sodium absorption. quantitative rt-pcr was used to evaluate expression levels for each of the 3 subunits of enac (alpha, beta, and gamma). epithelial monolayers were treated for 18-24hours with serca pump inhibitors (thapsigargin, 100-500 nm; dbhq,10-50 um; and curcumin, 10-50 um). treatment with serca pump inhibitors decreased amiloride-sensitive short circuit current by 50-80%. in contrast, campstimulated short circuit current was not reduced by treatment with serca pump inhibitors. the steady-state levels of alpha, beta, and gamma enac were reduced by 60-80% in renal epithelial monolayers treated with thapsigargin whereas beta and gamma enac were reduced by 80-90% with no significant change in alpha enac in airway epithelial cells. the results of these studies demonstrate that sustained depletion of endoplasmic reticulum calcium stores and/or elevation of intracellular calcium by inhibition of serca pump activity reduces enac mrna expression and enac-mediated sodium absorption. thus, inhibition of serca pump activity in airway epithelial cells of cf patients that carry the delta f508 mutation may provide dual benefit by promoting delivery of mutant cftr to the membrane 1 1. pediatrics, national jewish medical and research center, denver, co, usa; 2. integrated department of immunology, national jewish medical and research center, denver, co, usa; 3. department of medicine, cystic fibrosis/pulmonary research and treatment center, the university of north carolina at chapel hill, chapel hill, nc, usa increased cytosolic calcium ([ca 2+ ] i ) initiated by the release of stored ca 2+ from the endoplasmic reticulum (er) is a key signal to elicit a wide range of essential cellular responses, including secretory functions of the respiratory epithelium that are modified in cf. serca pumps are responsible for (re)filling the er ca 2+ stores, and serca blockers such as thapsigargin are very potent and commonly used pharmacological triggers of ca 2+ -signals. modulation of the activity of sercas can profoundly affect ca 2+ homeostasis. although defective calcium homeostasis is a characteristic of several pulmonary diseases including cf, the role of serca is unknown. lung tissue samples (bronchus and distal lungs) from normal (n=2) and cf subjects (n=2) were evaluated by immunohistochemistry. serca2 expression was decreased in the bronchial and bronchiolar epithelia of cf. non-cf and cf bronchial epithelial cell line pairs including calu-3 and jme/cf15, c-38 and ib3-1, 16hbe14o-(16hbe), cfbe41o-(cf41o-) and cfbe45o-(cf45o-) were also probed. given certain limitations of such cells, several consistent findings still emerged. a 65% and 75% decrease in serca2 expression was observed in cf41o-and cf45ocells as compared to 16hbe cells. immunocytochemical studies in these cells confirmed that the serca2 was localized in the er and that the decreased serca expression was not associated with decreased er content. reduced serca2 expression and activity (3.51±0.27 vs 1.76±0.23 and 2.59±0.29 pmol/min/mg protein in 16 hbe vs. cf41o-and cf45ocells respectively) was observed in the purified er membranes from cf cell lines. northern blot analysis revealed a parallel reduced mrna expression as well. decreased serca2 was accompanied by increase in the low affinity isoform serca3 (0.95±0.20 vs. 1.39±0.10 and 1.68±0.32 arbitrary serca2 intensity/b-actin in respectively) . we have also evaluated a limited number of primary airway epithelial cells isolated from lung samples of normal and cf subjects for serca2 expression. serca2 expression in polarized tracheobronchial epithelial cell lysates from cf subjects was decreased by about 60% as compared to those from normal subjects (1.86±0.56 vs. 5.68±1.24 arbitrary serca2 intensity/b-actin units in cf and normal respectively, n=3). expression of serca2 could also be suppressed by inhibiting cftr with cftr inh 172 in normal human bronchial epithelial cells ( various studies indicate that the airway surface fluid possess an antibacterial system based on the combined action of lactoperoxidase, h 2 o 2 , and scn -(thiocyanate). the enzyme lactoperoxidase (secreted by submucosal glands) utilizes h 2 o 2 and scnto generate oscn -(hypothiocyanite) a molecule with antimicrobial activity. h 2 o 2 is produced by dual oxidases expressed on the apical membrane of airway epithelial cells whereas scnis transported across the epithelium through anion transporters and channels. in particular, scntransport seems to occur through cftr and other ca 2+dependent clchannels. therefore, reduced scntransport in the airways of cystic fibrosis patients may contribute to impaired antimicrobial activity. in a recent study, we have found that cytokines, in particular il-4, cause a strong increase in the ability of cultured bronchial epithelia to transport scnfrom the basolateral to the apical membrane. this effect is mediated by upregulation of ca 2+ -dependent clchannels and of the slc26a4 (pendrin) anion transporter. these findings suggest that under proinflammatory conditions the activity of the scn -/h 2 o 2 /lactoperoxidase system is potentiated. we evaluated the antimicrobial activity of airway surface by seeding bacteria on the apical membrane of human bronchial epithelia grown with an air liquid interface on a porous membrane. an inoculum corresponding to 1,000 cfu of s. aureus was added to the cells in 25 µl of saline solution with and without lactoperoxidase (6.5 µg/ml). experiments were performed in the presence or absence of scn -(100 µm) in the basolateral solution. bacteria were recovered after four hours and plated on agar plates for colony counting. our preliminary results show that simple addition of lactoperoxidase to the apical surface decreases bacterial survival. in addition, bacterial killing is strongly enhanced by prestimulation of cells for 24 hours with il-4 (10 ng/ml). the effect of lactoperoxidase is dependent on h 2 o 2 as it is prevented by addition of catalase and is absent in cell-free experiments when h 2 o 2 is omitted from the reaction mixture. surprisingly, the presence of scnin the basolateral compartment did not increase bacterial killing and, in some cases, appeared to generate a protective effect. our results suggest that lactoperoxidase, in the presence of h 2 o 2 , is an effective antimicrobial molecule. the contribution of scnand the mechanism of the potentiation caused by cytokines is less clear and requires further investigation. a possibility is that, in the absence of scn -, lactoperoxidase generates an oxidant molecule that is more toxic to bacteria than oscn -. elucidation of this mechanism and comparison between cf and non-cf epithelia is in progress to assess the role of cftr and of other anion channels. supported by cfft and telethon-italy. cf patients become infected with pseudomonas aeruginosa, which release flagellin into the airway surface liquid to activate toll-like receptor 5 and proinflammatory signaling. flagellin has been shown to inhibit na absorption by airway epithelia. we tested flagellin on cl secretion and proinflammatory signaling (nf-κb activation) by calu3 cells, a cftrexpressing, serous-like airway gland cell line. calu3 cells were grown on filters and either mounted in ussing chambers (clamped to zero mv) in the presence of a serosa-to-mucosa gradient of [cl] for measurements of transepithelial cl secretion (i cl ) or treated with an adenovirus expressing nf-κb-controlled luciferase to assay nf-κb activation. flagellin (10 -7 g/ml) on either the apical or basolateral surface of cells increased apparent anion secretion that began in 3-10 mins and increased over 20-30 mins by 20-50 µa/cm 2 . this i cl was blocked by glibenclamide and glyh101, indicating that it resulted at least in part from activation of cftr. flagellin also stimulated fluid secretion by intact human tracheal glands. flagellin activated nf-κb (luciferase assays) in both calu-3 cells and in the cf cell line cf15, while i cl was stimulated in calu3 but not in cf15 cells. flagellin-stimulated i cl in calu-3 cells was blocked 50% by sb202190 (a p38 mapk blocker) and by a similar amount by wortmannin (pi3 kinase blocker). interleukin 1β and the tlr2-agonist pam3cys also activated i cl . the effect of flagellin was not due to increases in cytosolic [ca 2+ ] (ca i ) because flagellin did not alter ca i . in contrast to the slow effects of flagellin, pam3cys and il1β, atp rapidly increased i cl (within secs, up to 80 µa/cm 2 ) followed by slower decrease to steady i cl = 20-40 µa/cm 2 that had a similar time signature as the increases in ca i . forskolin (to increase cytosolic [cyclic amp]) increased i cl within 2 mins to a steady value = 50-80 µa/cm 2 . flagellin had small or no effects on i cl following maximal stimulation with either atp or forskolin. atp and forskolin on their own had no effect on nf-κb. flagellin increased phosphorylation of p38 mapk and of akt (down-stream kinase phosphorylated by pi3k) with a time course similar to the increase in i cl . flagellin-activated nf-κb was reduced by roughly 50% by either wortmannin or sb202190. these results indicated that tlr agonists and inflammatory cytokines stimulate both nf-κb and proinflammatory processes and also cftr-dependent cl and fluid secretion by airway gland cells. these responses are mediated in part by activation of both p38 mapk and pi3 kinase. flagellin-tlr5-activated increases in cftr-mediated cl secretion and reduction in enac-mediated na absorption will increase fluid secretion into the airways, which may facilitate bacterial removal by the mucociliary escalator and thereby reduce the proinflammatory stimulus. in cf it is expected that bacteria will activate tlr signaling to trigger innate immune responses, but cl and water secretion and the resulting "bacterial flush," will be missing, leading to sustained inflammation. (1, 2) . treatment with hs improved several measures of lung function including mucociliary clearance. in one study amiloride was seen to have a negative impact on the benefit of hs (2) . the long duration of action of hs and the paradoxical effect of amiloride are surprising and require further investigation. aims: our goal was to study the effect of exposure to a hypertonic challenge (hc) on amiloride sensitive na+ transport (ina) in primary cultures of human bronchial epithelial (hbe) cells from non-cf and cf patients. methods: hbe cells were grown at an air liquid interface and studied under short circuit current conditions. cells were challenged with hs by exchange of the apical or basolateral baths. in some experiments the timedependent effect of a small (30 µl) isosmotic or hs volume at the airway surface was tested on ina and apical osmolality. results: application of hypertonic nacl and mannitol solutions from either the mucosal or serosal sides of the epithelium inhibited ina. the degree of inhibition was a saturable function of the imposed hypertonicity with an ic50 of 379 (mannitol) and 398 (nacl) mosmol/kg h2o and a maximal inhibition of 97%. the inclusion of amiloride (10-100 µm) did not affect the hc-induced reduction in ina. the inhibition in ina by hc was rapid in onset and accompanied by a fall in the transepithelial resistance and the total transepithelial capacitance as expected for cell shrinkage. the inhibition in ina was only partially reversible (~20%) after returning to isotonic solutions for 45 min. exposure of the airway surface to hypertonicity (680 mosmol/kg h2o) inhibited ina by 72-79% at 30 min of exposure. as osmotically-driven water moved from the serosal to the mucosal side, the apical volume increased concomitant with a decrease in its osmolality and a recovery of ina. the osmolality declined exponentially and reached the isosmotic value after ~4 hrs. the recovery of ina lagged behind the recovery of apical osmolality by 80, 20 and 10% at 0.5, 2 and 3 hrs of incubation, respectively. after 4 hrs of airway hc exposure, the osmolality and ina recovered completely. our results demonstrate that hc causes a rapid, dramatic and prolonged decrease in na+ absorption and that continuous presence of amiloride had no effect on the inhibition of ina or the recovery of apical osmolality. hc also causes the epithelium to shrink. no appreciable differences were observed between cf and non-cf hbe cells. conclusions: we propose cell shrinkage together with the continued influx of na+ increases intracellular na+ ([na]i) and lead to an inhibition of na+ transport. elevated [na] i is a known inhibitor of epithelial na+ channels (3). the sustained nature of the inhibition in na+ transport to a hc may help explain the longer than expected duration of action of hs in the clinical trials (1, 2) . explanation of the paradoxical effects of amiloride observed in a clinical trial (2) requires further investigation. references: 1. n engl j med 354:229-240,2006; 2. n engl j med 354:241-250, 2006; 3. physiol rev 77:359-396, 1997 reduced airway surface liquid (asl) volume resulting from enac mediated na+ hyperabsorption and cftr-mediated hyposecretion plays a critical role in cf lung disease pathogenesis. however, the mechanisms involved in enac/cftr regulation are poorly understood. using human bronchial epithelial cultures (hbecs) under thick film conditions, i.e. in the ussing chamber with the asl washed away, adenosine (ado) directly stimulates cl-secretion. however, in a cl-free environment, ado has no effect on the amiloride-sensitive current, which is mediated by enac. mean change in i sc following 100µm ado to the apical chamber was 0.05 µa/cm 2 from baseline compared to vehicle alone (-0.17 µa/cm 2 ;ns;n=3) in hbecs. mean delta amiloride (100µm) was -5.32 and -5.38 µa/cm 2 respectively (ns; n=3 each). in contrast, under thin film conditions we have previously shown that ado stimulates sustained asl secretion via activation of cftr in the presence of the protease-inhibitor aprotinin, whereas trypsin pre-treatment abolished this secretion, likely by activating enac and abolishing the electrical driving force for cl-secretion 1 . further, ado-mediated asl secretion is significantly increased if hbecs (thin film conditions) are left with intact asl for 24 h, an action that is abolished by acutely washing the apical surface 1 . thus, we hypothesised that a soluble enac inhibitor was secreted into the asl under thin film conditions, which could accumulate sufficiently to inhibit enac and provide the necessary driving force for cftr-mediated cl-secretion after ado-addition. to search for such an inhibitor, we incubated asl with trypsin-coated beads and identified bound proteins by mass spectrometry (albumin-coated beads were used as a control to exclude non-specific binding). the major identified protein was plunc, a protein that is secreted into the asl both in vivo and in vitro which has no current known function. secretion of plunc into hbec asl was confirmed by western blot. to test whether plunc could alter asl volume homeostasis, we made an anti-plunc shrna retroviral construct which we used to infect hbecs. unlike the control anti-luciferase shrna infected hbecs, anti-plunc shrna-infected hbecs exhibited >90% knockdown of plunc and a failure to regulate asl volume (ctrl, 9.0±1.8µm; 5±0.9µm; n=5) . when co-expressed with enac in oocytes, plunc inhibited enac currents by 78% (n=12). cftr function in contrast was unaffected by plunc expression (n=10). thus,it is likely that ado stimulates cftr mediated cl-secretion but is not involved in enac regulation. however, experiments were performed in a very simplified environment i.e. static cultures with little mucosal atp or mucus and these findings will need to be expanded and further evaluated in native tissues. we conclude that plunc, rather than ado may be the soluble mediator in the asl which regulates enac function by reducing its activity. 1. tarran, r. et al., j. gen. physiol., 2006. 127 (5) airway surface liquid (asl) absorption is mediated by epithelial na + channels (enac), which establish an osmotic gradient that drives fluid absorption. we and others have recently reported that a protease / anti-protease balance regulates enac in normal human bronchial epithelial cells (hbe) and provides a mechanism for auto-regulation of asl volume. in cf, this balance is disturbed, leading to constitutive proteolytic activation of enac and the pathological na + hyper-absorption characteristic of the disease. to determine if channel activating protease expression is regulated by changes in asl volume and is altered in cf, we examined prostasin expression in control and cf hbe under basal and asl volume expansion conditions using western blotting. prostasin migrates as 37 kda and 40 kda bands in apically biotinylated proteins, apical secretions, and whole cell lysates of primary hbe. following apical aprotinin exposure, only the 40 kda prostasin molecular species was present in the biotinylated proteins, suggesting that the proteolytic conversion of prostasin zymogen to active enzyme occurs on the cell surface. following asl volume expansion, cell surface prostasin expression increased by > 55% (p=0.007, n=12 tissue donors, >2 cultures from each). as our recent studies indicated that increased proteolytic activation of enac occurs in cf airway epithelia, we next compared prostasin expression in cf and control hbe cells. prostasin expression in the apical biotinylate of cf hbe was 1.5 fold greater than in control hbe (p=0.025, n=9 tissue donors). furthermore, the ratio of the 37 kda (active enzyme) to 40 kda (zymogen) prostasin molecular species was 2.5 fold greater in cf (p=0.009), indicating that increased activation of prostasin occurs in cf airway epithelium. we next determined whether increased prostasin activation in cf may reflect the deficiency of a protease inhibitor. serpin e2, also known as protease nexin-1 (pn-1), forms an ~82 kda complex with prostasin and permanently inactivates its protease activity. while no significant difference in pn-1 expression was observed between control and cf hbe, pn-1 was found to contribute to prostasin regulation by (i) forming an inactive prostasin complex, (ii) inhibiting the amiloride-sensitive short-circuit current across hbe, and (iii) preventing conversion of prostasin zymogen to active enzyme. these findings demonstrate that cellular mechanisms coordinate prostasin expression and activity with asl volume. accordingly, at times when the asl volume is high, prostasin expression increases, and this presumably augments na + and asl absorption to absorb the excess luminal fluid. these regulatory mechanisms govern the apical membrane expression and processing of zymogen to active enzyme. because prostasin is incapable of autocatalysis, these findings support the existence of a proteolytic cascade that controls enac activity during asl volume homeostasis, perhaps reflecting the recently reported matriptase-prostasin cascade. furthermore, the increased expression of prostasin in cf suggests that abnormal regulation of prostasin contributes to na + hyper-absorption in cf airways. supported by the nih, cff, and ala. we report that annexin 2 (anx 2)-s100a10 forms a functional camp/pka/calcineurin (can)-dependent complex with cftr. cell stimulation with forskolin/ibmx significantly increases the amount of anx 2-s100a10 that reciprocally co-immunoprecipitates with cell surface cftr and can a. pre-inhibition of the cells with pka or can inhibitors attenuates the interaction. furthermore, we find that the acetylated peptide (stvheilcklsleg, ac1-14), but not the non-acetylated equivalent n1-14, corresponding to the s100a10 binding site on anx 2, disrupts the anx 2-s100a10/cftr complex. analysis of dids and cftr inh172 -sensitive currents, taken as indication of the outwardly rectifying cl-channels (orcc) and cftr-mediated currents, respectively, showed that ac1-14, but not n1-14, inhibits both the camp/pka-dependent orcc and cftr activities. can inhibitors (cypermethrin, cyclosporin a) discriminated between orcc/cftr by inhibiting the cftr inh172 , but not the dids, sensitive currents, by more than 70%. furthermore, peptide ac1-14 inhibited acetylcholine-induced short-circuit current measured across a sheet of intact intestinal biopsy. our data suggests that the anx 2-s100a10/cftr complex is important for cftr function across epithelia. the content and water (h2o) mobility in airway epithelium is tighly coupled to airway function. in cystic fibrosis (cf), h2o epithelial permeability is reduced in airways. the demand of noninvasively imaging techniques with high spatial resolution potential is rising because such imaging tools would expedite anatomical and functional phenotyping in the genetically altered mice. magnetic resonance microscopy (mrm) is a noninvasive, inherently three-dimensional (3d) imaging technique capable of visualizing anatomical structures in the mouse and allows for interpretation of complex spatial relationships between substructures and h2o. in this study, we explore different mr contrast parameters and signal-to-noise ratios at a 30 µm pixel size to characterize microstructure and h2o mobility in ex vivo trachea of cf transmembrane conductance regulator (cftr)-deficient (cftr knockout, cftr tm1unc ) mice and their aged-matched wt littermates. this study is performed using a bruker mrm system at 11.7 tesla. we demonstrate for the first time the ability of 3d-mrm to map the h2o content and mobility in trachea epithelium. from the 3d-mrm videoimages, differential h2o content was visualized in different levels of trachea in wt and cf mice. t2 mrm images depicting the h2o rotational mobility which is related to environmental viscosity of trachea epithelium will be also shown. finally, this 3d-mrm imaging method is a valuable method for measuring h2o content and permeability in airways and can serve for assessing the effects of drugs on h2o mobility in cf airways. supported by grants from inserm, upmc-paris6, cnrs, and the french cystic fibrosis association (vlm). cystic fibrosis (cf) is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (cftr) protein resulting in progressive lung oxidative damage. in this study, we evaluated the role of cftr in the control of ubiquitin-proteasome activity (ups system) and the nf-κb / iκb-αsignaling after lung oxidative stress. we exposed cftr deficient (cftr-/-) and wild type mice for 64 h to hyperoxia-mediated oxidative stress. cftr deficient mice exhibited significantly higher lung proteasomal activity than cftr+/+ animals after oxidative stress. this was accompagnied by a strong reduction of lung caspase-3 activity and an absence of degradation of nf-κb inhibitor iκb-α. in vitro, human cftr-deficient lung cells also exhibited higher proteasomal activity and a lack of increased nf-κbdependent transcriptional activity compared to cftr-sufficient lung cells after oxidative stress. furthermore, inhibition of the proteasomal activity by mg132 in cftr-deficient lung cells restored the nf-κb/iκb-α signaling to that of cftr-sufficient lung cells. inhibition of caspase-3 by z-dqmd in cftr-sufficient lung cells mimicked the response profile of increased proteasomal degradation and lowered nf-κb transcriptional activity of cftr-deficient lung cells when exposed to oxidative stress. all together, these results suggest that cftr is a crucial molecule in regulating proteasomal degradation and nf-κb activity in lung epithelium under oxidative stress. staphylococcus aureus, one of the major pathogen involved in airway infections, releases in the airway lumen virulence factors that may impair the airway epithelial functionality. to date, the effect of s. aureus virulence factors on the loss of electrolyte homeostasis of the airway epithelium has not been investigated. we have previously shown that the combination of a corticosteroid and a long-acting beta 2 agonist attenuated the airway epithelial cell inflammatory response induced by s. aureus virulence factors. the aim of the present work was to investigate the effect of s. aureus virulence factors on the airway epithelial tightness and on the chloride efflux of airway epithelial cells incubated or not with salmeterol hydroxynaphthoate 2×10 -7 m and fluticasone propionate 1×10 -8 m (sm/fp). the airway epithelial tightness was assessed by immunocytochemistry, western blotting and transepithelial resistance measurement. the chloride efflux was evaluated by dynamic imaging using the 6-methoxy-n-(3-sulfopropyl) quinolinium (spq) fluorescent probe. s. aureus (strain 8325-4) virulence factors were obtained by growing bacteria at 5×10 8 cfu/ml for 18h at 37°c. the bacteria supernatant containing s. aureus soluble virulence factors was then centrifuged, filtered and diluted at 2% in cell culture medium (a concentration that did not alter cell viability). human airway epithelial cells (mm39) were incubated with 2% s. aureus virulence factors for 1h and then co-incubated or not with sm/fp for 4h. we observed that the incubation with 2% s. aureus virulence factors alone did not significantly alter the epithelial integrity assessed by the expression of tight junction proteins and transepithelial resistance measurement. however, when the cells were incubated with sm/fp alone or with 2% s. aureus virulence factors plus sm/fp, the expression of tight junction proteins was significantly increased (p<0.05) as compared to cells incubated with s. aureus virulence factors. interestingly, the chloride efflux in airway epithelial cells was significantly decreased in a time-dependent way by s. aureus virulence factors (3 fold decrease after a 4h incubation). the co-incubation of airway epithelial cells with s. aureus virulence factors and sm/fp prevented the s. aureus virulence factorsdependent chloride efflux decrease. our results demonstrate that bacterial virulence factors induce the loss of the electrolyte homeostasis and suggest that the treatment by the combination of a corticosteroid and a long-acting beta 2 agonist may preserve the airway epithelial functionality. supported by association vaincre la mucoviscidose and glaxo-smithkline nilsson, h.e. 1 ; dragomir, a. 1 ; ahlander, a. 1 ; johannesson, m. 2 ; roomans, g.m. 1 1. medical cell biology, uppsala university, uppsala, sweden; 2. women's and children's health, uppsala university, uppsala, sweden inhalation of hyperosmotic solutions (salt, mannitol) has been used in the treatment of patients with cystic fibrosis or asthma, but the mechanism behind the effect of hyperosmotic solutions on mucociliary clearance (mcc) is unclear. one explanation has been suggested, namely that hypertonic solutions open tight junctions, which may lead to increased water transport followed by an increased airway surface liquid (asl) volume. furthermore, the role in cftr-mediated hco3-conductance in regulating asl ph has led us to investigate if ph changes may have an effect on the tightness of tight junctions. the effect of osmolarity was investigated on the 16hbe14o-cell line by the addition of nacl, nabr, licl, mannitol or xylitol (295-700 mosm). the effect of ph was investigated on the 16hbe14o-, calu-3 and t84 cell lines as well as the cystic fibrosis cell line cfbe41o-. transepithelial resistance was measured as indicator of the tightness of the cultures. cell-cell contacts and morphology were investigated by immuno-fluorescence and by transmission electron microscopy, with lanthanum nitrate added to the luminal side of the epithelium to investigate tight junction permeability. the electrolyte solutions caused a significant decrease in transepithelial resistance from 450 mosm on, when the hyperosmolar exposure was gradually increased from 295 to 700 mosm, whereas the non-electrolyte solutions caused a decrease in transepithelial resistance from 700 mosm on. immunofluorescence micrographs showed weaker staining for the proteins zo-1, claudin-4 in treated samples compared to the control. the ultrastructure revealed an increased number of open tight junctions as well as a disturbed morphology with increasing osmolarity, and with electrolyte solutions opening a larger proportion of tight junctions than non-electrolyte solutions. it was noted that during the exposure of the cultured cells to the hyperosmolar solutions, the ph of the medium increased from 7.4 to 8.0. 16hbe14o-cells exposed to both a rise in ph and hyperosmotic stress showed an overall lower teer and a significantly reduced ability to recover from stress compared to cultures at a ph hold constant at 7.4. without exposure to hyperosmolar solutions, a rise in ph caused a significant decrease in transepithelial resistance in 16hbe14o-cells, calu-3 and t84 cell lines but not in the cfbe14o-cell lines, where the reaction was significantly less and delayed. in conclusion, hyperosmolar solutions caused a reversible opening of the tj in 16hbe14o-cell cultures with electrolytes having stronger effects than non-electrolytes, where one of the effects on mcc may be due to increased water transport across the leaky paracellular space. an increase in ph caused a significant decrease in teer in the healthy cell lines compared to the cfbe41o-cell line. we speculated that an impaired alkalinisation of the apical fluid due to a defective cftr also will cause the tight junctions to react different to other external stimuli, such as osmolarity, compared to healthy cells, which is also indicated by preliminary experiments on the effect of nacl on teer in cfbe41o-cultures. extracellular nucleotides regulate surfactant secretion in alveoli and mucociliary clearance in airway epithelia, but the mechanism(s) of their release and their regulatory pathways remain incompletely understood. previously, we showed that hypotonic swelling of a549 epithelial cells induces ca 2+ -dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. in this study, we examined sources of intracellular ca 2+ ([ca 2+ ] i ) elevation evoked by acute 50% hypotonic stress and the role of autocrine purinergic signaling in ca 2+ -dependent atp release. we found that atp release does not directly involve ca 2+ influx from extracellular spaces, but depends entirely on ca 2+ mobilization from intracellular stores. the [ca 2+ ] i response consisted of slowly-rising elevation representing mobilization from thapsigargin (tg)-insensitive stores and a superimposed rapid spike due to ca 2+ release from tg-sensitive endoplasmic reticulum (er) stores. the latter could be abolished by hydrolysis of extracellular tri-and di-nucleotides with apyrase; blocking p2y 2 /p2y 6 receptors of a549 cells with suramin; blocking udp receptors (p2y 6 ) by ppads; emptying tg-sensitive stores downstream with 1 µm tg or 10 mm caffeine in ca 2+ -free extracellular solution; or blocking the ca 2+ -release inositol 1,4,5-triphosphate (ip 3 ) receptor channel of the er with 75 µm 2aminoethyl diphenylborinate. these results demonstrate that the rapid [ca 2+ ] i spike results from the autocrine stimulation of ip 3 )/ca 2+ -coupled p2y 2 /p2y 6 receptors, which accounts for ~70% of total ca 2+ -dependent atp release evoked by hypotonic shock. our study reveals a novel paradigm in which atp release is amplified by the synergistic autocrine/paracrine action of co-released uridine and adenosine nucleotides. we suggest that a similar mechanism of purinergic signal propagation operates in other cell types. (this study was supported in part by the canadian institutes of health research and the canadian cystic fibrosis foundation (ccff). s.t. was the recipient of a ccff studentship). the maintenance of a thin liquid layer on the airways and alveoli surfaces is essential for normal lung physiology.yet, the mechanism sensing the height of the layer remains obscure. in this study, we examined atp secretion from human lung a549 epithelial cell monolayers mounted in a closed, flowthrough chamber (0.5 mm height) and found that passage of an air bubble over the monolayer caused transient (<1.5 min) but significant atp release (600 to 2,000 pmoles/10 6 cells). air bubble-induced atp release was reduced by~70% from cells loaded with the intracellular ca 2+ chelator bapta-am, and was completely abolished in n-methylmalemide-treated (1 mm) cells, suggesting the involvement of ca 2+ -dependent exocytosis. fura-2 intracellular ca 2+ ([ca 2+ ] i ) imaging experiments revealed transient [ca 2+ ] i elevation during the passage of an air bubble over the cell monolayer, but the [ca 2+ ] i response did not involve non-specific ca 2+ influx from the extracellular space, e.g. due to cell damage, since similar responses were observed in ca 2+ -free extracellular solution. ethidium bromide staining did not disclose any cell damage in these experiments. confocal fluorescence microscopy study showed reversible cell deformation (flattening) of 10% to 40% in height in the monolayer part in contact with the air bubble and confirmed that cell integrity was entirely preserved. these experiments demonstrate that in close proximity to the air-liquid interface (i.e. between the air bubble and the wet cell surface), surface tension forces are transmitted directly on cells, causing their mechanical deformation, elevation of [ca 2+ ] i and subsequent atp release. we propose that a similar mechanism may operate in vivo in the airways, where surface tension forces acting directly on exposed epithelial cell surfaces may provide a fail-proof mechanism to protect proper airway surface liquid volume via mechanosensitive, ca 2+ -dependent atp release and purinergic modulation of fluid secretion. this mechanism may be defective in cystic fibrosis due to excessive mucus layer covering the airways. introduction : cystic fibrosis is a genetic disease that reflects the consequences of mutations in the cystic fibrosis transmembrane conductance reg-ulator (cftr) gene, affecting anionic transport in epithelia. slc26 family members are anionic transporters involved in cl-and hco3-absorption or secretion in epithelia. in addition, the activation of some slc26 family members by cftr has been demonstrated (nature cell biology, 2004, 6: 343-350) . slc26a9 is a poorly characterized member of this family, being the solely expressed in lung. putative interaction domains with cftr are also present in the slc26a9 protein. in this study, we have investigated the transport properties of slc26a9 (human) to determine the functional and pharmacological characteristics of this transporter. methods : to this aim, electrophysiological studies (two-electrode voltage clamp or current clamp methods and intracellular ph measurements using ph sensitive microelectrode) were performed in xenopus laevis oocytes expressing slc26a9 proteins. complementary (36cl) transport studies were also undertaken. results : the protein expression results in the appearance of an anionic current showing a linear current/voltage relationship. 36cl influxes experiments confirmed the induced cl-permeability following slc26a9 protein expression. the sequences of conductivity, cl->i-> no3-≥ gluconate > so4 2-and selectivity (px/pcl), i-> cl-= no3-> gluconate > so42-are found. the hco3-conductance mediated by the slc26a9 protein expression is low. using co2/hco3-containing ringer solutions, no intracellular phi changes were detectable in conditions (low chloride in external medium) favoring the cl-/hco3-exchange whereas phi changes (alkalinization) were observed with the expression of the ae1 exchanger. however, phi changes could be detected in conditions largely favoring the driving force for hco3-entry. dids and ns 3623 inhibited slc26a9 associated currents. the specific cftr inhibitor (cftrinh172) or glybenclamide had little effect. elevation of intracellular camp (a cftr activator) was also ineffective while maneuvers increasing intracellular calcium blocked the slc26a9 associated currents. conclusions : our study demonstrates that slc26a9 presents an electrogenic anion conductance (characteristics of an anionic channel) when expressed in xenopus oocytes (however a transporter functioning as a ncl-/hco3-exchanger can not be excluded). this function (channel) has also been described for another member of this family, slc26a7 (j biol. chem, 2005, 280: 6463-6470) . the physiological role of slc26a9, present in the bronchial cells of airway epithelia and its potential interaction with cftr has to be precised in situ or in oocytes and in mammal expression systems. medicine, women's & children's hospital, nth adelaide, sa, australia; 2. cf research & treatment center, university of nth carolina at chapel hill, chapel hill, nc, usa; [3] [4] [5] [6] [7] [8] jasri, hyogo, japan; 4. physics and synchrotron science, monash university, clayton, vic, australia; 5. paediatrics, university of adelaide, adelaide, sa, australia non-invasive imaging of lung (e.g. hrct, mri, pet) is a valuable modality for detection and monitoring of the effects of cf in human airways, but resolution is limited; e.g. airway hrct detects airways no smaller than ~ 1.5mm dia. detection of wall or lumen changes in smaller airways, or the detection and monitoring of induced airway disease in live rodent models, demands significantly greater resolution coupled with rapid image capture. we report substantially increased airway resolution is achievable using synchrotron phase-contrast x-ray and can produce three-dimensional reconstructions encompassing the smallest mouse lung airways. nembutal-anaesthetised c57bl/6 mice (~ 20 gm) were imaged at the spring-8 synchrotron in hyogo, japan. 2-d phase-contrast airway images were obtained on ccd detectors (1.1 µm pixels) with a 90 cm phase-contrast propagation distance at 25 kev; nose and trachea were imaged at 15 or 30 sec intervals (100-300 ms exposures) over 45 mins. mice killed with nembutal overdose were imaged in axially aligned segments to obtain 3dimensional ct data voume of approximately 20x20x15 mm3 with 12x12x12 um3 voxels of nose, trachea, and lung (hammamatsu ccd detector). volume renderings were produced using volview or osirix software. live, 2-d nasal or tracheal imaging revealed airway-surface activity in some live mice consistent with dynamic mucociliary clearance activity. individual ct slices revealed the fine detail in the mouse lung, with dynamic ("fly through") sequences of lung ct slices permitting visual identification and tracking of lung tree branching from trachea (~ 1mm dia) into small airways (approx 100 um dia). adjustment of x-ray contrast, opacity, and range in these volume reconstructions permitted selective display of the mouse lung conducting-airway tree and airway surfaces. selected regions of the lung could be examined statically, or in rotation through different orientations in high-resolution 3-d. synchrotron phase contrast x-ray provides a new option for non-invasive imaging of intact mouse lung, at a resolution that permits examination of small conducting airways in mice. combined with volume-reconstruction software these ultract images can provide a powerful option for understanding the structure-function relationships produced by airway disease. ultra-ct 3-d studies are underway to compare lung airways at high resoltuion in normal and transgenic mice having altered airway pathophysiology. the epithelial sodium channel, enac, has a vital role in the function of the pulmonary epithelia and significantly contributes to the pathophysiology of the cf airway. thus, strategies to repair mutant cftr dysfunction must also consider the influence of such repair on enac functional expression. the ∆f508 trafficking repair agent 4-phenylbutyrate modulates the expression of the 70 kda molecular chaperones hsc70 and hsp70 in cf epithelial cells. we therefore assessed the role of these chaperones in the regulation of enac trafficking. in xenopus oocytes, we previously observed that overexpression of hsc70 inhibits murine enac (menac) functional and surface expression. in contrast, hsp70 can either enhance or inhibit menac functional and surface expression depending on the extent of overexpression [goldfarb et al. (2006), proc. natl. acad. sci. 103:5817-22 ]. here, we tested the hypothesis that these differential effects of hsc70 and hsp70 on menac expression also occur in epithelial cells. mdck cell lines with stable expression of α-ha, β-v5, γ-myc-menac and tetracyclineinducible expression of either hsc70, atpase-deficient hsc70, hsp70 or atpase-deficient hsp70 containing myc/his epitope-tags were selected. all epitopes were fused to the respective c-termini. these cells were grown as high-resistance (>500 ω/cm 2 ) monolayers on permeable supports. doxycycline-induced overexpression of hsc70 decreased amiloride-sensitive i sc and the whole cell content of α-haand β-v5-menac. in contrast, lower amounts of tetracycline-induced hsp70 overexpression increased amiloridesensitive i sc and whole cell α-haand β-v5-menac expression; these effects were absent at higher levels of hsp70 overexpression. these data are consistent with our previous data in xenopus oocytes. interestingly, the atpase-deficient chaperones had the opposite effect on menac functional expression. modest doxycycline-induced expression of atpase-deficient hsc70 increased amiloride-sensitive i sc in these cells, while modest overexpression of atpase-deficient hsp70 decreased amiloride-sensitive i sc . these effects may result from "dominant negative" interference with hsc70 and hsp70 function, respectively. these data are consistent with hsc70 and hsp70 having different effects on menac functional expression in epithelial cells, and that these effects are dependent upon the atpase activity of the respective chaperones. supported by grants from niddk. we have studied survival, airway epithelia bioelectrics and lung pathology of mice over-expressing various combinations of the 3 epithelial na + channel (enac) subunits (α, β and γ, genes scnn1a, scnn1b and scnn1g). we generated double-transgenic mice and crossed them with single-transgenic mice to obtain litters comprising all 8 possible genotypes. survival analysis revealed that overexpression of βenac in combination with either αenac or γenac significantly reduced survival in comparison to wild-type (wt) littermates. strikingly, at 3 days of age, all genotypes were represented according to the expected mendelian proportion, except for the triple transgenic αβγenac, which was significantly under represented. in utero studies are ongoing to understand if over-expression of αβγ affects the fetuses or the newborn pups during the first hours post-partum. due to the high mortality of mice over-expressing βenac subunit combinations, we studied tracheas from pups at 3 days of age. as previously reported, mice overexpressing β, but not the α or γ enac subunits, exhibit airways hyperabsorption of na + and lung pathology (mall et all 2004) . the βenac tracheas exhibited na + absorption [as measured by the change in short circuit current (isc) in response to amiloride in the ussing chamber] that was ~5.4 fold greater (∆isc 24.6±3.9 µa×cm -2 , n=9, means±sem) than wt (4.6±0.8 µa×cm -2 , n=7). over-expressing αβ or αγ enac subunits resulted in rates of na + absorption that did not differ significantly from those of βenac or γenac subunit alone, respectively. however over-expressing the β and γ enac subunits together resulted in an amiloride sensitive isc that was ~13.7 fold greater (∆isc 63.1±8.2 µa×cm -2 , n=9) than wt. we also studied the tracheal bioelectrics of the few 3 day-old αβγenac pups available. tracheas from the αbγ pups exhibited na + absorption that was ~16.7 fold greater (∆isc 77±8.2 µa×cm -2 , n=3) than wt. due to the small sample size, we cannot determine if this response is significantly different from the response of the βγenac pups. analysis of lung pathology in 3 day-old pups revealed that all the combinations that exhibited increased airway na + absorption and decreased survival in comparison to wt littermates, e.g. αβ, βγ and αβγ, also presented with alveolar space enlargement, maybe due to postobstructive air trapping, and airway epithelia necrotic degeneration associated with ab-pas negative bronchial obstruction. from these data, it appears that the rate of airway na + absorption negatively correlates with the survival of the pups. thus, it is likely that the mucus and airway surface liquid is more dehydrated as a function of the rates of na + absorption (aβγ > βγ > αβ, β > wt) which produces gradual failure to survive due to asphyxia secondary to airway obstruction. supported by nih scor p50 hl060280 choi, j. 1,2 ; joo, n. 1 ; wu, j. 1 ; krouse, m. 1 ; wine, j.j. 1 1. cystic fibrosis research laboratory, , stanford, ca, usa; 2. otorhinolaryngology, yonsei university, seoul, south korea submucosal glands, which produce most airway mucus, are mainly controlled by parasympathetic pathways that stimulate glands via airway intrinsic neurons distributed along the airway walls. the intrinsic neurons can be stimulated via axon reflexes from receptors in the mucosa. in previous experiments, it was shown that capsaicin applied to the mucosa stimulated gland secretion in the upper tracheas of wt but not cftr -/-mice (ianowski et al., j physiol, 2007, 580, 301) . it seemed important to assess a possible role of capsaicin-sensitive pathways in pigs and humans. we obtained pig tracheas following acute experiments carried out for other reasons, and human airways following lung transplants from donor tracheas and from the excised lungs of the transplant recipients. secretion from individual submucosal glands was quantified optically by time-lapse digital imaging of the growth of spherical bubbles of mucus in an oil layer in humans, commercial chili oil (5 µm dispersed in 20 µm mineral oil) stimulated mucus secretion from submucosal glands from both donors (n = 46 glands from 9 subjects, 0.30 ± 0.24 nl/min/gland) and disease control subjects (n = 45 glands from 7 subjects, 0.25 ± 0.16 nl/min/gland). however, there were no responses to chili oil in submucosal glands from cf subjects (n = 36 glands from 5 subjects, 0.01 ± 0.01 nl/min/gland). in pigs, we determined concentrationresponse relations for gland secretion in response to purified capsaicin (sigma). the threshold was ~1 µm and the ec50 was 19.01 ± 4.9 µm. the response to 100 µm capsaicin was partially blocked by high dose ttx (>1 µm). as in humans, the capsaicin response required cftr, because it was blocked ~70% by cftrinh-172, (n = 29 glands, from 4 pigs, p < 0.05). cftrinh-172 is presently the most specific inhibitor of cftr that can be used with glands, where we do not have access to the apical membrane. in ferrets, intrinsic airway neurons express acetylcholine, vip and sp, often colocalized within the same neurons (dey et al., am j respir cell mol biol, 1996, 14, 207) . in pigs, all three of these transmitter systems appear to be involved in gland secretion to capsaicin, because the response was partially blocked by the nk-1 receptor blocker l703606 (oxalate salt, 1µm), the vip receptor inhibitor l8k (10µm), and the muscarinic receptor blocker atropine (1µm); each of these blocked 36 to 59 % of the response (n = 21 to 39 glands from 3 to 5 pigs). indeed, most of the local pathways are probably damaged, which may account for the relatively small responses to strong stimulation. relatively few experiments have been carried out in intact airways with uninterrupted innervation and circulation, but in those experiments the glands were highly responsive to modest mucosal stimuli (reviewed in wine, auto neurosci, 2007, 133, 35) . hence, we hypothesize that (1) reflex stimulation of glands plays an important role in lung innate defenses; (2) defects in these responses is an important reason that cf airways are prone to infection, and (3) local reflexes may assume greater importance for maintaining the mucosal defenses of transplanted lungs. supported by niddk ro1-51817 (jjw), cff and cfri. most airway mucus arises from submucosal glands, which express cftr in their ciliated ducts and in serous cells. the airway glands secrete to agonists that either increase intracellular ca2+ (e.g. acetylcholine) or camp (e.g. vip).substance p(sp) also stimulates airway gland liquid secretion in pig (trout l et al., am j physiol lung cell mol physiol. 2001, 281,:l639) , suggesting that sensory afferents or local neurons that express sp play a role in stimulating submucosal glands. we used pharmacological methods to dissect the secretory mechanism of sp-induced gland secretion in comparison with responses to carbachol. tracheas were obtained from pigs after acute surgeries carried out for other reasons. the ventral mucosa with underlying glands was dissected from the cartilage, pinned mucosal side up at the gas/bath interface of a physiological chamber, and covered with oil. secretions from individual glands could be visualized as spherical bubbles in the oil, and secretion rates determined by optical monitoring of bubble diameters. concentration-response relations for gland secretion were determined for sp (19-42 bubbles at each concentration from 9 pigs). the threshold was ~100 nm for sp and the ec50 was 1.76 ± 0.57 µm. the maximum secretion rate to sp was 1.17 ± 0.21 nl/min.gland, which is only ~ 1/3 of the maximal secretion rate to carbachol (3.43± 0.29 nl/min.gland). the inhibition profile for responses to sp (10 µm) was quite different from those to carbachol (200 nm, a value chose to mimic the secretion rate to 10 µm sp). gland secretory responses to 10 µm sp (0.75 ± 0.17 nl/min.gland, 41 glands from 4 pigs) were strongly inhibited by 20 µm cftrinh-172 (0.34 ± 0.10 nl/min.gland, 36 glands from 4 pigs, p<0.05), and by 25 µm clotrimazole (0.28 ± 0.06 nl/min.gland, 31 glands from 4 pigs), whereas 100 µm niflumic acid did not inhibit (0.81 ± 0.25 nl/min.gland, 32 glands from 3 pigs). in contrast, secretory response to 200 nm carbachol (0.61 ± 0.22 nl/min.gland, 36 glands from 3 pigs) were only weakly inhibited by cftrinh-172 (0.49 ± 0.13 nl/min.gland, 29 glands from 3 pigs, p<0.05), and by clotrimazole (0.53 ± 0.16 nl/min.gland, 33 glands from 3 pigs, p>0.05), but niflumic acid, which was ineffective with sp, inhibited the carbachol response (0.44 ± 0.13 nl/min.gland, 41 glands from 3 pigs, p<0.05). gland secretion to sp depends at least partially on intracellular ca2+ release, because the response was partially inhibited by 50 µm bapta-am (0.33 ± 0.09 nl/min.gland, 21 glands from 2 pigs, p<0.05). there was no additional effect of sp on top of 1 µm carbachol, but a subthreshold level of sp (50 nm) showed synergic response (0.44 ± 0.22 nl/min.gland, 21 glands from 3 pigs) with subthreshold level of vip (10 nm). we are presently testing how sp activates cftr-dependent mucus secretion. in initial experiments to determine if pkc was being recruited, however the pkc inhibitor (gf 109203, sigma) did not suppress sp-induced mucus secretion. in contrast, the nkcc inhibitor bumetanide (100 µm) markedly suppressed secretion to sp (0.22 ± 0.06 nl/min.gland, 23 glands from 2 pigs, p<0.05). supported by niddk ro1-51817 (jjw), cff and cfri. mucus obstruction of airways is considered the most vicious agent of morbidity and mortality in cystic fibrosis (cf). to provide optimal defense of the small airways the volume/thickness of airway surface fluid must be controlled according to physiological demand. to date we have no clear understandings of how these fluids are maintained in a steady state between two pathological extremes (too little or too much) in native small airways. earlier studies have attempted to measure the ion transport properties of small airways of sheep (al-bazzaz et. al. 2001 ) and pigs (ballard et. al. 1992 ), but the complicated branching structure of small airways may have compromised electrical signals. to avoid dissection trauma wang et. al. (2005) examined the electrophysiological properties of undissected intact small airways from pigs in vitro by microperfusing bronchioles (diam.1-2 mm) embedded in the lung parenchyma; however, these studies were limited to luminal manipulations and transbronchiolar electrical potentials only. to more rigorously define small airway properties, we designed a special micro ussing chamber ( area ≈1 mm 2 ). we excised and opened small airways (diam.1-2mm) of pig lung that we mounted as a flat sheet over a pvc supporting filter (10-20 µm holes). both sides of the tissue could then be bathed with different solutions at 37°c. the luminal side was isolated by pressing a pipette (≈1 mm diam., tip fire polished) on to the apical surface of the tissue until it sealed electrically. constant current pulses (0.5-3.0 µa) were passed across the tissue. transepithelial potential (tep) and resistance in bilateral 150 mm nacl ringers were, -1.3 ± 0.1 mv and 83.1 ± 8.1 ω.cm 2 (mean ± se) respectively. when 150 mm naglu ringers replaced the luminal solution, tep increased significantly to -36.7 ± 1.5 mv (n=30, p< 0.0005) and resistance increased to 173.8 ± 21.0 ω.cm 2 (n=30, p< 0.0005. adding forkskolin (fsk, 10 µm) plus ibmx (0.1 mm) hyperpolarized the tep to -40.22 ± 1.99 mv (n=16, p<0.002), but decreased the resistance to 142.9 ± 28.2 ω.cm 2 (n=16, p< 0.0001). luminal amiloride (10 µm) depolarized the tep to -32.6 ± 1.6 mv (n=25, p<0.0001) but significantly increased resistance to 215.7 ± 26.6 ω.cm 2 (n=25, p<0.0005). these results show that this system supports measurements of ion transport properties of airways smaller than 1 mm diameter where cf lung pathology is thought to originate. these airway epithelia express a very high constitutively active clconductance and are apparently capable of secretory as well as absorptive functions. amiloride sensitive na + conductance (≈ 43 ms/cm 2 ) was not significantly affected by fsk added to activate secretion. the observation that stimulation with fsk did not significantly suppress na + conductance (≈ 41 ms/cm 2 ) (p= 0.4) may suggest that separate groups of reabsorptive and secretory cells comprise small airway epithelia. mucins are class of proteins uniquely characterized by large glycosylated domains. these proteins likely play a key role in cystic fibrosis (cf) lung disease as major constituents of mucus. in airways of both humans and mice, the gel-forming secreted mucins are represented by muc5ac and muc5b, and the transmembrane mucins are represented primarily by muc1, muc4, and muc16. we have been interested in the role of the transmembrane mucins in airways and have previously demonstrated that lack of muc1 in a knock-out mouse model (provided by s. gendler) shows increased susceptibility to adenoviral-mediated gene transfer (stonebraker, j. virology, 2004) . to expand upon these findings, mice deficient for muc4 were developed using embryonic stem cell technology by deleting exon 1 of the mouse muc4 gene, which contains the atg start codon and signal peptide. rna analysis reveals loss of exon 1 in homozygous mutant mice and immunohistochemistry using a vntr antibody indicated a loss of full length muc4 protein. muc4 homozygous mice are viable and do not develop any spontaneous, readily detectable disease phenotype. although the muc4 deficient mice do have reduced fertility consisting of both reduced litter size and number, all major tissue systems where the muc4 protein is easily detected, including the eye, respiratory tract, intestine, and reproductive tracts, appear normal in year old mice after histological analyses. in contrast to the results we reported in the muc1 mice, the muc4 deficient mice do not show an increase in adenoviral mediated gene transfer, suggesting that muc4 is not involved in the barrier aspect of the airway glycocalxy, at least for adenoviral infection. interestingly, quantitative real-time pcr analysis revealed up-regulation of muc16 protein in the trachea of the muc4 deficient mice, suggesting that muc16 may be able to compensate for loss of muc4. the expression of muc4 in human ciliated cells as revealed by recent antibody studies prompted us to evaluate the potential role of muc4 in ciliary function. ciliary beat frequency (cbf) was monitored in a shear chamber using microscopy image analysis. cbf under baseline conditions for this study (low shear stress) from muc4 deficient trachea (c57bl/6j congenic n5) was significantly lower than that for littermate controls (8.86 vs. 11.53, sem ±1.31, p = 0.046). application of shear resulted in increased cbf in both groups, although the wt response to shear was smaller in magnitude. thus, muc4 may have a role in supporting basal cbf and may make cbf less vulnerable to stimulation by shear, perhaps by reducing the inherent friction of ciliary motion. this finding underscores the usefulness of this model for dissecting out the normal roles that the transmembrane mucins play in airway biology. supported by nih (hl066973) and cff (r026-cr02). enac (joo et al. jbc 279, 2004 and jbc 281, 2006) . in a search for a model to test the hypothesis we found that ussing chamber short-circuit current(i sc ) measurements with freshly obtained airway mucosa provided useful information about enac activity in response to various drug treatments. in these studies we observed an as yet unidentified pathway that regulates surface enac activity. airway mucosa from sheep tracheas displays a large, amiloride/benzamil-sensitive i sc prior to stimulation. when stimulated with basolateral carbachol, the enac i sc was almost abolished, as indicated by a sustained decrease (77.1 ± 3.4%, n=14) in i sc after an initial increase and left a small effect to subsequent amiloride or benzamil. in contrast, when carbachol was given after benzamil, it again produced a peak response followed by a sustained increase (~30%, n=4) in the i sc . enac inhibition by carbachol was dose dependent with a minimal sensitivity between 0.1~1 µm and was slowly reversible. apical 100 µm atp also inhibited enac i sc by 48.4 ± 8.1% (n=3) and carbachol on top of atp further reduced the enac i sc by 93.6 ± 3.9% (n=3). however, 10 u/ml of apyrase pretreatment, in attempt to breakdown atp, failed to inhibit carbachol-induced enac inhibition, indicating that atp release is not on obligatory component of carbachol inhibition of enac. carbachol inhibition of enac could result from a direct action of carbachol on the surface epithelia, by activation of airway intrinsic neurons, or by activation of the glands, which were intact in these preparations and are strongly stimulated by carbachol. rabbit tracheas, which lack airway submucosal glands, displayed large enac currents and these were not inhibited by cholinergic stimulation (n=3). carbachol-induced enac inhibition, like gland stimulation but unlike stimulation of neurons, was mediated by muscarinic receptors since it was eliminated by atropine but not by the nicotinic receptor antagonist, hexomethonium bromide (n=3). however, inhibition of gland secretion with hepes/bumetanide did not eliminate the ability of carbachol to inhibit enac. the effects of carbachol on surface na + transport in our study are consistent with previous observations in microperfused sheep bronchiole preparations (al-bazzaz, ajp-lung 11, 1994). although we do not yet know the mechanism, it would be physiologically efficient if parasympathetic, cholinergic activation of airway gland secretion was accompanied by enac inhibition, which would minimize the fluid absorption by surface epithelia and increase clearance. it will be important to determine if cholinergic inhibition of enac is present in human tissues, and if so, if it is defective in cf. supported by cff and nih. introduction: lung disease accounts for more than 95% of the current morbidity and mortality associated with cf. the majority of research aiming to elucidate the reason(s) of severe lung disease has focused on airway surface liquid (asl) layer homeostasis or composition. the cftr gene encodes a camp-regulated clchannel located on the apical side of the epithelium and is instrumental in maintaining a hydrated asl layer and promoting effective mucociliary clearance (mcc). in patients with class i or class ii cftr genetic mutations, normal cftr clchannel activity in the epithelium is impaired resulting in na + hyperabsorption and consequently in the generation of a thick and static overlaying asl layer creating an environment that favors progressively worsening bacterial infections. in this study we investigated the potential contributory role of altered respiratory cilia function to inefficient mcc in cf airways allowing for bacterial infestations. our ex vivo model system was the nasal airways of the cftr gene knockout (cftr tm1unc ) mouse in which key aspects of the phenotype of human cf lung disease, such as dehydrated asl layer and goblet hyperplasia, are observed. methods and results: ciliary beat frequency (cbf) was measured using a dual temperature controlled perfusion chamber, differential interference contrast microscopy and high speed digital video analysis system. we evaluated the cbf of nasal septa explants from homozygote cf (affected) and non-affected littermate wild type (wt) and heterozygote mice congenic on the c57bl/6 background. the basal cbf of homozygote cf mice (12±3 hz, n=7) was significantly lower than that of wt mice (28±5 hz, n=7) and also heterozygote mice (19±3 hz, n=17) (p<0.05, anova, snk test). to determine whether the dehydrated asl layer was still present on the surface of the cf mouse nasal airway epithelium and as such impeding normal cilial beating, we fixed nasal septa (n=3) of cf and wt mice immediately following isolation using the pfc/oso 4 fixation method to preserve the fragile asl layer and compared the height of the asl layer to nasal septa (n=3) from cf and wt mice fixed with pfc/oso 4 immediately following cbf analysis. electron microscopy analysis revealed that although the asl layer was present at the time of isolation, there was no asl layer following the perfusion of solutions necessary for the cbf measurements. this finding demonstrated that the activity of cilia was not impaired in the cf nasal airway epithelium due to the presence of a dehydrated asl layer. cbf frequency was also significantly reduced in three different codes of mouse ciliated nasal airway epithelial cultures derived from affected cf mice compared to those derived from non-affected wt mice. conclusion: we show that the absence of the cftr gene product in the cf knockout mouse nasal airway epithelium is associated with significantly decreased basal cbf compared to the non-affected wt and heterozygote mice. this observation potentially provides an additional mechanism as to why lung disease persists in human cf lungs. ml and mba contributed equally. clinical studies have linked increased sputum and peripheral blood neutrophil mpo activity with increased airflow obstruction in cystic fibrosis (cf) patients of the same age, gender, airway bacterial flora, and cftr genotype. variations in the tgf-β1 gene associated with increased tgf-β1 production have been linked to worse airflow obstruction in cf patients of similar age, gender and cftr genotype. we hypothesized that in the presence of mpo, tgf-β1 production would increase in airway epithelial cells. we obtained normal human airway epithelial cells (hae) and cultured them in trans-wells. tgf-β1 mrna was measured by pcr. tgf-β1 protein was detected by immunofluorescence staining and immunoprecipitation. we found increased tgf-β1 mrna after hae were exposed to mpo (at activities found in cf sputum) normalized to mrna of constitutive beta actin. under identical conditions, tgf-β1 protein expression was increased in hae. we conclude that neutrophil mpo present on the apical surface of cultured hae induces transcription and protein synthesis of tgf-β1. these results suggest that mpo induction of airway epithelial tgf-β1 is one molecular system linking the chronic neutrophilic endobronchitis seen in cf and subsequent airway wall fibrosis. are more oxidatively stressed than normal. as oxidative stress (os) is a potent activator of nf-κb, these data supported our hypothesis that cftr dysfunction causes alterations in the expression and/or modification of redox related proteins thereby enhancing activation of nf-κb. delineation of this interaction will produce good therapeutic targets. in this report we examine the role of os in cf inflammation. methods: to address our hypothesis, we used both in vitro and in vivo models of cf. our airway epithelial cell models are the 9hteo-pcep and pcep-r cell pair; and the 16hbeo-sense (s) and antisense (as) cell pair. additionally, we examined whole lungs from 8-12 week old b6.129s6-cftr tm2mrc mice (r117h mutants back-crossed into the c57bl6j background). proteins were prepared from homogenates of whole samples, run on 2-d gels, and their densities compared. the gel spots of differentially expressed proteins were excised, subjected to in-gel trypsin digestion, and identified by lc ms/ms. for biochemical analysis, we tested for the levels of h 2 o 2 , glutathione, and lipid peroxidation. we correlated these data with measurements of nf-κb activity (elisa and cytokine production), and the activity of nrf-2, an antioxidant (ao) response pathway transcription factor. we studied cultured cells in the presence and absence of inflammatory stimulation (tnf-α/il-1β), and compared non-stimulated cf mouse lungs to lungs from normal litter mates. results: our proteomic analysis revealed that in the absence of inflammatory stimulation, a paradoxical decrease in ao proteins exists in cf cells by 2 or more fold compared to matched pairs. this is despite a significant increase in intracellular os, which we confirmed by 3 different biochemical measures. activity of nrf-2 was decreased by ~30% in cf cells vs. normal. the data predict an increase in h 2 o 2 , which is a potent activator of nf-κb, and we confirmed both a significant elevation in h 2 o 2 (up 3-5 fold in cf) and a related significant increase in nf-κb activity, as assessed by elisa for nuclear p50, and il-6 and il-8 production. when we analyzed whole lungs from cf mice for antioxidant protein levels, we similarly observed decreases vs. normal littermates. when cells were stimulated, differences in protein expression and oxidative stress between cf and normal were further enhanced, corresponding to an excessive activation of nf-κb and increased production of il-6 and il-8. treatment with the antioxidants nac or selenium decreased the activation of nf-κb to normal levels. members of the clc family of proteins form either voltage-gated chloride channels or cl -/h + exchangers; nine subtypes are found in mammals. among these, clc-2 is one of the most ubiquitous variants, which is expressed in several of the types of epithelial cells that also express the cystic fibrosis transmembrane conductance regulator (cftr). it has been postulated that clc-2 may be a suitable alternative chloride pathway in these cells; however the contribution of clc-2 to chloride transport in these cells is poorly understood. this is partly due to the lack of appropriate pharmacological tools that are capable of specifically inhibiting clc-2. we describe here the isolation, from venom of the scorpion leiurus quinquestriatus hebraeus, of the first peptide inhibitor of any clc channel. this toxin, which we have named gatx2, specifically inhibits the clc-2 channel. gatx2 bears primary sequence identical to that of a toxin thought to serve as a k + channel inhibitor, although no molecular target for this toxin was previously known. a homology model of gatx2 reveals that it adopts a similar fold to other scorpion toxins. gatx2 was prepared via solid-phase chemical synthesis, and the synthetic toxin inhibits clc-2 with higher affinity than any other inhibitor of clc-2 or any other chloride channel. the k d for gatx2mediated inhibition of clc-2 in multichannel patches is only 11 pm at v m = -100 mv, and 80 pm at -160 mv. single channel experiments show that gatx2 does not alter single channel amplitude, but may alter channel gating. these experiments provide the basis for developing gatx2 into a useful tool that can be used to define the role of clc-2 in airway and intestinal epithelial cells. furthermore, the high affinity and specificity of inhibition suggest that gatx2 interacts intimately with the channel, and thus may the exploitation of alternative chloride channels to bypass the defect in cftr-mediated chloride secretion is an established therapeutic strategy in cf. in search of such potential therapeutic targets, we assessed camp/pkadependent chloride secretion across excised nasal epithelium of cftr null mice and wildtype controls, by measuring short-circuit currents (isc) in an ussing chamber set-up. all experiments were performed in the presence of amiloride to block the contribution of epithelial sodium channels to the isc. in cftr null mice of mixed background (c57bl/6;129), the camp agonist forskolin activated a bumetanide-sensitive rise in isc that was comparable in magnitude to the cftr-mediated isc response of littermate controls (118±30 vs. 90±21 µa/cm 2 , respectively). the forskolin-induced isc in these null mice was inhibited by the general chloride channel inhibitor dpc, but appeared insensitive to the cftr inhibitors cftrinh-172 and glibenclamide, the orcc and calcium-activated chloride channel inhibitor dids, and the clc-2 inhibitors cadmium and pre-activated omeprazol. moreover, the isc response to forskolin greatly exceeded the response to the calciumlinked agonists ionomycin and carbachol. intriguingly, in nasal epithelium of cftr null mice with a different genetic background (fvb), no such forskolin-inducible secretory pathway was evident (cftr-/-: 7±4 vs. cftr+/+: 98±23 µa/cm 2 ). when, in nasal epithelium of cftr null mice (c57bl/6;129), the basolateral membrane electrical resistance was negated by nystatin treatment, and in the presence of a transepithelial chloride gradient, forskolin failed to elicit a rise in isc, suggesting that camp/pka signaling targets a basolaterally located ion transport system. indeed, in intact epithelium, addition of chromanol 293b, a selective inhibitor of camp/pka-activated potassium channels, to the serosal bath, blocked the forskolin-induced rise in isc. we tentatively conclude that the hyperpolarization of nasal epithelial cells, which ensues from camp/pka-induced potassium efflux across the basolateral membrane, drives chloride exit across the apical membrane via a constitutively active anion conductive pathway that is absent from fvb cftr null mice. rt-pcr and immunostaining demonstrated that, among the candidate anion channels that fit the above pharmacological profile, clc-ka, but not clc-kb, was expressed in nasal epithelium of both cftr null mice and controls (c57bl/6;129), together with the accessory protein barttin. importantly, immunostaining suggested a strong increase in clc-k protein abundance in the cftr null mice, as compared to wildtype animals. on the basis of these findings, we postulate that clc-ka channels may serve as cftr bypass channels in nasal epithelium of cftr null mice that, albeit indirectly, are responsive to camp/pka signaling. future studies aim to characterize clc-ka and barttin in the airways from cf patients and controls, and to identify the post-transcriptional mechanism resulting in the up-regulation of clc-ka protein in cftr null mice. in the disease cystic fibrosis (cf), the most common mutation f508del results in endoplasmic reticulum retention of misfolded cf gene proteins (cftr). the endoplasmic reticulum (er) f508del-cftr protein retention is dependent upon chaperone proteins, many of which require ca 2+ for optimal activity. an increase in cytosolic free ca 2+ concentration is dependent on the activity of ion channels in the membrane of er and on extracellular ca 2+ influx. this influx, named capacitative ca2+ entry (cce), induced by calcium store depletion, represents the major ca 2+ influx mechanism in cells (putney et al., 1986) . during the cce, ca 2+ influx can be mediated by one or several of the 7 isoforms of transient receptor potential canonical channels (trpc) (putney et al., 1986) . aims & methods: in the present work, we studied the cce in human cf tracheal gland serous cfkm4 cells compared to non cf human tracheal mm39 cells. for this, we characterized the molecular identity of trpc channels responsible of cce by rt pcr technique and we compared global ca 2+ influx in cf and non cf cells by using fluo4-am probes. moreover, we measured the single channel activity of plasma membrane trpc channels by cellattached patch-clamp configuration in cf and non cf cells. we also studied the cce in cf cells after correction of the abnormal f508del-cftr trafficking by miglustat (2h at 100 µm of n-butyldeoxynojirimycin, nb-dnj) (norez et al., 2006) and low temperature (24h at 27°c) (denning et al., 1992) . results: we detected by rt-pcr technique the presence of trpc1 and trpc6 in both cell lines and we observed that cce was increased 2-fold in cf-km4 cells compare to non-cf mm39 cells. following stimulation by histamine, we recorded elementary ca 2+ currents with similar amplitude (0.5pa at -60mv) and conductance (8ps) whatever the cells tested (cf-km4 or mm39 cells). however, we observed a 2-fold increase of the npo (number of channels in each patch × open probability of single channel) in cf cells compare to non-cf cells. in f508del-cftr corrected-cf cells, the ca 2+ influx stimulated by the ca 2+ stores depletion was reduced to a similar level to non cf mm39 cells. the permanent contact of the airway epithelium with the external milieu induces frequent injuries caused by inhaled pathogens and particles. regeneration of the wounded airway epithelium needs to respect its prior structure in order to restore its defence functions. the underlying molecular mechanisms associated with the regeneration of the human airway epithelium are poorly described. the aim of our study was to determine, using a global expression approach, the transcriptional profile of human airway cells during the epithelial regeneration process. human airway epithelial cells collected from nasal polyps were seeded on type iv collagen-coated porous membranes, cultured up to confluence in liquid-liquid(ll) conditions, then put at the air-liquid interface (ali) allowing epithelial mucociliary differentiation. rna were extracted from epithelial cells at 70% confluence in ll conditions (epithelial cell proliferation and migration stage, step i), after 5 days in ali conditions (beginning of epithelial cell differentiation, step ii), when first ciliated cells appeared (step iii) and when cultures were completely differentiated (step iv). our results demonstrated that between step i and step ii, 127 genes were significantly modulated (ratio r<-3 or r>3, p<0.001), among them 74 were activated and 53 were repressed. between step ii and step iii, 127 transcripts were up-regulated (tubulin, dynein, stath, microtubule associated proteins…) and only one gene (fatty acid desaturase-1) was repressed. finally, between step iii and step iv, except stath which was~12-fold down-regulated, we observed only few genes differentially and slightly modulated. all along the regeneration process, between step i and step iv, 305 genes were significantly modulated with 84% of activated and 16% of repressed genes, among them a family of genes encoding proteins involved in the ciliary differentiation (foxj1, tektin, dynein, tubulin, …), extracellular matrix proteins (collagen, laminin, …) and inflammatory-related genes like cytokines, growth factors and their receptors (ccl20, vegfc, csf3, il32, il13ra2…). by elucidating the specific transcriptome involved in each step of the human airway epithelium regeneration, our study could allow to better understand the different cellular and molecular mechanisms, as well as the chronological events involved in these processes, and will further be applied to the study of the regeneration process in cystic fibrosis airway epithelium. supported by french association vaincre la mucoviscidose. electrolytes may be transported either through cells, the transcellular pathway, or between cells, the paracellular pathway. most research has focused on the channels and transporters of the transcellular pathway, while much less is known about paracellular transport. paracellular transport is regulated by tight junctions. claudins, the main component of tight junctions, are necessary and sufficient for tight junction formation and are important in establishing ion selectivity. the goal of this study was to further investigate the role of tight junctions in human airway epithelia. real-time pcr identified claudins 1, 3, 4, 7, 10, and 16 in primary human airway epithelia. immunocytochemistry localized claudins 1, 3, 4, and 7 to tight junctions. to measure the relative ion selectivity of tight junctions, we studied well-differentiated human airway epithelia in ussing chambers. to minimize transcellular transport, we used cf epithelia, which lack cftr and blocked enac with amiloride. we found the pna/pcl is 1.55 ± .34, suggesting tight juctions are more permeable to cations than anions. the relative cation selectivity was na+ > k+ > cs+ > tma+ > mg2+ and the relative anion selectivity was cl-> br-≥ i-> no3-> gluconate-. we compared these in vitro results to in vivo studies in people with cf and measured nasal voltage in the presence of amiloride. we found the pna/pcl is 1.2 ± 0.1. tight junctions were slightly more selective for cations than anions in vitro and in vivo. to determine the effect of specific claudins on the selectivity of the paracellular pathway, primary human airway epithelia were infected with adenovirus expressing claudin 4 or gfp. epithelia overexpressing claudin 4 had a higher transepithelial resistance (ter) and the pna/pcl was 1.11. however, infection efficiency was only 22%. this lead us to investigate if transformed human airway epithelial cells lines may be a better model to study permeability. cufi cells also express claudins 1, 3, 4, 7, 10, and 16. furthermore, like the primary human airway, cufi cells are more selective for cations; pna/pcl = 1.38 ± .14. future studies will investigate the effect of claudin expression on the paracellular permeability of these cells. the results will help identify the properties of a pathway critical to epithelial transport and barrier functions. altered adenosine (ado) signaling is associated with chronic lung disease and increased asl ado levels are thought to drive inflammation by stimulation of a2b receptors (a2b-r) in airway epithelia, which can activate nfκb. when measured by lavage, asl ado is ≤200 nm. due to its relatively low affinity to ado, a2b-r are thought to be inactive under normal conditions (ec50 is ~1.5 µm), and only stimulated during chronic lung disease. however, we have previously demonstrated that the ado-receptor (ado-r) antagonist 8-spt inhibits asl volume regulation, and that a2b-r is in close proximity to cftr. thus, we hypothesized that the asl ado concentration close to the apical membrane is higher than measured by asl lavage in bulk solution. we tested this hypothesis by looking for ado-r expression and function in well-differentiated human bronchial epithelia cultures (hbecs) and in vivo. by pcr analysis, we found that a2b-r was the only ado-r expressed in hbecs. we then measured asl height over 8 h ± ado-r antagonists by xz confocal microcopy when asl height was preset to ~5 µm at t=0. at 30 µm, caffeine is a non-specific ado-r antagonist, which inhibited asl volume regulation (8 h asl height was 4.1 µm vs. 8.2 µm for controls, n=6). similar inhibition was observed with alloxazine, which is relatively specific for a2b-r (n=6). in contrast, dpcpx, zm241385 & mrs241385, which inhibit a1, a2a & a3-r respectively had no effect on asl volume regulation. asl height regulation was also ablated by the addition of adenosine deaminase (ada) to the asl (n = 6). we next attempted to recapitulate the changes in asl height seen under control conditions, i.e. increase from 5 to 8 µm over the initial 2 h followed by a plateau (the remainder of the 8 h) by adding the non-hydrolyzable ado analogue neca to the asl in the presence of ada. 100 nm neca was insufficient to regulate asl volume and height remained at 5 µm over 8 h. in contrast, addition of 1 µm neca to the asl resulted asl volume regulation that was indistinguishable from control conditions (n=6), suggesting that near-membrane asl is much higher than has been measured by lavage. to confirm that a2b-r stimulates cftr chloride secretion, we measured transepithelial potential differences of hbecs mounted in ussing chambers. a dose-dependent increase in cl-secretion was observed by ado addition with an ec50 of 1.6 +/-0.09 µm, similar to the concentration of neca required to regulate asl height. to demonstrate which ado-r are expressed in human airways in vivo, we used laser capture to obtain ciliated cells from frozen tissue sections of excised donor bronchi. relative expression of a2b-r by qpcr was found to be 4.42 +/-0.30 fold higher than the a1, a2a-r, or a3 receptors (n=3). to confirm that ado-r activate cftr in vivo, we measured nasal potential differences. we found that ado activated chloride secretion at similar levels to isoproterenol. the effect of ado was inhibited by pre-exposure to 30 µm caffeine (ado, 20.2 +/-2.5 mv; ado + caffeine, 5.6 +/-4.9mv). we conclude that a2b-r has an important role in asl homeostasis through regulation of cftr and it is active in human airway epithelia under normal physiological conditions. through osmotic forces, hypertonic saline (hs) may increase the volume of cf airway surface liquid, restore mucus clearance, and improve lung function. the na + channel antagonist amiloride is predicted to increase the duration of action of hs. however, patients taking amiloride with hs showed less clinical benefits than patients taking placebo with hs. in vitro data suggested that amiloride inhibits transepithelial water flux (pf) in cystic fibrosis airway epithelia. we have extended this study by looking at the effects of amiloride on aquaporins in a heterologous expression system (mdck cells) and have used immunofluorescence to determine which aquaporins are expressed in human airways in vivo and in vitro. inhibition of enac by aprotinin pretreatment had no effect on hs-induced pf (n=6), suggesting that amiloride did not exert its actions on pf by inhibiting enac. however, since hs-induced pf was also inhibited with hgcl 2 , we speculated that amiloride directly blocked an aquaporin. to test this hypothesis, we measured the rate of cell shrinkage of wild-type mdck cells, and those stably-expressing aqp1 or 5, or expressing aqp3 (after 24 h exposure to a mildly hypertonic media; 400 mosm). following mucosal exposure to a 450 mosm hypertonic ringer solution, mdck cultures grown to confluency on permeable filters shrunk by 8% after 30 s (n = 6), as measured by xz confocal microscopy following staining with calcein-am. in contrast mdck/aqp1 or mdck/aqp3 cultures shrunk by 20% over the same period and mdck/aqp5 cultures shrunk by 35% (all n=6). a 10 min pretreatment with 200 µm amiloride abolished hypertonic ringer-induced cell shrinkage in mdck/aqp5 cultures (93% at 30 s; n=6) but had no affect on cell shrinkage in aqp1 or aqp3 expressing cultures. amiloride is fluorescent and can be imaged in epithelia by xz confocal scanning under uv excitation. amiloride was not internalized immediately (<10 s) before hypertonic ringer addition and did not block aqp5-mediated cell shrinkage (69% at 30 s; n = 6). however, a 10 min pretreatment with amiloride caused it be internalized and at this time aqp5-induced cell shrinkage was blocked, suggesting that amiloride blocks aqp5 intracellularly. to confirm that aqp5 is present in airway epithelia, we performed immunostaining with an aqp5 antibody and found that aqp5 is highly expressed at the apical membrane of ciliated cells in human bronchial tissue and well-differentiated bronchial cultures. we conclude that amiloride blocks aqp5, which is abundantly expressed in the superficial airway epithelia. supported we have previously observed that cftr, annexin 1 (a1) and cytosolic phospholipase a2 (cpla2) are partially recruited in detergent insoluble microdomains (dim) upon proinflammatory stimulation. this is prevented by specific inhibition of cftr. a1 participates in the regulation of inflammation by inhibiting cpla2 activity. we have also shown that a1 expression is decreased in cftr-/-mouse tissues. we postulate that cftr, a1 and cpla2 participate in the regulation of inflammation by their dynamic interaction within dim. in this study we aimed to (i) demonstrate and characterize this interaction, and (ii) identify other potential partners of this complex in dim. by coimmunoprecipitation, we examined the interaction between cftr, a1 and cpla2 in human pulmonary epithelial cells calu3. to search for additional partners we developed a new proteomic approach based on double sds-page (dsds-page), which is compatible with membrane protein analysis. calu3 cells and human epithelial bronchial cfbe cells (expressing either wt or f508delcftr) were subjected or not to proinflammatory conditions (tnfα). dim were isolated by density gradient and analyzed by dsds-page. cftr immunoprecipitated with a1 and cpla2 in calu3 cells. dsds-page analyses showed significant differential expression of cytokeratin 18, actin and protein disulfide isomerase (pdi, involved in protein folding) in dim between control and proinflammatory conditions in calu3 cells, and between wt and f508del cfbe cells, as determined by mass spectrometry. in conclusion, cftr, a1 and cpla2 interact in basal conditions. proinflammatory treatment causes differential expression of cytoskeletal proteins and pdi depending on the expression of normal and mutated cftr. this work suggests that cftr could participate in the regulation of inflammation within a complex with a1 and cpla2. cytoskeletal proteins and pdi could be involved in the dynamics of this complex. this could be related to the abnormal inflammatory response characteristic of cf. we acute and chronic inflammation, common features of cf airways disease, have been linked to higher atp release and changes in atp metabolic patterns in vivo, e.g., in induced sputum and exhaled breath condensate from cf patients and bronchoalveolar lavage fluid from inflamed lobes (esther, ped pulm sppl 29, 2006) . we, therefore, investigated the contribution of airway epithelia per se to the raised atp concentrations on cf airway surface during inflammation. when exposed to supernatant of mucopurulent material (smm) from cf airways for 48 h, or infected with respiratory syncytial virus (rsv) 72 h prior to the study, basal atp release from well-differentiated primary human bronchial epithelial (hbe) cultures did not differ from that of control cultures. however, basal atp release was increased 6 weeks after rsv infection when inflammation had waned (as indexed by il-8 secretion). the increase in basal atp release correlated with increased basal udp-glucose and mucin release, and goblet cell metaplasia. these data indicate that the increased basal atp release following rsv infection reflected increased goblet cell secretion. a different pattern was observed for stimulated atp release. hbes exposed to smm for 48 h, or infected with rsv 72 h prior, released 2-3 times more atp following hypotonic challenge than control cultures (okada, ped pulm sppl, 2006) . the increase in peak atp concentrations correlated with the degree of inflammation as indexed by il-8 secretion and the increase in intracellular calcium (ca 2+ i ) mobilization in response to hypotonic challenge, but not with the goblet cell number. ca 2+ i mobilization in response to hypotonic challenge in control hbes was almost completely due to extracellular ca 2+ i influx which was inhibited by antagonists of a transient receptor potential v4 (trpv4) ca 2+ channel. in contrast, an additional component of atp-induced ca 2+ i release from endoplasmic reticulum (er) was observed in smm-treated hbes. the increase in hypotonicity-induced atp release in smm-treated hbes compared to control hbes was almost completely sensitive to chelation of ca 2+ i by bapta (10 µm; 1 h), or inhibition of exocytosis by brefeldin a (5 µm; 2.5 h), monensin (10 µm; 18 h) or n-ethylmaleimide (1 mm; 15 min), whereas these reagents were ineffective against hypotonicity-induced atp release from control hbes. these data suggest a positive feedback loop between the increased atp release linked to ca 2+ i -dependent exocytosis and the increase in atp-induced ca 2+ i mobolization linked to increased er ca 2+ i stores (ribeiro, jbc 280, 2005) in inflamed hbes. in conclusion, we propose that basal and hypotonicity-induced atp release from hbes are regulated in distinct mechanisms during inflammation. basal atp release reflects continuous exocytosis processes independent of ca 2+ i mobilization and differs as a function of cell type (ciliated vs goblet cell), whereas acute inflammation upregulates hypotonicity-induced atp release via increased ca 2+ i mobilization and ca 2+ i -dependent exocytosis. we conclude that airway epithelia exhibit multiple mechanisms of increasing atp release into the lumen in response to inflammation. supported by cff grant okada06i0 to sfo and grants from nih. the main biological role of vitamin d is in control of bone and mineral metabolism, but it has also been discovered to have many immune system functions. vitamin d, a fat soluble vitamin, is poorly absorbed in cf leading to low serum levels. we sought to investigate the involvement of vitamin d in the control of inflammation in a cf model. methods cf tracheal epithelial cells (cfte) were pretreated with vitamin d (1, 25 [oh]2d3) or control and exposed to p aeruginosa lipopolysaccharide (lps) for 24 hrs before collecting the cell supernatants. cytokine levels in the supernatants were assessed by cytokine array and subsequent elisas specific for il-6, il-8 and mcp-1. vitamin d was measured in the plasma of 37 children with cystic fibrosis aged 3 months to 18 years. neutrophil numbers, neutrophil elastase (ne) activity and il-8 concentrations were measured in the bronchoalveolar lavage (bal) of these children. children with cystic fibrosis were deemed to be non-colonised, intermittently colonised or chronically colonised with p aeruginosa on the basis of the leeds criteria. pretreatment with vitamin d significantly reduced secretion of il-6, il-8 and mcp-1 from cftes after challenge with p aeruginosa lps in a dose dependant manner. plasma vitamin d levels were highest in the noncolonised group and lowest in the chronically colonised group with intermediate levels in the intermittently colonised group. bal neutrophil counts, ne activity and il-8 concentration showed a reciprocal pattern. conclusions vitamin d acts on cf lung epithelial cells to protect against excessive cytokine production in response to lps. levels of vitamin d are inversely related to both p aeruginosa colonisation status and bal markers of inflammation. low levels of vitamin d may contribute to advancing lung disease in children with cystic fibrosis. more aggressive correction of vitamin d deficiency may have a role in protecting against excessive inflammatory response to infection. sleeping in a mist tent has been used as a treatment for cystic fibrosis (cf) patients, in order to hydrate the viscous mucus and make it easier to be removed by coughing (matthews et al., 1968) . however, the efficiency of the method has been questioned, and its use was largely discontinued. with a new method to measure the ion content of human nasal fluid the effect of sleeping in a moisture tent on the ion content of nasal airway surface liquid (asl) in cf-patients and healthy controls was determined. the cf-patients and healthy controls spent a night (8h) in a mist tent, and samples of the nasal asl were taken before the experiment, after the period in the tent, and then at each hour during 4h after the persons had left the tent. samples of nasal fluid were collected on sephadex g-25 beads that had been mounted on strips of filter paper, which were then put into the nasal cavity of the cf patients or controls for 10-15 min. the strips were removed, and the beads were isolated, dried, and analyzed by x-ray microanalysis (vanthanouvong et al., 2006) . the concentration of na, cl, and k in the nasal fluid of cf patients was 132, 147, and 50 mm, respectively, before the patient entered the tent, significantly higher than the levels in the nasal fluid of the controls (na 118 mm, cl 107 mm, and k 19 mm). during the period in the tent, the ion content decreased, to levels of na 44 mm, cl 88 mm, and k 19 mm (cfpatients) and na 32 mm, cl 45 mm, and k 9 mm (controls) immediately after leaving the tent. after leaving the mist tent the ion levels in the nasal fluid increased, reaching after 4 h values of na 131 mm, cl 176 mm, and k 59 mm (cf-patients) and na 133 mm, cl 157 mm and k 37 mm (controls), which was for both groups higher than before entering the tent. no major changes in the ion content of the asl occurred after 4h. the salt content of the asl may be relevant in cf, since the asl is known to contain anti-bacterial proteins, defensins, which are sensitive to the salt concentration (bals et al., 1998) . hence, the higher salt concentra-tions in the asl of cf-patients may have negative consequences for the anti-bacterial defense system in the lungs, and conversely, the decrease in ion concentrations, caused by spending time in a mist tent, may have positive effects. (we have previously shown that none of our patients was chronically colonized with pseudomonas aeruginosa while regularly sleeping in mist tents.) however, with currently used procedures, the effect of sleeping in a mist tent on the ion content of the nasal asl is short-lived. bals background: antibody microarrays for clinical applications have been anticipated for more than a decade but the technology has only recently become sufficiently mature for reproducible, robust detection of low abundance proteins. the advantage of this new technology is the high throughput, parallel detection of identified, known, low abundance proteins. antibody microarray results are usually given in terms of ratios between two samples (e.g., "experiment" and "control"). however, given the printing and calibration issues inherent with antibody microarrays, this ratio'ing approach precludes statistically valid inter-array comparisons of the individual protein concentrations. we have therefore developed and implemented a different calibration strategy using a benchmark mixture that is applied to every array as an internal standard, thus controlling for any differences in antibody activities as well as printing imperfections. the amount of protein bound to each spot is determined relative to the respective protein in the internal standard. since all samples in the study are compared to the same internal standard, this semi-quantitative approach permits the comparison of multiple samples. all data for the same antibody on multiple arrays can then be used to calculate the average and standard deviation for a given population (such as patients or control groups) in a parallel fashion. methods: cf lung epithelial cultured cells ib3-1 and the cftrrepaired ib3-1/s9 were biosynthetically labeled with a 1 hour pulse of 35 [s]methionine, and then chased with cold methionine for 2, 4 or 6 hours. we then isolated total protein from each sample, and labeled the proteins with cy3. labeled proteins were applied to clontech® antibody arrays (507 antibodies printed in duplicates) according to the manufacturer's protocols. the internal standard, consisting of a mixture of cell cultures and tissue extracts known to contain the cognate protein for each antibody on the array, was labeled with cy5. the data were then used to compare the cf and the corrected cells in terms of amount, biosynthesis rate and degradation half-life (t 1/2 ) for each one of the 500 features on the array. data analysis: for each spot on the array the ratio of cy3/cy5 (after background subtraction) was calculated, normalized and stored for further analysis. for "pulse-chase" experiments the ratio of 35 [s] counts to cy3 at each time point gives a measure of the fraction of newly synthesized (radiolabeled) to total protein (dye labeled) bound to each given antibody on the array. the addition of the cy5 labeled internal standard enables the calibration of these totals, allowing the comparison all the spots for any given antibody on multiple arrays. furthermore, the internal standard calibration makes it possible to compare not only t 1/2 values but also to calculate the relative degradation rates in different cell lines even when the amounts of that protein are different in the "control" and "experiment" cells. the new approach quantitatively identifies and subjects to analysis the effect of the cftr mutation on protein biosynthesis and degradation of the cellular signaling proteome. supported by nih no1-hv-28287 (hbp) and ro1-53051 (hbp) in cf patients, defective apical chloride secretion, due to lack of functional cftr, leads to dehydration of airway surface liquid (asl) and mucus plugging. this defect is likely ameliorated, in part, by an alternative route of chloride conductance, the calcium activated chloride channel (cacc) . female gender appears to be associated with more rapid decline of lung function and earlier death in cf. this suggests that gender differences in hormonal environments might influence asl height regulation, thus modifying the pulmonary phenotype. we hypothesized that changes in estrogen concentrations might alter asl hydration by affecting cacc. consistent with this, we have shown that estrogen attenuates increases in intracellular calcium and asl height that follow purinergic receptor activation in primary cultures of human bronchial epithelial cells (hbecs, see accompanying abstract by hengrui sun et al). here, we report correlations between estrogen levels and nasal ion transport in vivo and further probe hormonal effects on ion transport and asl height regulation in vitro. in vivo studies: in 10 healthy, spontaneously menstruating females, nasal potential difference (npd) was measured (both nostrils) on two occasions during a single monthly cycle. based on a history of the 3 most recent menstrual cycles, we measured npd on a day when estrogen levels were predicted to be low (generally 8-9 days following onset of menses) and subsequently on the day when the pre-ovulatory estrogen surge was predicted. the recording electrode was positioned at the location where maximal stable basal pd was detected and changes in pd recorded continuously during sequential perfusion with 1] ringer's solution, 2] ringer's solution containing amiloride (to inhibit sodium absorption via enac), 3] modified (low chloride) ringer's containing amiloride and finally 4] a similar solution also containing utp (to activate cacc). as predicted, blood estrogen levels were higher on the "pre-ovulatory" day (0.44±0.01 vs 0.10±0.09 nm, p<0.001). utp-stimulated change in pd was significantly lower on the study day when estrogen levels were elevated (13.5±1.77 vs 18.9±0.7 mvs, p<0.05). further, the amiloride-sensitive component of basal pd was higher on this day (14.1±1.7 vs 9.4±2.8 mvs, p<0.05). the change in pd in response to perfusion with a low chloride solution was not different. in vitro studies: expression of estrogen receptor (er) α (but not β) was confirmed in non-cf and cf hbecs by real time pcr. erα was detected in hbecs from males and female donors. in cf and non-cf hbecs mounted in modified ussing chambers, exposure to 10 nm e 2 was associated with a significant (~25%) reduction in utp-stimulated short circuit current. e 2 inhibited the atp-stimulated increase in asl height in male and female hbecs and this asl regulatory phenotype was not reproduced by testosterone or progesterone. in conclusion, estrogen inhibits utp-activated chloride secretion in vivo and in vitro suggesting that elevated pre-ovulatory estrogen levels may further impair mucociliary clearance in females with cf. all data mean±sem. supported by cff leroy matthews award [1] . data demonstrating an upregulation of caccs when cftr is absent or defective [2, 3] have triggered further studies of these channels and prompted further elucidation of the functional (and possibly also physical) interactions between these two channels. although searched for long, the molecular identity of caccs is still under debate. the role of bestrophin family proteins as putative candidates for caccs is discussed controversially. bestrophin 1 (best-1) has been proposed to form ca 2+ activated clchannels in epithelial cells. moreover, caccs have been shown to support cell proliferation, namely in the development of cancer [4] . here, our goal is two-fold: 1) to investigate the correlation between expression levels of best-1 and epithelial cell proliferation; and 2) to pursue a search for interacting protein partners of best-1 to better understand the role and function of bestrophin(s) in epithelial cells. to study the correlation between best-1 expression and cell proliferation, we analyzed two populations of the t84 colonic carcinoma cell line with very different proliferation rates: the original (t84-slow) and the spontaneously transformed t84 cells (t84-fast). we observed that t84-slow cells have a small proliferation rate and express low amounts of best-1, while t84-fast cells express high levels of this protein. best-1 is spontaneously active in t84-fast cells. best-1-rnai inhibited ca 2+ activated clconductance and cell proliferation, therefore establishing a novel role of bestrophins in cell proliferation, which may be of relevance for the regeneration of the epithelia in cf and also for alternative therapies for cf. for the second objective, two putative cytoplasmic domains of best1, polyhistidine -tagged (phis) n-term (aa 1-30, best1-n) and c-term (aa 291-584, best1-c), were cloned into the pet-sumo bacterial expression vector. these domains were then immobilized onto metal-affinity resin to capture interacting proteins from human t84 whole cell and sub-cellular lysates. protein-containing fractions recovered from best1-n/c-coated and blank resins were subjected to 2d-gel and protein identification. it is expected that functional characterization of the interaction between the captured proteins and best-1 will give new insights into the biological role, plausibly of relevance to cf. work supported by dfg-sfb699-a7 grant (germany) and pluriannual funding of cigmh (feder-eu and fct, portugal extracellular nucleotides acting on epithelial cell surface purinergic receptors regulate the mucociliary clearance process that removes noxious materials from the lung. however, how epithelial cells release nucleotides into the airways remains largely unknown. cftr has been suggested to mediate atp release in airway epithelia, but conclusive demonstration remains elusive. calu-3 is a human airway derived cell line often described as a model of airway gland serous cells. at least 70% of cells in a monolayer expressed high levels of cftr protein and activity at the apical membrane. atp, adenosine, and udp-glucose, the naturally occurring agonists for the p2y2, a2b, and p2y14 receptors, respectively, were quantified in airway surface liquid with nanomolar sensitivity using radiolabeling and fluorescence hplc-based techniques, and real-time atp measurements. atp (and udp-glucose) was found to be released constitutively and selectively onto the mucosal surface of calu-3 cells. however, the specific cftr inhibitor 172 (10 µm, 10-30 min) failed to decrease atp concentration in the mucosal bath of resting calu-3 cells. forskolin (10 µm, 10 min) stimulated cftr channel activity, but did not affect basal levels of atp (or udpglucose) release. in contrast, elevation of intracellular calcium using ionomycin (1-10 µm, 1-10 min) significantly increased atp (and udp-glucose) release into the mucosal (but not serosal) bath of calu-3 cells. calciummediated nucleotide release enhancement was not abolished by cftr inhibitor 172. maneuvers like pre-incubation in nominally free-calcium solution, bapta (10 µm, 30 min), or cytochalasin d (5µm, 30 min) to disrupt actin cytoskeleton, which inhibited regulated exocytosis in calu-3 cells, also reduced calcium-mediated increase in nucleotide release. calu-3 monolayers presented a fraction of cells (<30%) exhibiting negligible cftr immunostaining signal, but displaying airway goblet cell mucin granules. remarkably, stimulation of atp and udp-sugar release with ionomycin resulted in concomitant enhanced exocytotic secretion of muc5ac. these results suggest that cftr is not involved in regulation of atp release in airway epithelial cells. more likely, nucleotides and nucleotide-sugars residing in the biosynthetic pathway involved in modification of apically targeted glycoconjugates (e.g. mucins) are released to the extracellular environment via calcium-triggered exocytosis. supported although the regulation of na+ transport is relatively well described in normal and cf airway epithelial cells, the impact of inflammation and infection, two major components of cf lung disease, on na+ transport has not been studied extensively. significant amounts of evidence suggest that bacterial by-products may influence na+ transport in airway epithelium. furthermore, the secretion of exoenzyme s by pseudomonas aeruginosa is enhanced during an exacerbation of lung disease in cystic fibrosis. in the past few years, our laboratory has shown that exposition of lung epithelial cells to p. aeruginosa lps leads to a rapid decrease in activity and expression of enac channel. the aim of our study was to evaluate the mechanism involved in the modulation of enac expression and activity by lps. primary culture of lung epithelial cells grown on filters were treated with lps (15µg/ml) for different time (30min, 1, 2, 4h). at each time point, the total and amiloride-sensitive short circuit current (isc) was determined in ussing chamber. in parallel, the cell proteins were collected to investigate by western blot the signalling pathways involved in this response to lps. a 4 hours lps treatment leads to a significant decrease in alpha and gamma enac mrna to 44% and 53% of control respectively. similarly, the amiloridesensitive current was decreased by 53% of the current in untreated cells after a 30 min exposure to lps. by western blot, we demonstrated the strong implication of the pi3k/akt pathway in response to lps in the regulation enac mrna and the potential implication of tyrosine/kinase receptors signalling pathway through mtor in the regulation of enac channel activity. in conclusion, we demonstrate that inflammatory molecules can modulate the expression and function of the na+ transport mechanism in lung epithelial cells and could contribute to the modulation of airway surface fluid in the chronic infectious process associated with cf. serous acinar cells are thought to be crucial for secretion of airway surface liquid by submucosal glands in large airways, but the molecular mechanisms of serous cell ion and fluid transport are not well understood. to address this, we isolated murine nasal serous acinar cells and studied them using simultaneous dic and quantitative fluorescence microscopy to track changes in cell volume and intracellular concentrations of ions involved in fluid secretion ([cl -] i ; spq) and its regulation ([ca 2+ ] i ; fura-2). previous results indicated that serous acinar cell volume changes were indicative of solute efflux (shrinking) and solute influx (swelling), reflecting the secretory state of the cell. acinar cells stimulated with 100 µm carbachol (cch) exhibited a rapid increase in [ca 2+ ] i and subsequent ~ 20 % cell shrinkage due to efflux of ~ 67 % of cell clcontent and parallel loss of k + and osmotically obliged h 2 o. because murine serous cells are sites of apical cftr expression in the airway, we tested the requirement for cftr in cch/ca 2+activated shrinkage/clefflux. acinar cells isolated from either cftr tm1unc-/mice or wt cells pre-treated with cftr inh 172 exhibited identical rates and magnitudes of ca 2+ -induced cell shrinkage and clloss compared to wt control cells. resting [cl -] i was also identical in wt and cftr tm1unc-/serous acinar cells (65 ± 4 and 66 ± 2 mm, respectively), suggesting similar levels of resting clconductance. thus, murine serous acinar cells can secrete kcl/h 2 o by ca 2+ -dependent mechanisms in the absence of functional cftr, in agreement with studies of intact glands suggesting cch-induced glandular fluid secretion is cftr-independent. wt serous acinar cells were stimulated with camp-agonists, including 15 µm forskolin, 100 µm isoproterenol, 200 µm adenosine, and 3 µm vip. however, no camp-induced shrinkage was observed, even in the presence of a cocktail of inhibitors to block solute uptake. to determine if cftr activation could enhance cch/ca 2+ -induced cell shrinkage at a sub-optimal [cch], serous acinar cells were pre-treated with forskolin followed by stimulation with 1 µm cch in the continued presence of forskolin. however, similar rates and magnitudes of cell shrinkage were observed in forskolin-treated and control cells. these results suggest that the magnitude of the cftr clconductance in murine serous acinar cells is much smaller than the ca 2+ -activated clconductance, below the resolution of these optical assays. more sensitive electrophysiological approaches are being used to determine the molecular identity of the non-cftr clconductance(s) and the role of cftr in murine serous acinar cell ion transport. females with cf exhibit more rapid decline in fev1 than males, which reduces their life span. normal airway epithelia regulate airway surface liquid (asl) volume and mucus transport by secretion through both cftr and the ca2+-activated cl-channel (cacc), and inhibition of either of these pathways in isolation does not abolish mucus transport. however, a loss of both pathways is predicted to result in mucus plugging which contributes to lung disease. despite a loss of cftr, cacc is functional in cf airways due to mechanical stimulation of atp release and activation of the p2y2 pathway which serves to raise intracellular ca2+. we hypothesized that e2 may inhibit cacc, leaving females with cf more prone to lung disease than males due to a greater depletion of asl volume. using confocal microscopy, we found that acute pre-exposure with e2 inhibited atp induced asl secretion in normal and cf human bronchial epithelial cultures (hbecs) from both male and female donors. the ic50 for e2-inhibition of atp-induced asl secretion was 0.75 nm. to better understand how estrogen receptors (ers) interact with the p2y2 pathway, we transiently transfected er-α or er-β linked to an orange fluorescent protein in an er null bhk cell line. with the co-transfection of ha tagged p2y2-r, we observed that e2 addition did not induce internalization of p2y2-r, neither did it alter the internalization induced by atp. we then measured total cell inositol phosphates (ip) in cf airway cells +/-e2 pretreatment. cell ip levels were not different between groups (n=9), suggesting that the effect of e2 is downstream of ip production. the investigation of the changes of the intracellular ca2+ (ca2+i) by recording fura-2 emission ratio over time in er-α or er-β expressed cells provided clues. after 10 nm e2 exposure, the fura-2 emission ratio in untransfected cells (n=7) or er-β transfected cells (n=7) increased by 0.6 following 10 um atp addition. this increase was significantly inhibited by~40% in er-α transfected cells (n=7), providing good evidence that er-α is respon-sible for inhibiting the p2y2 pathway. we next measured ca2+i in normal hbecs. while acute 10 nm e2 addition had no affect on basal ca2+i levels, which were around 370 nm, this maneuver reduced the ca2+i response to 70 nm following atp addition (both n=9), suggesting that e2 addition altered ca2+ signaling in response to atp. thapsigargin induces ca2+i release from the endoplasmic reticulum, but the magnitude of ca2+i release after thapsigargin addition was not altered by e2 pretreatment, indicating that e2 works further up stream to affect the p2y2 signaling pathway. together, these results suggest that e2 directly interacts with the p2y2 signaling pathway through er-α by altering ca2+i. we propose that this interaction leaves females more vulnerable to infections due to a reduced asl volume during periods of high e2, such as occur prior to ovulation. the nasal potential difference (or npd) is a sensitive, real time bioelectric assay of cftr-dependent and independent ion transport that is a commonly used study endpoint in early clinical stages of drug development. technical limitations of the assay must be controlled to facilitate its use in multicenter trials and limited intra-site variability. two forms of equipment are commonly employed to evaluate nasal potential difference in humans: silver-silver chloride electrodes (agcl) with saline bridges or calomel electrodes with agar bridges; however, these techniques have never been directly compared. we repeatedly evaluated (within 7 days) the basal potential difference of five normal subjects using the two available techniques, then compared variance and repeatability of the measures. basal pd was measured during perfusion of ringer's solution, and measured at 0.5, 1.0, 1.5, 2.0, and 3.0 cm within the inferior meatus, as indicated in the cf-tdn standard operating procedures. mean basal pd were within 0.8 mv using the two techniques, with very similar coefficients of variation (cvar; 0.36 vs. 0.32 in agcl vs. calomel, respectively). maximum basal pds were also similar between the two methodologies (mean difference 1.5 mv; cvar 0.32 and 0.28, respectively). the correlation between first and second measures of mean pd was highly reproducible, and similar between the two techniques (r=0.94 vs. 0.91 in agcl vs. calomel, respectively); however, when each individual basal pd measure was compared, correlation between first and second pd using the agcl device appeared superior (r=0.90 vs. 0.60 respectively; n=25 measures per device). comparison of values obtained in cf subjects are currently in progress and will also be reported. next, we examined diagnostic npd tracings in 12 cf subjects for whether the effect of amiloride might influence values obtained in the contralateral nare. an effect of cross-contamination by amiloride or other agent (gene therapy vectors or other nasal administered compounds, for example) could influence accurate measure of npd values. when basal pds were performed in both nostrils prior to amiloride exposure in either nostril, mean basal pd between nares remained stable (<1 mv, p=ns for change). however, if the basal pd in one nostril was measured after amiloride exposure in the contralateral side, mean pd depolarized by 12 mv (from -50 to -38 mv; p<0.01), indicating the presence of a 'cross-nostril' amiloride effect that might potentially affect interpretation of this endpoint if not performed in a sequence designed to avoid amiloride exposure. in summary, we show that calomel and agcl electrodes perform similarly in repeated measures from the same normal subjects, and that improper npd sequence can adversely affect basal pd measures. other potential impediments to the pd measurement (e.g. solution temperature, tonicity, ionic content, and sources of electrical offset) will also be discussed. these findings are relevant to the optimizing the use of the npd assay in clinical trials. supported by nih, cff, and cfft. the gene defective in patients with cystic fibrosis (cf) is the cystic fibrosis transmembrane conductance regulator (cftr), a camp-activated chloride channel in the apical membrane of epithelia, but alteration of expression and function of many genes undoubtedly contributes to the pleiotropic manifestations of cf. a hallmark of cf in airway epithelia is the hyperabsorption of sodium through the epithelial sodium channel (enac) in the apical membrane followed by extrusion through the na,k-atpase in the basolateral membrane. this results in airway surface liquid (asl) dehydration, mucus buildup and bacterial infection characteristically observed in the lungs of cf patients. previous studies have shown that the na,k-atpase is increased in cf airway epithelia, consistent with increased sodium transport along the enac/na,k-atpase axis. we have previously demonstrated that fxyd5, a member of a small family of proteins known to regulate the na,k-atpase, is increased in s489x cf mouse lung and nasal epithelia and in primary human tracheal epithelial cells (hte) after treatment with an inhibitor of cftr (172). recent evidence has indicated that enac activation occurs through modification of the serine protease-protease inhibitor balance in asl. thus, we hypothesized that enac-mediated increases in sodium absorption may be partially responsible for the increase in fxyd5 expression observed in cf airway epithelia. we now report that increasing asl volume on hte cells upregulated fxyd5 expression (p<0.01) after 24 hours, an effect that was further increased after trypsin addition and abrogated by the serine-protease inhibitor aprotinin. treatment of hte cells with nystatin also increased fxyd5 expression measured by quantitative rt-pcr (p<0.01). based on these results and the observation that mice overexpressing enac beta-subunit exhibit cystic fibrosis-like lung pathophysiology, we examined fxyd5 expression in the lungs of mice overexpressing the scnn1b-transgene. contrary to our expectations, fxyd5 lung expression was decreased as assessed by quantitative rt-pcr and immunoblot analysis (p<0.005), indicating fxyd5 may be alternatively regulated in epithelia under conditions of acute versus chronic sodium hyperabsorption. therefore, we measured fxyd5 expression in an epithelial cell culture model using an inducible, tri-cistronic vector capable of expressing enac alpha, beta and gamma subunits. we determined that fxyd5 expression is decreased after chronic enac induction. we conclude that fxyd5 expression is coordinately regulated based on acute versus chronic enac-mediated sodium absorption and suggest that fxyd5 is increased in cystic fibrosis airway epithelia as a result of an imbalance between increased inflammatory signaling and altered ion transport. picher, m.; van heusden, c. cystic fibrosis center, university of north carolina, chapel hill, nc, usa airway infection by the respiratory syncytial virus (rsv) is considered an important cause of pulmonary exacerbation and hospitalization in cystic fibrosis (cf) children. this disease is characterized by functional mutations in the cf transmembrane regulator (cftr). the inability of cftr -/-mice to clear rsv is associated with exaggerated inflammatory responses. a relationship was recently established between airway inflammation and chronically-elevated airway adenosine. this signaling molecule regulates key aspects of lung defenses, including bacterial clearance and inflammatory cells. high adenosine levels measured in bronchoalveolar lavage fluid of asthmatic or cf patients cause airway inflammation, remodeling and fibrosis in animal models. the objective of this study was to investigate the longterm effects of rsv infection on adenosine regulation and inflammatory responses in human airway epithelia. methods. polarized primary cultures of bronchial epithelial cells from healthy subjects and cf patients were exposed to vehicle, uv-inactivated or active green fluorescent protein (gfp)-rsv. the cultures were monitored 21 days for infection (fluorescence microscopy), adenosine levels (fluorescence chromatography), the release of cytokines regulating adenosine tnfα) and enzyme activities (ecto 5'-nucleotidase [ecto 5'-nt: amp ç adenosine] and adenosine deaminase 1 [ada1: adenosine ç inosine]). results. (a) both normal and cf cultures exhibited detectable rsv infection over 21 days, with peak density on day 3-4. (b) we demonstrate that rsv enhances the release of all five cytokines from normal cultures over 21 days. in contrast, cf cultures responded to rsv by a transient decrease in il-1β, il-4 and tnfα (peak on day 3), but a sustained decrease in il-12, and a sustained increase in il-13, secretion. (c) on normal epithelia, rsv transiently stimulated ecto 5'-nt (peak on day 3) but caused a sustained increase in ada1 activity, resulting in chronically-low adenosine levels. in cf, rsv raised adenosine levels by the combined effects of a sustained increase in ecto 5'-nt activity and the lack ada1 response. (d) chronically-elevated adenosine generated by ada1 inhibition [erythro9-(2-hydroxy-3-nonyl)adenine] stimulated the secretion of all cytokines, except il-12. conclusion. these data support a complex relationship between adenosine and cytokine regulation in human airway epithelia. the il-13-adenosine amplification pathways identified in rsv-infected cf epithelia are in agreement with animal models and their key role in chronic airway inflammation/remodeling. on the other hand, the data also suggest that ada1 activity is influenced by il-12, known to stimulate the expression of a major protein anchoring soluble ada1 to epithelial surfaces: cd26. altogether, this study suggests that an rsv infection in cf children may accelerate the progression of the disease by setting the stage for airway adenosine-cytokine amplification pathways. supported by the cystic fibrosis foundation (picher 05g0). cystic fibrosis (cf) airway submucosal glands are defective in mucus secretion rate (jayaraman, pnas, 2001, 98, 8119; joo, j biol chem, 2002, 277, 50710; joo, pediatr. pulmonol., 2003, suppl. 70, 281; wine, proc ats, 2004, 1, 47; thiagarajah, faseb j, 2004, 18, 875; song, ajp cell, 2005; salinas, faseb j, 2005, 19, 431) . one hypothesis is that loss of cftr results in reduced secretion of electrolytes leading to underhydration of mucins, thereby altering the mucus properties. it has been generally believed that the cf mucus in response to cholinergic agonists is thicker and more viscous. however, comparisons of pure gland mucus are difficult because of variability in gland secretion rates and a decline in mucus solids with repeated stimulations (wu, pediatr. pulmonol. 2006, suppl. 29, 252) . in the present study, we directly compared the solid content of airway submucosal gland mucus between control and cf individuals. human donor tracheas (hn) or disease control bronchi (dc) were dissected and mounted as previously described but without mineral oil. tissue was kept warmed and humidified in a chamber with two layers of saturated 37 degree c 95/5% co 2 /o 2 water vapor. the mucosal surface was blotted dry and coated with a thin layer of dimethylpolysiloxane to prevent re-absorption by the surface epithelium. the tissue surface was covered with a cover slip, leaving minimal air space between it and the surface to further reduce evaporation. a 12 µl droplet of 2% nacl was placed at the side of every tissue as a standard to assess evaporation and measurement error. any basal secretions were removed prior to stimulation with 10 µm carbachol. mucus secretion from individual glands was pooled using forceps and then drawn into a constant bore microcapillary (drummond) that had been alcohol-cleaned, dried, and weighed. stimulation time was allowed to vary so that at least 2 µl of mucus was collected. after mucus collection, both the standard and the bath solution were refreshed. mucus filled capillaries were weighed with a mettler microbalance before and after overnight 80 degree c oven drying to obtain nonvolatile solid content. the average solids measured in six repeated measures of 2% salt solutions was 2 ± 0.2 % with no trend over time. results for hn (n=5) and dc (n=3) did not differ and were pooled as 'control'. the initial control secretion yielded 6.5 ± 0.8 % solids (mean ± sem, n=8 subjects) and required 24 ± 3 min to produce adequate mucus. the initial cf secretion gave 9.1 ± 0.5 % solids (n=4) in 31 ± 2 min. the second control secretion yielded 5.6 ± 0.5 % solids (n=8) and required 40 ± 5 min, whereas the second cf secretion gave 7.4 ± 0.9 % solids (n=4) and required 56 ± 7 min. in summary, cf gland mucus solids content was 40% higher than control following the first stimulation (p = 0.05). after the second stimulation, cf solids content was 32% higher than the control (p = 0.08). solids contents of controls observed in our experiment are much higher than was found for mucus collected from airway lumens after 2 hr stimulation (c. 2%, trout, am j physiol, 1998, 274, l258) . supported by niddk ro1-51817 (jjw) and cff. in patients with cystic fibrosis (cf) as well as in some animal models of cf lung disease, higher levels of prostaglandin e2 (pge2) have been detected compared to control subjects. it is not clear whether this excess of pge2 contributes to the airway pathology of cf, or whether it may serve a protective function against airway constriction. inhaled pge2 has been tried in asthma and may be of benefit as a bronchodilator. interestingly, pge2 has shown benefit in aspirin-sensitive asthmatics, a population that shares some features with the cf airway phenotype including nasal polyposis. the mechanism of airway relaxation to pge2 is mediated primarily by ep2 receptors signaling through camp. in cf, beta-adrenergic bronchodilators are commonly used, but are often only partially effective at reversing bronchospasm. beta-agonists and pge2 both signal through g-proteins coupled to camp and pka signaling, thus pge2 may offer another treatment when beta-agonists are ineffective at reversing bronchospasm in cf. in mouse tracheas, the beta-agonist isoproterenol (iso) will partially relax a pre-contracted airway, but 47 ± 2% of contractile force remains (n=13). in contrast to the limited relaxation with iso, constricted mouse airways demonstrate significantly greater relaxation to pge2, with only 18 ± 3% of force remaining (n=9, p<0.001 compared to iso). this difference in relaxation responses suggests different intracellular signals are activated by each agonist despite both signaling through pka. to investigate phosphorylation events in response to iso or pge2, we developed a new method for perfusing isolated mouse lungs. using this technique, tissues were perfused with buffered solution containing 32p for radiolabeling. lungs were then stimulated with either iso or pge2, and phosphoproteins were compared from each condition. radiolabeling was effective under all three conditions. targets were chosen from each group (unstimulated, iso, or pge2 treated) that showed phosphorylation or dephosphorylation, and proteins were identified by mass spectrometry. over 20 differentially phosphorylated proteins were identified, and two of these (rho gdi 1 and inositol-requiring protein 2) were selected as candidates to be tested in the context of relaxation to pge2 compared to iso. by identifying signaling targets involved in airway relaxation to pge2, it may be possible to activate beneficial airway relaxation responses without the limitations seen in iso relaxation and without activating pge2 responses other than relaxation that may link pge2 to the airway pathology seen in cf. the parental ∆f508/∆f508 cf bronchial epithelial cell line (cfbe41o-) was compared to clones of cfbe41o-complemented with 6.2kb wild type (wt) or 4.7kb ∆f508 cftr cdna in terms of their ion transport characteristics. the transepithelial resistance (rt) (~310 (cm2) observed in the parental cfbe41o-was maintained in the complemented cells. two clones complemented with wt cftr cdna (c7-6.2wt and c10-6.2wt) demonstrated rt ~315 ωcm2, while another clone (c4-4.7∆f) complemented with the ∆f508 cftr cdna showed an rt of ~340 ωcm2. two stable clones expressing wt cftr were selected based on electrophysiological characteristics and stability of cftr expression. forskolin-stimulated cl secretion in clone c7-6.2wt was on average 13.4±2.5 µa/cm2 and 41.3±25.3 µa/cm2 in clone c10-6.2wt. furthermore small camp-stimulated transepithelial current of 4.7±0.7 µa/cm2 was observed in clone c4-4.7∆f indicating that ∆f508 cftr can traffic to the plasma membrane if present at high enough levels at physiological temperatures. the role that cftr plays in ca-activated cl conductance was investigated following treatment of the cells with atp in parental cfbe41o-and wtcftr6.2-cfbe41o-monolayers. in symmetrical cl-containing solutions, mucosal application of atp (100 µm) elicited a small, transient cl secretory response in the parental cfbe41ocells (8.5±2.1 µa/cm2), while the magnitude of the atp-stimulated cl current was markedly enhanced in cftr-corrected cfbe41o-cells with peak currents at 29.8±6.4 µa/cm2. recordings done in presence of a serosal-tomucosal cl gradient to increase the driving force for cl secretion showed a transient atp-dependent activation of cl currents in parental cfbe41ocells with peak cl currents of 33.3±3.8 µa/cm2 (n=30). cftr corrected cfbe41o-cells showed a sustained activation of cl currents of similar magnitude (42.7±10.5 µa/cm2; n=16). atp-stimulated cl currents were effectively blocked by the cftr inhibitor glyh101 (20µm). histochemical analysis showed prominent epithelial characteristics in all clones. these observations indicate that i) the apical driving force is limiting for atp-stimulated cl secretion and that ii) cftr participates in the cl secretory response to atp. furthermore, the findings suggest that these novel clonal isolates of the cfbe41o-bronchial epithelial cell line can be useful for the investigation of cf therapies. this work was supported by grants from the cff, cfri, nih, elizabeth nash foundation, and pacfi. the lactoperoxidase (lpo) system functions in normal airways to prevent infection and may be compromised in cystic fibrosis (cf) due to defective transport of its substrate thiocyanate. a computational model of normal airway surface liquid suggests that hydrogen peroxide levels regulate lpo system activity and that the lower thiocyanate in cf airways may result in elevated levels of hydrogen peroxide that is likely produced by duox the major nadph oxidase in airway epithelial cells. to investigate the regulation of duox by bacterial products, passage 1 normal human airway epithelial cells were grown and redifferentiated at the air liquid interface and treated for 12, 24 or 48h with either pseudomonas aeruginosa flagellin or lps, applied apically. treated and mock treated cultures were analyzed for levels of duox and lpo mrna and protein, and for hydrogen peroxide production. quantitative pcr was performed using taqman kits, protein was measured by western blots, and hydrogen peroxide was quantified using amplex red-hrp coupled fluorescence. the data showed the duox 2 and lpo mrna but not duox 1 mrna, were significantly upregulated at all measured time points following apical treatment of the cultures with purified flagellin compared to mock treated. at 24 h duox 2 was increased 7.7 +/-3.8 fold and lpo was increased 2.3 +/-0.7 fold (means +/-s.e.m., n = 5 lungs, 1-3 cultures of each lung). muc5ac mrna was also upregulated by flagellin treatment (4.9 +/-1.6 fold, n = 5 lungs, 1-3 cultures of each lung). apical treatment with lps had no effect on any tested mrna and induced minimal il-8 secretion in these cultures. western blots using anti-duox antibodies (courtesy of f. miot, brussels, belgium) showed corresponding increases in duox protein following flagellin treatments. hydrogen peroxide production by flagellin treated cultures was increased in proportion to increases in duox 2 mrna. application of flagellin alone to the apical surface of previously untreated cultures did not increase hydrogen peroxide production. thus, pseudomonas flagellin up-regulates duox 2 and results in higher activity in normal human airway epithelial cells perhaps serving to increase innate host defense activity of the lpo system. chloride ion in phagolysosomes is an essential substrate for neutrophils to produce hypochlorous acid (hocl), an important bactericidal agent. we have previously shown that cftr was present in the phagolysosomal membrane of neutrophils and the cftr defect led to deficient chlorination of ingested pseudomonas aeruginosa (pa) (painter et al, biochemistry, 45:10260-10269, 2006) . these results suggested that chloride accessibility to the phagolysosomal lumen might be impaired in cf neutrophils thereby affecting neutrophil-mediated bacterial killing. to understand the role of chloride ion in the process, we first assessed the effect of extracellular chloride concentration on pa killing by normal neutrophils under iso-osmotic conditions. surprisingly, bacterial killing was strongly dependent on extracellular chloride concentration. the initial rate of killing of pa was~2 fold less in a chloride-free medium than a physiological chloride medium. killing efficiency increased as the chloride concentration was raised in a dosedependent fashion and plateaued at~60 mm chloride concentration. to determine what percent of the chloride-dependent killing was oxidantdependent, we applied the cftr inhibitor glyh-101, the myeloperoxidase (mpo) inhibitor aminobenzoic acid hydrazide (abah) and the nadph-oxidase inhibitor diphenylene iodium chloride (dpi), respectively to bacterial killing assays. the results showed that the killing of pa could be divided into three discernible components: (1) a component accounting for about 20-25 % of the total killing rate that was inhibited by glyh-101, abah or dpi; (2) a second component amounting to~25-30% that was chloride-dependent and partially sensitive to glyh-101 and; (3) a third accounting for~50% of the total killing activity that was not sensitive to oxidant inhibitors, or extracellular chloride concentration. dpi and abah had no effect on killing of pa in the absence of extracellular chloride suggesting that most of the oxidant-mediated killing activity toward pa was due to hocl. none of the inhibitors had any significant effect on phagocytosis. finally, the rate of pa killing by cf neutrophils was found to be significantly lower (30-40 %) than that seen for normal neutrophils and was comparable to the effect of glyh-101 on killing of pa by normal neutrophils. the data suggest that cftr plays a significant role in killing of pa by normal neutrophils and, when defective as in cf, may compromise the ability of neutrophils to efficiently kill pa. this research was supported by a cff grant to gw. 1%. the cf group demonstrated a significantly higher frequency of pseudomonas respiratory infections than the non-cf group. interestingly, no significant differences were detected in any infections from other systems including blood, sinuses, skin, wounds, oral cavity, bowel, eyes, peritoneal cavity, and urinary tract. moreover, the cf lung-transplant patients had significantly less time free from pseudomonas infections in the transplanted lungs. conclusion normal lungs transplanted into cf patients had significantly higher susceptibility to pseudomonas infections than those transplanted into non-cf patients, which strongly suggests that defective host defense mechanism(s) beyond the respiratory system contribute to cf lung pathogenesis. pyocyanin (n-methyl-1-hydroxyphenazine, pcn) is produced by pseudomonas aeruginosa and is found in pulmonary secretions of infected cf patients at concentrations up to 100 µm. pcn is a redox-active compound that generates superoxide and hydrogen peroxide in the presence of cellular reductants. since cftr was reported to be modulated by h 2 o 2 , we hypothesized that pcn, through its ability to redox cycle and to produce h 2 o 2 , would affect cftr-mediated cl transport. we therefore tested pcn and h 2 o 2 for effects on cltransport (transepithelial short-circuit currents in ussing chambers measured with serosal-to-mucosal clgradient) and cytosolic redox potential (imaging microscopy on cells transfected with a redox sensitive gfp, rogfp1) using cftr-corrected cf bronchial epithelial cells (wtcftr6. . under resting conditions, acute exposure of the apical epithelial surface to 100 µm pcn elicited a small clsecretory response (7.3±0.9 µa/cm 2 , n=10) that was completely blocked by the cftr inhibitor glyh101 (20 µm). in contrast, in presence of the camp-elevating agonist forskolin, pcn caused a time-dependent decline of forskolin-stimulated cftr clcurrents by 86%. exposure to h 2 o 2 (100 µm) elicited a more pronounced clsecretory response than pcn in resting cells and h 2 o 2 -stimulated clcurrents peaked at 47.4±5.8 µa/cm 2 (n=10). h 2 o 2 was less effective than pcn at inhibiting forskolin-stimulated cftr clcurrents and currents declined by 33%. both pcn and h 2 o 2 oxidized the cytosolic redox potential from a resting value of -270 mv by similar amounts (48±8 mv for pcn vs. 44±10 mv for h 2 o 2 ), but rates of oxidation were faster for h 2 o 2 (2.5±0.1 mv/min) than for pcn (1.1±0.2 mv/min). inhibition of camp-stimulated cftr clcurrents by oxidation was a linear function of the cytosolic redox potential, but pcn (2 µa×cm -2 ×mv -1 ) was more potent than h 2 o 2 (0.69 µa×cm -2 ×mv -1 ). cf bronchial epithelial cells homozygous for ∆f508-cftr did not show a clsecretory response to pcn or h 2 o 2 . these data suggest that cftr-corrected cf bronchial epithelial cells respond acutely to oxidative stress by turning on cftr activity probably as part of the innate host defense response and this mechanism is absent in cf airways. furthermore, prolonged exposure to pcn and h 2 o 2 is detrimental for the proper function of the camp-regulated cftr clconductance. we conclude that oxidative stress in p. aeruginosa-infected airways is a key factor that needs to be considered for successfully correcting cftr function in cf airways. supported by nih (nccam p01 at002620), cff (fischer07g0) the lung is continually exposed to environmental agents and pathogens. the lung epithelial lining fluid (elf) is first to encounter these agents and gsh is a major antioxidant found in this apical fluid. the cystic fibrosis transmembrane conductance regulator protein (cftr) is the only known gsh transporter that maintains lung elf gsh levels. cystic fibrosis (cf) patient have low levels of gsh in their elf and have copious and viscous mucus. hypertonic saline is used in cf patient to help clear mucous and improve lung function however the mechanism(s) by which it does so are poorly understood. the purpose of these studies was to examine whether hypertonic (3.0%) saline can modulate apical gsh levels and protect lung epithelial cells against the oxidant, t-butyl hydroperoxide (t-booh). we used human lung epithelial cell lines sufficient (c38) and deficient (ib3) in cftr. the cftr deficient ib3 cells have 40% lower basal levels of apical gsh as compare to cftr sufficient c38 cells. cells were exposed separately and in combination to 3% saline, and t-booh (100 um) for 48 hours. the cftr deficient ib3 cells were much more sensitive to t-booh-mediated oxidative injury as measured by lactate dehydrogenase (ldh) release. hypertonic saline exposure was associated with an increase in apical gsh levels in both cftr sufficient and deficient cells and decreased t-boohmediated oxidative injury in both cells lines. hypertonic saline increased gsh in the apical compartment, which appeared to be largely cftr mediated. we examined this issue in mice given a clinically used dosing regimen of 7% hypertonic saline nebulized twice daily and examined changes in elf gsh levels 12 hours after last treatment. mice receiving the hypertonic saline treatment had significantly higher gsh levels in their elf than untreated controls. this data suggests that cftr and gsh adaptive responses play an important role in lung's reaction to oxidants. we propose that factors, which interfere with the lung's capability to mount and maintain an adequate adaptive apical gsh response, may compromise and sensitize the lung to oxidative injury. (supported in part from nih grant hl075523). the absence of functional cftr in airway epithelia of cf patients leads to abnormal airway surface liquid, which favors chronic bacterial infection. this chronic infection in cf lungs is associated with an exaggerated inflammatory response characterized by abnormal cytokines secretion and massive lung neutrophils infiltration. we have previous recapitulated the abnormal inflammatory response in cftr-/-(cf) mice by repeated administration of pseudomonas aeruginosa lipopolysaccharide (lps) (ped. pulmonology, supp. 29, 2006) . we demonstrated that the bronchoalveolar lavage (bal) fluid of cf and heterozygous (het, cftr+/-) littermate control mice when compared to the bal from wt mice shows 1) higher numbers of neutrophils and 2) higher concentrations of 6 cytokines (il-1alpha, il-6, kc, mcp-1, gm-csf, m-csf) as assessed using multiplex analysis of 22 selected cytokines. of note, airway epithelial cells from cf patients are known to have elevated secretion of some of these cytokines. however to date, the contribution of immune cells to these abnormal cytokine levels has not been well characterized. this study aims to investigate the contribution of cf macrophages in this altered cytokine profile/response. we examined cytokine secretion from two different macrophage populations: alveolar macrophages (am) obtained from the bal fluid and bone marrow (bm)-derived macrophages differentiated in presence of m-csf. both macrophage populations were cultured in the presence or absence of lps, and the supernatants were tested for the concentration of the 6 cytokines that we had shown to be abnormal in vivo. these macrophage populations were examined from cf, het and wt mice. in absence of lps exposure, bm-derived macrophages from cf mice have lower levels of il-6 and kc compared to wt and het mice. in contrast, am of cf mice have higher levels of il-6 and kc than am of wt mice. this difference may be due to am's prior exposure to antigens in vivo whereas the bm-derived macrophages are a truly naïve population. interestingly, after lps stimulation, il-1a, il-6, mcp-1, kc, g-csf and gm-csf are higher in cf macrophages compare to wt and het mice suggesting that cf affected macrophages display an altered inflammatory response when compared to wt and het. the abnormal production of these cytokines was also detected at the transcriptional level using quantitative rt-pcr analysis. in conclusion, our data support the hypothesis that cftr-null macrophages may have an impaired cytokine profile at baseline as well as after stimulation. furthermore, cftr deficient macrophages may contribute directly to the abnormal inflammatory response in cf. (supported by cff, nih, niddk) cystic fibrosis lung disease is associated with an excess of free neutrophil elastase (ne) in the airway arising from persistent neutrophil inflammation. dx-890 is a potent small protein inhibitor of neutrophil elastase that could be a useful therapy for diseases involving neutrophilic inflammation such as cystic fibrosis and copd. dx-890 effectively inhibits ne activity in cf sputum sol (ic50 = 766 pm). however the physical barriers posed by cf whole sputum may prevent dx-890 accessing and inhibiting ne ensconced within the mucus. the mucolytic dnase acts by cleaving extracellular dna in mucus thereby reducing viscosity. however dna binds and inactivates ne and therefore dnase treatment of mucus results in an acute increase in active ne. the lipid portion of pulmonary surfactant acts as a lubricant and our group has previously shown that bovine lung extract surfactant (bles) increases the fluidity of whole sputum. ( aim we aimed to show that, by reducing the surface tension of sputum, bles would enhance the anti-elastase effect of dx-890 in whole sputum. methods whole sputum (0.2g per well in a 12-well plate) was pre-incubated with 20 µl saline as control, 20 µl dnase (100µg/ml) or 20 µl bovine lung extract surfactant (bles, 100 µg/ml) for 30 minutes at 37 °c followed by addition of 20 µl dx-890 (50 µm or 100 µm) for 1 h at 37 °c. the contents of each well were aspirated, added to 1 ml nacl (0.9%) and centrifuged at 10,000 g for 30 min. the resulting sputum sol was assayed for ne activity using the colorimetric substrate n-meosuc-aapv-pna. results ne activity in whole sputum was significantly inhibited by 50 µm dx-890 (p=0.0071) and 100 µm dx-890 (p=0.0009). 50 µm dx-80 inhibited significantly more ne when sputum was pre-treated with bles (p=0.0173) treatment of sputum with dnase caused a significant increase in ne activity compared to untreated sputum (p=0.0398) whereas treatment of sputum with bles did not have this effect. conclusion dx-890 effectively inhibits elastase in cf sol. pre-treatment of cf whole sputum with bles enhanced the anti-elastase effect of dx-890 and treatment of sputum with bles alone did not cause an increase in ne activity in contrast to treatment with dnase. dx-890 inhibitory capacity in whole sputum could be improved by co-treatment with surfactant which increases fluidity of sputum and could allow greater access of drugs to sputum components. bovine lung extract surfactant, bles™ was a kind gift from bles biochemicals inc., on, canada. dx-890 was a kind gift from dyax corp, ma who also funded part of this research. table 1 : results are summarised as neutrophil elastase activity µg/ml and show the mean and sem of n=27 experiments. cystic fibrosis lung disease is associated with an excess of free neutrophil elastase (ne) in the airway arising from persistent neutrophil inflammation. dx-890 is a potent small protein inhibitor of neutrophil elastase that could be a useful therapy for diseases involving neutrophilic inflammation such as cystic fibrosis and copd. dx-890 effectively inhibits ne activity in cf sputum sol (ic50 = 766 pm). however the physical barriers posed by cf whole sputum may prevent dx-890 accessing and inhibiting ne ensconced within the mucus. the mucolytic dnase acts by cleaving extracellular dna in mucus thereby reducing viscosity. however dna binds and inactivates ne and therefore dnase treatment of mucus results in an acute increase in active ne. pulmonary surfactant acts as a lubricant and our group has previously shown that bovine lung extract surfactant (bles™) increases the fluidity of whole sputum. ( we aimed to show that, by reducing the surface tension of sputum, bles™ would enhance the anti-elastase effect of dx-890 in whole sputum. methods whole sputum (0.2 g per well in a 12-well plate) was pre-incubated with 20 µl saline as control, 20 µl dnase (100 µg/ml) or 20 µl bovine lung extract surfactant (bles™, 100 µg/ml) for 30 minutes at 37 °c followed by addition of 20 µl dx-890 (50 µm or 100 µm) for 1 h at 37 °c. the contents of each well were aspirated, added to 1 ml nacl (0.9%) and centrifuged at 10,000 g for 30 min. the resulting sputum sol was assayed for ne activity using the colorimetric substrate n-meosuc-aapv-pna. results ne activity in whole sputum was significantly inhibited by 50 µm dx-890 (p=0.0071) and 100 µm dx-890 (p=0.0009). 50 µm dx-80 inhibited significantly more ne when sputum was pre-treated with bles™ (p=0.0173) treatment of sputum with dnase caused a significant increase in ne activity compared to untreated sputum (p=0.0398) whereas treatment of sputum with bles did not have this effect. conclusion dx-890 effectively inhibits elastase in cf sol. pre-treatment of cf whole sputum with bles™ enhanced the anti-elastase effect of dx-890 and treatment of sputum with bles alone did not cause an increase in ne activity in contrast to treatment with dnase. dx-890 inhibitory capacity in whole sputum could be improved by co-treatment with surfactant which increases fluidity of sputum and could allow greater access of drugs to sputum components. we would like to thank prof. f. possmayer, on, canada and bles biochemicals inc., on, canada for the bovine lung extract surfactant, bles™. dx-890 was a kind gift from dyax corp, ma who also funded part of this research. table 1 results are summarised as neutrophil elastase activity µg/ml and show the mean and sem of n=27 experiments. cf patients infected with p. aeruginosa (pa) have increased levels of proinflammatory cytokines, such as tnf-α, il-1β, il-6, and il-8; but have low levels of the anti-inflammatory cytokine, il-10 in their airways. the proinflammatory mediators in the cf airway recruit a large number of polymorphonuclear neukocytes (pmns), which release high levels of neutrophil elastase and human neutrophil peptides and damage the lungs. the intense neutrophil-dominated inflammatory response associated with chronic pa infection results in progressive airway obstruction, the cause of death of over three-quarters of cf patients. recent studies highlight the significance of sphingolipid metabolism in cell growth, differentiation, membrane traffic, apoptosis, senescence, and immune regulation. acid sphingomyelinase (asm) plays a critical role in sphingolipid metabolism by hydrolyzing sphingomyelin to ceramide. ceramide self-associates to form ceramide-enriched membrane platforms(also called lipid rafts). these platforms have been shown to be involved in the internalization of pa; induction of apoptosis and immune regulation. our in vitro and in vivo studies of asm confirm that there is a loss of induction of asm after pa infection in both cf bronchial epithelial cells and cftr knock out mice as compared with controls. in vitro, we examined the asm activity in human bronchial epithelial cells with normal cftr expression (s9 cells) and with mutant cftr expression (ib3 cells ) after pa infection at 0, 10, 30, 60, 180, and 300 min. asm activity was up-regulated in s9 cells following pa infection after 60 min and kept increasing until 300 min, while asm induction was lost in ib3 cells. asm protein and ceramide levels also increased in s9 cells following pa infection compared with ib3 cells. in our in vivo studies, cftr knock out mice and control c57bl/6 mice were both administered various doses of pa orally (2x106 cfu to 5x107 cfu) . six hours after pa infection, lung homogenates from the c57bl/6 mice indicated increasing asm protein and ceramide production, which corresponded to the dose of pa given. in contrast, asm protein and total ceramide production remained at baseline levels in the lung homogenates of cf mice regardless of the dose of pa given. rna interference (sirna) assays were performed by transfecting a 21 bp pre-designed sirna specific for human asm in s9 cells. these cells were then infected with pa for 5 hrs to see if they had similar il-8 level compared with infected ib3 cells. we demonstrated a significantly elevated il-8 expression in s9 cells tranfected with asm sirna compare with s9 cells transfected with a scramble sirna (p<0.005). our results suggest that the loss of asm induction, caused by mutant cftr, is involved in pa clearance and contributes to the exaggerated proinflammatory cytokine response in cf airway. we are conducting research on defining the roles of asm and the sphingolipids in pa infection; cell signaling; as well as cytokine induction, we are also looking at the therapeutic potential of aav.asm vectors in pa-infected cf bronchial epithelial cells and cftr knock out mice. work supported by nhlbi introduction: new therapies are critically needed to treat cystic fibrosis (cf) lung disease, which is characterized by chronic infection and inflammation. novel therapies to target airway inflammation are likely to improve the clinical outcome in cf. as yet there is no consensus regarding the molecular pathway(s) mediating inflammation in the cf lung. objective: to establish whether toll-like receptor (tlr) signalling is the key molecular pathway responsible for the inflammation seen in cf lung disease and to determine whether inhibitors of tlr signalling can reduce this damaging inflammatory response. methods and results: to quantify the airway inflammatory response, we used established cf and cf-corrected lung epithelial cells (ib3-1 and c38). since artefacts may alter the inflammatory phenotype of cell lines, we validated all findings with fresh peripheral blood mononuclear cells (pbmcs) from cf patients (n=23) and healthy controls. we measured the inflammatory response (il6, il8, tnfα) of these cells following stimulation with: (i) cf pathogens (p. aeurginosa, b. multivorans, b. cenocepacia); (ii) purified tlr ligands. both cf respiratory epithelial cells and cf pbmcs had a greatly increased inflammatory response compared to matched controls when stimulated with whole bacteria and purified tlr ligands (p<0.05). interestingly, the cf airway cells responded most vigorously to the tlr5 ligand, flagellin (p<0.001). to investigate tlr5 as a potential anti-inflammatory target, we found that tlr5 mrna (p<0.001) and protein (p<0.05) were expressed at a higher level in cf compared to cf-corrected lung epithelial cells. finally, to determine the contribution of tlr5 activation to cf lung inflammation we exposed the airway cells to wild-type (pak & pao1) and isogenic flagellin-deficient strains of p. aeruginosa (pak∆flic, pao1∆flic, pao1∆flge & pao1∆flim) . strikingly, the absence of the tlr5-flagellin interaction significantly reduced the proinflammatory response of cf respiratory epithelial cells to p. aeruginosa (p<0.001). we show that tlr-mediated innate immune responsiveness is increased in both cf respiratory epithelial cells and fresh blood cells from cf patients. moreover, inhibition of the tlr5-flagellin interaction markedly reduced proinflammatory response of cf respiratory epithelial cells following exposure to predominant cf pathogen p. aeruginosa. these data suggest that tlr5 activation may represent a novel anti-inflammatory target for improving cf lung disease. supported by the canadian cf foundation. cf airway cells (ib3-1) were stimulated with wild-type (pak) and flagellin-deficient p. aeruginosa (pak∆flic). introduction: genetic polymorphisms for tgf-β 1 have recently been identified as a significant modifier of cf lung disease severity. however, at present little is known about protein measurements of tgf-β 1 in bronchoalveolar lavage fluid (balf) or serum obtained from pediatric cf patients during an acute respiratory exacerbation. hypothesis: tgf-β 1 in cf balf and serum is increased in association with markers of cf lung disease severity, and is decreased after completion of iv antibiotic (iv abx) therapy. study design and methods: in a descriptive, prospective study of children with cf presenting for hospital admission to treat a pulmonary exacerbation, balf specimens were obtained from patients undergoing clinically-indicated flexible bronchoscopy on admission. balf was immediately analyzed for total and differential cell counts with the cell-free balf supernatant stored at -80°c. serum specimens were obtained at the initiation and conclusion of iv abx and similarly stored at -80°c. measurement of total tgf-β 1 utilized a commercial elisa kit. t-test statistical analysis assumed significance at p<.05. results: balf was obtained from 8 cf patients with a mean age of 8.5±1.9 years (range 1-16 years). three balf cultures were pseudomonas aeruginosa positive (psa+). balf tgf-β 1 levels ranged from 88-309 pg/ml (mean 168±26.4 pg/ml) with the highest tgf-β 1 measured in a patient with abpa and culture positive for m. abscessus. in balf, higher neutrophil counts (greater than the median, 2.58x10 6 pmn/ml) were associated with increased balf tgf-β 1 (214±31.9 pg/ml vs. 121±28.8 pg/ml, p<.05). serum tgf-β 1 was collected in 8 cf patients with a mean age of 7.5±2.3 years (range 1-18 years), four of whom were psa+. serum tgf-β 1 ranged from 21.4-99.7 ng/ml (mean 56.6±9.23 ng/ml). in both balf and serum, neither patient age nor psa culture status was associated with increased tgf-β 1 at admission. however, previous hospitalization in the last 12 months was significantly associated with an elevated admission tgf-β 1 in both balf (233±38.2 pg/ml vs. 122±23.8 pg/ml, p<.05) and serum (75.8±8.19 ng/ml vs. 37.3±9.09 ng/ml, p<.02). after completing the clinically-indicated course of iv abx, 3/4 psa-cf patients had a reduction in serum tgf-β 1 (mean 69.5±11.4 ng/ml to 48.1±4.52 ng/ml, p=ns). in contrast, 3/4 psa+ cf patients had an increase in serum tgf-β 1 (mean 43.7±12.5 ng/ml to 62.3±17.1 ng/ml, p=ns) after completing iv abx therapy. conclusions: tgf-β 1 is measurable in balf and serum from pediatric cf patients and varies in association with clinical parameters. these preliminary results support our hypothesis that increased tgf-β 1 may be associated with more severe lung disease as evidenced by 1) association of increased tgf-β 1 in balf and serum obtained from cf patients with previous hospitalization in the last 12 months, and 2) association of increased tgf-β 1 in balf with increased cellular inflammation. the direction of change in serum tgf-β 1 after iv abx therapy may be influenced by the presence or absence of pseudomonas infection. acknowledgments: supported by cff (harris07ao). paula murphy and cassidy henegar provided technical support. background: cystic fibrosis is one of the leading genetic causes of end stage lung disease for which the treatment is lung transplantation. however, mortality due to chronic rejection known as obliterative bronchiolitis (ob) remains high due to small airway obliteration. ob is associated with increased levels of matrix metalloproteinase-8 (mmp-8), an interstitial collagenase, expressed by polymorphonuclear cells (pmns). pmns are among the first inflammatory cells to be detected in ob but very little is known about the role of pmn mmp-8 or the mechanisms used by pmns to migrate through extracellular collagen matrices. objective: to identify the molecular mechanism for neutrophil recruitment into the airway. we hypothesize that mmp-8 promotes accumulation of pmns in ob lesions by promoting their migration through the extracellular matrix protein barriers by its pericellular collagenase activity. method: the heterotopic airway transplant model was used to study the role of neutrophil mmp-8 in promoting pmn migration through extracellular matrices. mhc-disparate balb/c tracheas were subcutaneously transplanted into c57bl/6 wild-type (wt) or mmp-8 deficient (mmp-8-/-) mice. to further dissect the mechanism of pmn migration in vitro their migration through collagen gels was studied. collagen gels were made in transwells using rat-tail collagen providing a highly cross-linked thick collagen barrier. bone marrow derived pmns from wt or mmp8 -/-mice were fluorescently labeled and stimulated to induce the surface expression of mmp-8. pmns were placed on thick collagen gels in traswells. the transwells had polycarbonate membranes at the bottom of the gels. the pore size of the membranes did not allow pmns to pass through into the lower chambers that had flmp a chemo attractant. at 4 hours and 24 hours the gels were fixed and the polycarbonate membrane at the bottom of the collagen gel was gently peeled off. pmns on the membrane were counted under a fluorescent microscope. results: significantly decreased migration of pmns into the airway lumen was noted in the mmp-8 deficient mice (p<0.05) 2 weeks post-transplantation compared to wt mice in the heterotopic airway transplant model. in addition there was more than a three-fold increase in the pmns outside the lumen in the extracellular collagen matix (p<0.02). wt-pmns were able to penetrate collagen barriers in greater numbers both at 4 and 24 hours compared to mmp8 -/-pmns in vitro (p < 0.002 for 4 hours and 0.001 for 24 hours). in addition, mmp inhibitor gm 6001-treated wt cells demonstrated decreased migration through collagen gels (4 hours p<0.008 and 24 hours p<0.008). conclusions: mmp-8 -/-mice has significantly less pmns in the airway lumen due to decreased migration through collagen matrices providing protection against ob. this has great significance for conditions such as cystic fibrosis, lung transplant rejection (obliterative bronchiolitis) and other inflammatory diseases mediated by pmns. mmp-8 blockade can potentially be a novel therapeutic target to decrease neutrophil efflux into the airways and thus reduce airway inflammation. background: our laboratory has demonstrated alterations in cf-related regulation of cholesterol processing. hydroxycholesterols (oxysterols) are oxygenated derivatives of cholesterol formed from autoxidation of cholesterol and from de novo synthesis. oxysterols are known to be key regulators of cholesterol metabolism and cellular signaling. the role of oxysterols in the cellular pathways involved in cholesterol processing in cystic fibrosis has not been elucidated. based on our previous findings of increased de novo cholesterol synthesis in cf tissues, we hypothesize that oxysterol production in cf would be positively impacted. increased oxysterol production may lead to abnormalities in cellular signaling associated with oxidative stress and may influence the inflammatory response in cf. the goals of this research are to investigate the effect of oxysterols on inflammation in cf and to determine if there is a difference in oxysterol production in cf compared to wild type controls. results: the goal of these studies is to determine if cf cells respond uniquely to oxysterols compared to control cells. initial studies demonstrate that cf-model 9/hteo-pcepr cells failed to respond to 25-oh cholesterol (25-oh) with regards to nfκb activation. control 9/hteo-pcep cells, however, exhibited a significant 2-fold increase in nfκb activity upon 25-oh stimulation (2.1-fold +/-0.1 increase from il-1, tnf alpha alone). however, il-8 promoter activation in response to 25-oh was not significantly altered in either cf or wt cells (2.5-fold +/-2.3 (cf) increase from il-1, tnf alpha alone, 1.8-fold +/-2.3 (wt) increase from il-1, tnf alpha alone). in order to verify il-8 promoter results, il-8 protein production was measured directly in response to pseudomonas aeruginosa. il-8 protein level in response to 25-oh was increased in cf cells (1.4-fold) and decreased in wt cells (1.2-fold), although not significantly in either group. oxysterol content and de novo synthesis are currently being examined in vivo in cf and wild type mouse models. mice lacking cftr on a c57bl/6 background and wild type controls were obtained from our animal core and the amount of oxysterol present in these mouse models is currently being studied. conclusion: these findings suggest that endogenous oxysterol production or other components of oxidative stress are pre-stimulating nfκb activation via the oxysterol-sensitive pathway. interestingly, although oxysterols robustly activate nfκb in wt cells, oxysterol-dependent pathways are not involved in il-8 production. these data suggest that il-8 is not a primary cause for increased oxysterol mediated inflammation. elucidating the role of oxysterols in cf will be an important contribution to our understanding of the inflammatory pathways in cf and will contribute to the growing body of evidence for aberrant cholesterol regulation in cf. this work is supported by grants from the cff and the nhlbi. cystic fibrosis (cf) is a common genetic disorder caused by a mutation in the cftr, a chloride transporter of the abc transporter family. innate immunity, the first line in host defense against infectious agents, is abnormal in cf patients. cf patients are commonly colonized with bacterial pathogens, which is the major clinical problem of this disease. despite a marked difference in the clinical outcome amongst cf patients infected with pseudomonas aeruginosa and burkholderia cepacia, the mechanisms under-lying these differences remain unclear. understanding the innate immune response, specifically the role of cftr, in p. aeruginosa and b. cepacia infections will lead to new insights into mechanisms that limit these infections. our objective is to develop a cf model in the simple organism, the worm, c.elegans, and to test the role of the cftr orthologue, mrp-4, in host defense. using this model, we have compared the survival of c. elegans between virulent p.aeruginosa (pa14) and b.cepacia (bc). our experiments have demonstrated that, when compared to growth on their natural food source, e.coli op50, worms fed on pa14 and bc die prematurely. specifically, worms survive up to 140 hours on op50, whereas pa14 fed worms die with in 40 hours. death on pa14 also requires active infection, as heat inactivation of these bacteria destroys their killing capacity. two bc strains have been tested, a virulent strain (25416) and an avirulent strain (17616). worms infected with bc show an intermediate phenotype, surviving approximately 100 hours. our future work will define whether synergy occurs during coinfection with p.aeruginosa and b.cepacia, as would be suggested by clinical data. in addition, we will define the transcriptional changes induced in c.elegans fed on pa14 and bc, and use these data to identify common and pathogen specific responses to these infections. finally, mrp-4 knock-down worms will be generated using rna interference, and the role of the cftr orthologue in infection will be tested. in conclusion, c.elegans offers a genetically tractable model organism in which the innate immune response to pseudomonas aeruginosa and burkholderia cepacia can be studied. in addition, this simple model system will allow us to define the role of the cftr orthologue in host defense. supported serine proteases released from neutrophils are considered central to the pathogenesis of cf lung disease, and have been obvious therapeutic targets. although intracellular serine proteases are important in host defense and bacterial killing, extracellular enzymes may contribute to bacterial persistence and promote airway inflammation. neutrophil elastase (ne) digests key opsonins present in the lung and has been shown to disrupt phagocytosis, allowing the bacteria to persist in the face of established pulmonary inflammation. in addition we have found that other proteases, like cathepsin g (cg), an abundant serine protease found in human and murine neutrophils, may also have other, critical roles in development of cf lung disease. indeed, using murine models of endobronchial inflammation, cg inhibits airway defenses and interferes with the host's ability to clear pseudomonas aeruginosa from the lung with effects quite distinct from ne. to test the hypothesis that differences in bacterial killing are due to defects in innate defenses created by excess, unopposed cg at the apical surface of the airway epithelium, we have examined profiles of proteins secreted into the airway lumen by epithelial cells, which are necessary for p. aeruginosa clearance and susceptible to cg proteolysis. analysis of lavage fluid utilizing 2-d page indicated a total of 88 spots which were increased in pooled lavage fluid from 6 infected cg-deficient mice as compared to 6 wild-type littermates. tandem mass spectrometry analysis identified several novel proteins that were cleaved by cg, including inter-alpha inhibitor protein, selenium-binding protein, sec14-like protein, beta-2-glycoprotein, and annexin a5. another protein candidate, serum amyloid p component (sap), is a novel opsonin and may be involved in airway defenses. immunoblot analy-sis demonstrates that cg cleaves sap. these results suggest that cg, like ne, plays an important role in the pathogenesis of lung diseases. we have previously demonstrated in ∆f508-cf mice that in vivo treatment with azithromycin (azm) attenuates cellular infiltration in baseline and lipopolysaccharide (lps)-induced inflammation, and inhibits proinflammatory cytokine release in induced inflammatory condition (legssyer et al, 2006) . this study aimed at investigating macrophages isolated from wild type (wt) or ∆f508-cf mice (van doorninck et al, 1995) and the effect of azm on these cells. purified peritoneal and alveolar macrophages were stimulated with lps (p. aeruginosa, 0.1µg/ml) plus ifnγ (0.1µg/ml), with or without azm (1µg/ml). macrophage inflammatory status was investigated by assessing different pro-and anti-inflammatory mediators at mrna level 6h after stimulation. pro and anti-inflammatory markers seemed to be reduced in naive alveolar cf macrophages. by contrast, a trend to overexpression of these markers was seen in naive peritoneal cf cells. pro-inflammatory status, as assessed by il-1β, was induced in stimulated alveolar and peritoneal cf macrophages and in naive peritoneal cf cells. moreover, nos-2 was found to be overexpressed in stimulated alveolar cf macrophages while il-10 was downregulated. azm significantly reduced il-1β expression in stimulated alveolar macrophages. in conclusion, peritoneal and alveolar macrophages exhibit distinct phenotype. a pro-inflammatory status was more prominent in stimulated cf alveolar and peritoneal cells. interestingly, azm modulates il-1β overexpression only in cf stimulated alveolar macrophages, supporting the antiinflammatory activity of this macrolide and identifying, at least partly, alveolar macrophages as possible target cells for its effects. supported it has been recently reported by perez and coll. that the inhibition of function of w/t cftr produces an inflammatory profile that resembles that observed in cf patients [am j physiol lung cell mol physiol, 2007] , whereas we demonstrated that correction of f508del cftr function with mpb-07 down modulates the pseudomonas aeruginosa (p.aeruginosa) dependent expression of the pro-inflammatory mediators il-8 and icam-1 in cf bronchial cells [am. j. resp. cell. mol biology, 2007] . since both evidence support a direct link between cftr function and inflammatory response in respiratory epithelial cells, we extended our investigation to other f508del cftr correctors, such as miglustat [norez et al, febs letters, 2006] , which is an approved drug for gaucher disease, in comparison with an isomer without any correcting effect, namely nb-dgj. human bronchial ib3-1 (cftr f508del/w1282x), cufi-1 (cftr f508del/f508del) and nuli-1 (cftr w/t) cells were exposed to the laboratory strain of p.aeruginosa (pao1) or tnf-alpha or il-1beta and the transcription of icam-1 and il-8 were quantitated by real time rt pcr. cftr function was assayed by single-cell fluorescence imaging, using the potential-sensitive probe disbac2 [renier et al, hum. gene ther. 1994 ]. analysis of binding of nf-kb and ap-1 transcription factors to labelled dna target was performed with electrophoretic mobility shift assay (emsa) [borgatti et al, j. biol. chem. 2003 ]. miglustat significantly reduced the expression of il-8 by 90% in ib3-1 and by 70% in cufi-1 cells and of icam-1 by 80% in ib3-1 and by 50% in cufi-1 cells, upon infection by pao1. in parallel, correction of f508del cftr function was observed after miglustat treatment in both ib3-1 and cufi-1 cells. miglustat had no major effects on overall binding activity of transcription factors nf-kb and ap-1, activated by pao1 in ib3-1cells. the inflammatory response induced by tnf-alpha or il-1beta was also significantly reduced in ib3-1 and cufi-1 cells treated with miglustat. nb-dgj, which is not a corrector of function of f508del cftr, down modulated the induction of il-8 and icam-1 in ib3-1 and cufi-1 cells. in addition, both miglustat and nb-dgj reduced the inflammatory response to pao1 in non cf nuli-1 cells. in conclusion, miglustat and nb-dgj have an anti-inflammatory effect in bronchial cells independently of the correction of f508del cftr, by interfering with the pro-inflammatory signaling downstream the receptors for pathogens and pro-inflammatory cytokines. since miglustat is already approved for the treatment of gaucher disease and other glycosphingolipidoses, it may be an interesting candidate to ameliorate lung inflammation in cf patients. background: the primary cause of mortality and morbidity in cystic fibrosis (cf) is lung disease, which is characterized as a chronic cycle of infection and inflammation dominated by a neutrophilic infiltrate and increased levels of circulating mediators such as il-8. a wide variation in the rate of progression of lung disease is observed in cf. hypothesis: we postulate the presence of a brisk systemic inflammatory response in cf patients and that this systemic response reflects the magnitude and progression of lung disease. methods: lung function and clinical data were obtained from 164 cf patients treated and followed in the adult cf clinic, st paul's hospital. forced expiratory volume at 1s (fev1) and forced vital capacity (fvc) values dating back to 2000 were used in regression equations to group patients into quartiles based on their decline in predicted fev1%. rate of decline in lung function was compared with clinical characteristics and various systemic inflammatory biomarkers, including wbc, band cell count, crp, il-1β, il-6, il-8, mcp-1, and gm-csf results: the quartiles exemplified significantly different rates of decline in fev1/fvc% values. subjects with a rapid decline in lung function were younger and were colonized/infected more commonly with methicillinresistant staphylococcus aureus (mrsa) (p<0.05). in addition, altered calcium, magnesium, and albumin levels were found in subjects with increased rates of decline in lung function (p<0.05). preliminary results show that patients with cf have significantly elevated levels of wbc (10.94e9/l), crp (8.708e3ng/ml), and il-8 (48.65 pg/ml) in serum when compared to control subjects (p<0.05). there were trends in relationships between levels of il-6, il-8 and mcp-1 and rates of decline in lung function, however none reached statistical significance. conclusion: there is a variable rate in the decline in lung function among cf patients and one factor may be a heterogeneous systemic inflammatory response. circulating pro-inflammatory mediators may not only impact progression of lung disease, but could also be novel biomarkers to monitor and assess disease severity in cystic fibrosis. nutritional status in addition effects the rate of decline in lung function perhaps by modifying the inflammatory response. this research is supported by the bc lung association. overexpression of the epithelial na + channel β subunit (protein = βenac, gene = scnn1b) in transgenic mice results in cf-like lung pathology, characterized by neonatal mortality, mucus obstruction and airway inflammation (mall et al., nat. med. 2004) . breeding βenac mice into gene-deleted mice enables quick and efficient determination of the specific pathways relevant to the development of lung pathology. tnfα is a pleiotropic pro-inflammatory cytokine released by many different cell types, including t cells, macrophages, granulocytes, and epithelial cells. tnfα levels are significantly elevated in bronchoalveolar lavage (bal) from βenac mice in comparison to wild-type (wt) littermates. we crossbred βenac mice (heterozygous, inbred line c57-6608) with tnfα knockout mice, generating four types of mice: wt, tnfα +/-; wt, tnfα -/-; βenac, tnfα +/-; and βenac, tnfα -/-. all mice had comparable, high survival, ranging between 80-90%. the lack of tnfα did not prevent neutrophil and eosinophil infiltration and did not significantly modify the lung pathology characteristic of βenac mice, namely mucus plugs, mucous secretory cell hyperplasia, airway inflammation and emphysema. the absence of tnfα in bal from βenac, tnfα -/-mice, in comparison to βenac, tnfα +/mice, was confirmed by luminex cytokine assay. the levels of kc, a potent neutrophil chemoattractant, were significantly elevated in bal fluid from βenac mice, regardless of the presence or absence of tnfα. in wt mice, no granulocytic infiltrate or morphologic changes were observed and tnfα and kc were undetectable. similarly, the pathologic changes in βenac mice were not mitigated by crossbreeding to knockout mice for tnfα receptor 1 (tnfαr1), the major mediator of biologic responses classically attributed to tnfα. these results suggest that tnfα per se does not have a critical pro-inflammatory role in the development of inflammation in the βenac mouse model. the production of alternative cytokines may compensate for the loss of tnfα bioactivity in the tnfα and tnfαr1 -/-mice. since tnfα has been suggested to play a role in the regulation of enac, ussing chamber studies are underway to test whether the bioelectric features of tracheobronchial epithelia from βenac mice are altered by the absence of tnfα in vivo. these data suggest that a tnfα-independent signaling cascade links airway surface dehydration to airway inflammation in the βenac mouse model. innovative pharmacological approaches to control the excessive neutrophil infiltrates into the bronchial lumen of cf patients are thought to be beneficial to reduce the extensive airway tissue damage. the activation of expression of proinflammatory genes by p.aeruginosa with bronchial epithelial cells is a central mechanism to be targeted with novel therapies. medicinal plants from the socalled traditional asian medicine are attracting a growing interest because of their potential safety, already tested in large scale applications in human diseases. however, due to the presence of different active principles in each plant extract, whose multifunctional effects may even result contradictory, understanding the effect of each component is mandatory to pursue selective and reproducible applications. a panel of medicinal plant extracts have been firstly screened for their capacity to interfere in the binding of nuclear transcription factor proteins (tf) with dna consensus sequences identified in the promoters of the pro-inflammatory genes, thus for their potential inhibitory action on gene expression. extracts from several medicinal plants have been screened for their ability to interfere with the tf nf-kb, ap-1 and creb, which are induced by p.aeruginosa, and some of them, such as emblica officinalis (eo), aegle marmelos (am), polyalthia longifolia (pl), have been shown to inhibit tf/dna interactions, opening the possibility of potential applications to downregulate expression of pro-inflammatory genes. extracts from eo, am, pl were tested in ib3-1 cf bronchial cells exposed to the p.aeruginosa laboratory strain pao1. eo, am and pl strongly inhibited the pao1-dependent transcription of il-8 in ib3-1 cells. pyrogallol, one active principle of eo, was tested in ib3-1 cells, where it inhibited the transcription of the neutrophil chemokines il-8, gro-alpha and gamma, of the intercellular adhesion molecule icam-1 and the pro-inflammatory cytokine interleukin 6, similarly to the whole eo extract, whereas a second active principle from eo, namely 5hydroxy-isoquinoline, had no effect. similar results were obtained with eo and pyrogallol in the monocyte-derived macrophage cell line thp-1 exposed to pao1. in conclusion, extracts from plants of the traditional medicine can inhibit expression of pro-inflammatory genes and screening active principles purified from medicinal plants could result useful to identify safe and innovative pharmaceutical molecules to control lung inflammation in the lung of cf patients. supported by italian cystic fibrosis research foundation and by fondazione cariverona -bando 2005 -malattie rare e della povertà. massive infiltrates of neutrophils in the mucosal wall and lumen of the conductive airways of cf patients contribute to the progressive lung func-tion decline by releasing different proteases responsible for the progressive airway tissue damage. bacterial products and pathogens themselves within the mucopurulent material of the airway surface fluid induce the activation of transcription factors such as nf-kappab, ap-1, sp1, nf-il6, nf-at, elk-1, creb resulting in expression of chemo/cytokine genes driving the recruitment of leukocytes inside the bronchial lumen. to find new treatment options focused on the reduction of neutrophil chemotaxis, without abolishing the immune response against pathogens, we are exploring the transcription factor (tf) "decoy" strategy, in which oligodeoxynucleotides (odn) mimicking the consensus sequences for the tfs proteins identified in the promoters of different chemo/cytokines are delivered inside the cell in order to interfere with gene transcription. cf bronchial epithelial cells ib3-1 have been exposed to the (i)p.aeruginosa(/i) strain pao1. a clear pao1-dependent activation of tfs such as nf-κb, sp1, ap-1, nf-at, nf-il6 was confirmed by electrophoretic mobility shift assay (emsa). in parallel, transcription of genes involved in innate immune response has been quantified by real-time rt pcr. transcription factor "decoy" odns directed against the consensus sequences identified in the promoters of different genes have been designed and validated by testing their interference in tf protein/dna binding assays. transfection of ib3-1 cells with hiv-1 ltr and igk chain nf-κb odn "decoys" complexed with lipofectamine, performed 30 hrs before challenge with pao1, was shown previously to inhibit strongly pao1-dependent transcription of il-8 but not gro, il-1β, il-6 and icam-1. therefore other tf "decoy" odns have been also tested: a) odn for nf-κb from il-8 promoter inhibited il-8, gro-gamma and il-6 by 50%, b) odn for sp1 from hiv-1 genome inhibited il-6 by 50%, c) odn for ap-1 from il-8 promoter inhibited both il-8 and gro-gamma by 50%. a tf "decoy" molecule designed as peptide-dna chimera mimicking the consensus sequence of hiv ltr nf-κb strongly and selectively inhibited il-8 transcription. in conclusion, transcription of chemo/cytokines induced by (i)p.aeruginosa(/i) in cf bronchial epithelial cells (i)in vitro(/i) can be inhibited with different efficiency and selectivity by tf "decoy" molecules. these results provide useful hints for a gene-targeted anti-inflammatory approach and add further information on the regulation of expression of pro-inflammatory genes induced by (i)p.aeruginosa(/i) in bronchial epithelial cells. supported by italian cystic fibrosis research foundation and by fondazione cariverona -bando 2005 -malattie rare e della povertà. background: cf is characterized by hypersecretion of the pro-inflammatory cytokine il-8 from airway epithelial cells. however the mechanism by which il-8 gene expression is dysregulated in cf is not known. the expression of cytokine and chemokine genes is known to be regulated at multiple mechanistic steps including transcription, mrna decay, translation, and various post-translational steps. sequence-specific mrna degradation is now recognized to be an important site controlling the expression of several chemokine mrnas. this selective behavior is conferred by cisacting elements in the mrna composed of au-rich sequences (ares). the regulatory function of ares is thought to be mediated via rna-binding proteins that specifically recognize the are motifs. the steps required for mrna decay is comprised of deadenylation, decapping and body decay. deadenylation or removal of the poly (a) tail by poly(a)-specific ribonuclease (parn) appears to be the first and perhaps the rate-limiting step. this is accompanied by decapping or enzymatic removal of the 5' methylate guanosine cap. subsequently, exonuclease activities in the 5' to 3' or 3' to 5' direction predominate to degrade the remaining mrna. hypothesis: we have hypothesized that defects in transcription as well as modulation of the post-transcriptional stability of il-8 mrna might contribute to hyper-production of il-8 protein in the cystic fibrosis. methods: il-8 mrna stability was assessed in cystic fibrosis ib3-1 lung epithelial cells and in aav-cftr-repaired ib3-1/s9 cells, by measuring residual il-8 mrna levels at various intervals of time after addition of actinomycin-d to the culture. protein s100 extracts, prepared from the cf cell line as well as the cftr-corrected cell line, were analyzed by western blot. the expression levels of the various factors known to participate in are-mediated mrna decay, including ttp (an are-binding protein), parn and the exosome were compared in the two sets of s100 extracts. results: we find that the levels of il-8 mrna in the cf cells are greater than the levels found in the cftr-repaired cells. in addition, we found that the rate of decay of il-8 mrna in cf cells was significantly less than that in the repaired cell line. the levels of ttp, parn and exosome proteins were significantly reduced in the cf cells compared to the cftrrepaired controls. however, expression of ttp in ib3-1 cells causes significant destabilization of il-8 mrna. conclusion: we conclude that the high levels of il-8 protein expression in cf lung epithelial cells can be partly due to enhanced stability of the il-8 mrna. consistently, the actual levels of il-8 mrna are higher in the cf cells compared to controls. understanding the mechanism by which ttp promotes enhanced destabilization of il-8 mrna in cf cells may be important for developing novel therapeutic targets to alleviate the pulmonary pathophysiology of this disease manifested by hyper-secretion of il-8 protein. understanding the mechanism of airway remodeling could lead to the identification of novel therapeutic targets for the prevention of irreversible lung damage in cf. we have investigated transcriptional responses to epithelial injury in a mouse model of cf that was developed in our laboratory (f508del, cftr tm1eur fvb backcross f12). to induce transient epithelial lung injury, homozygous normal and mutant age matched littermates were treated in parallel with naphthalene (200 mg/kg ip) or carrier (corn oil) as control (n= 4x12). naphthalene causes an almost complete selective ablation of clara cells overnight, followed by migration of resistant ciliated cells and rapid proliferation of undifferentiated progenitor cells. two and seven days after treatment, lungs were collected for histological and transcriptome analysis. based on a previous affymetrix microarray analysis (n=6), a quantitative pcr array was designed containing 56 genes differentially expressed after naphthalene injury. as expected, naphthalene injury results in a transient increase of cell proliferation markers (mki67, cdc20, cdc2a etc), and a strong (90%) reduction of clara cell markers (cc10/scgb1a1; claudin10/cldn10; cyp2f2). in addition, we have now identified twenty-five genes that are increased (p< 0.001) two-to fifty-fold after naphthalene injury in both normal and mutant mice. this includes known and novel markers of epithelial tissue injury such as timp1, retlna, mmp8, serpin3n, and lipocalin. in particular, a group of egf receptor agonists is strongly induced: amphiregulin (areg, 13 fold), epiregulin (ereg 50 fold) and heparin binding egf (3 fold). amphiregulin mrna can be detected by in situ hybridization only in airway epithelial cells after injury. these egfr agonists are involved in stimulating epithelial repair, but can also activate cells in the underlying mesenchyma. indeed, we observe a substantial increase of major extracellular matrix mrna's two days after injury (co1a1: 5-fold, col3a1: 10-fold, elastin: 3-fold). whereas expression levels were substantially reduced in normal animals seven days after injury, in cf mutant animals two to three fold higher levels were observed for these three genes at day seven (p<0.04). conclusions. we have designed and validated a gene array that can be used to study distal lung injury and repair. egfr agonists produced by epithelial cells are the most prominent growth factors after injury, involved in both epithelial repair and extracellular matrix production by mesenchymal cells. ecm production in cf mouse lung is sustained compared to normal mice, suggesting an inherent tendency towards fibrosis in cf lungs. our data suggest that the mesenchymal egf receptor and regulation of its agonists are important future targets of novel therapeutic strategies. supported by eec 6th fw projects eurocarecf, improved precision the mucosa of the proximal airways defends itself and the lower airways from inhaled irritants, allergens, and microbial and viral infections by several mechanisms. sensory nerves monitor the luminal microenvironment and trigger reflexes in the central nervous system (cns) that alter breathing, induce cough and stimulate mucus secretion when challenged with noxious stimuli. sensory nerves also release the tachykinins substance p (sp), neurokinin a and calcitonin gene-related peptide through axon reflexes in neighboring tissues, and these locally released tachykinins stimulate mucus secretion by binding to neurokinin 1 receptors on submucosal glands. recently we reported that local fluid secretory responses to noxious stimuli are dependent on the clchannel cftr in mouse airway submucosal glands and are defective in glands from cftr knockout mice (ianowski et al., j physiol, 2007, 580, 301) . we have now tested the effects of sp directly and examined the possible role of cftr in mediating these responses using tracheas from congenic wild-type and cftr knockout mice (cftr m1unc /cftr m1unc ) that had been bred onto c57bl/6 j or balb/c genetic backgrounds. we compared single gland secretion rates using optical methods as described previously. the cftr genotype of each mouse was assessed by using pcr to amplify genomic dna from tail clippings obtained at age 18 days. after the cftr knockout mice were weaned, intestinal obstruction was minimized by supplementing their water with peglite. capsaicinoids (chili pepper oil) increased fluid secretion from glands of wild-type mice from 10 ± 2 pl/min (n=9 tracheas, 17 glands) to 50 ± 10 pl/min (n=4 tracheas, 9 glands, anova p<0.05, tukey-kramer multiple comparison test p<0.05). this response was abolished by exposing the basolateral surface of the tracheas to l-732,138 (10 µmol/l), a known sp (nk-1) receptor antagonist (15 ± 5 pl/min, not different from control, tukey-kramer multiple comparison test p>0.05). secretion was stimulated from 4 ± 1 pl/min to 100 ± 30 pl/min (n=12 tracheas, 55 glands, student's t-test p<0.05) by the direct application of sp, and this response was strongly inhibited by pre-incubation with the cftr inhibitor cftrinh172 (saturating concentration, nominal 100 µmol/l; n=6 tracheas, 21 glands). finally, submucosal glands from cftr knockout mice failed to secrete when exposed to sp (1 µmol/l) whereas wild-type littermates were responsive. these results indicate that sp mediates local responses to capsaicinoids through a cftr-dependent mechanism. loss of this local regulation in cf may contribute to the susceptibility of cf airways. support: canadian cf foundation, canadian institutes of health research, nih (dk51817), and the cff (usa). chronically infected/inflamed cf human bronchial epithelia (hbe), or normal hbe exposed to supernatant from mucopurulent material (smm) from human cf airways, exhibit expansion of the endoplasmic reticulum (er) ca 2+ stores and amplification of ca 2+ -mediated inflammatory responses. we have shown that infection/inflammation of hbe triggers an unfolded protein response (upr) coupled to mrna splicing of x-box binding protein-1 (xbp-1). spliced xbp-1 (xbp-1s) is a transcription factor that promotes er expansion to augment the protein folding capacity during increased protein synthesis. because we have shown that hbe inflammation couples to increases in protein synthesis, we hypothesized that hbe infection/inflammation-induced er ca 2+ store expansion is mediated by xbp-1s. to test this hypothesis, we constructed four retrovirally-tranduced stable 16hbe14o-cell lines containing empty vector (ev), xbp-1 unspliced (xbp-1u), xbp-1s, or dominant negative xbp-1 (dn-xbp-1). cells were grown to confluence under an air-liquid interface and er ca 2+ store expansion and il-8 secretion studied in the absence or following 24 hr mucosal pbs or smm exposure. consistent with our hypothesis, expression of xbp-1s in the absence of stimulation induced er expansion and increased mucosal utp-sensitive er ca 2+ stores ( to confirm that these effects were mediated by upr activation, cells were transfected with a upr response element luciferase reporter plasmid, and luciferase activity was measured in the absence or presence of the upr inducer tunicamycin (tm). cells expressing xbp-1s exhibited higher baseline luciferase activity than cells expressing ev or xbp-1u, whereas luciferase activity was decreased in the dn-xbp-1 expressing cells. furthermore, while tm increased luciferase activity in cultures expressing ev or xbp-1u, its effect was blocked in dn-xbp-1 expressing cells. these findings suggest that xbp-1s is the major trigger of er ca 2+ store expansion, which mediates the amplified ca 2+ -dependent inflammatory response in infected/inflamed airway epithelia. although it remains to be established whether upr-dependent xbp-1s is a beneficial or a maladaptive response in infected/inflamed airways, therapies aimed at manipulating this upr pathway may be beneficial for patients with chronic inflammatory lung diseases. luminal exposure of well-differentiated normal human bronchial epithelia (hbe) to supernatant from mucopurulent material (smm) from human cf airways increases the secretion of inflammatory mediators and triggers an unfolded protein response (upr) mediated by the mrna splicing of the x-box binding protein-1 (xbp-1). spliced xbp-1 (xbp-1s) is a transcription factor that expands the endoplasmic reticulum (er) and protein secretory pathway during er stress induced by accumulation of unfolded proteins due to increases in protein synthesis. our previous studies in primary cultures of hbe and 16hbe14o-cells stably expressing empty vector, inactive unspliced xbp-1 (xbp-1u), xbp-1s or a dominant negative xbp-1 (dn-xbp-1) suggest that induction of xbp-1s by smm exposure promotes er ca 2+ store expansion, which mediates a ca 2+ -dependent hyperinflammatory response. the present studies were designed to determine if pseudomonas aeruginosa pak strain (p.a.) would reproduce the effects of smm in both in vitro and, importantly, in vivo models. for in vitro studies, hbe were exposed to broth (tsb) or to a 20% p.a. extract for 24 hr, and inflammation, xbp-1s, and er ca 2+ store expansion investigated by il-8 secretion, southern blots and calreticulin expression, respectively. similar to smm, p.a. induced a 4.5 fold increase in il-8 secretion coupled to a 2 fold increase in xbp-1s and larger er ca 2+ stores as compared with tsbexposed hbe. these data demonstrate the link between p.a. infection, upr activation and xbp-1s in vitro. we next tested the relevance of these findings in vivo. wild-type mice airways were challenged with pbs or 10 6 cfu of p.a., and airway epithelial er density was assessed by calreticulin staining 24 hr later. in comparison with pbs challenges, p.a.-challenged mice exhibited airway epithelial er expansion, which correlated with the degree of airway inflammation, based on the presence of inflammatory cells. to test whether this p.a.-induced er expansion was linked to xbp-1s, we utilized "er stress activated indicator" (erai) mice expressing a fusion protein consisting of xbp-1u and the fluorescent protein venus. in erai mice, upr activation-dependent xbp-1 splicing leads to venus expression; hence, venus fluorescence is an index of xbp-1s. consistent with our hypothesis that p.a. infection-triggered inflammation would induce upr-dependent xbp-1s, venus fluorescence, after 24 hr challenge with 10 6 cfu of p.a., was increased in inflamed as compared with non-inflamed airways. these findings suggest that 1) airway epithelia respond to bacterial infection-induced inflammation by up-regulating the er ca 2+ stores and 2) activation of the xbp-1s pathway by bacterial infection may be relevant to airway inflammatory responses in vivo. funded by the cff. we have shown that luminal exposure of well-differentiated primary cultures of normal human bronchial epithelia (hbe) to supernatant from mucopurulent material (smm) from human cf airways increases total cellular protein synthesis, which reflects the increased secretion of inflammatory factors induced by the infectious and inflammatory process. in the present studies, we first investigated whether these hbe responses to smm were linked with an increased metabolic rate by measuring lactate accumulation into the serosal media. smm increased lactate production, and this effect was maximal within 24 hrs (9.0+1.0 vs. 13.2+0.8, and 7.1+0.9 vs. 13.1+0. 4 mmol/l in 24 and 48 hr pbs vs. smm, respectively; n=5-10), suggesting that the increase in protein synthesis couples to a hyper-metabolic state in infected/inflamed hbe. in agreement with this notion, 6 or 24 hr smm, as compared with pbs exposure, induced the expression of genes associated with amino acid transport and metabolism (n=4). in addition, 6 or 24 hr smm exposure up-regulated genes involved in oxidative stress (n=4). we hypothesized that these hbe responses were linked to an unfolded protein response (upr) mediated by activation of the pkr-like er kinase/pancreatic eif2α kinase (perk)-induced activating transcription factor 4 (atf4), since this pathway has been shown to confer protection against amino acid loss and oxidative stress in other cells. we first tested whether smm induced activation of perk/atf4 in hbe by performing western blot analyzes of the components of this pathway. twenty-four hr smm exposure induced perk activation, as indexed by phosphorylation of perk, in comparison with pbs-exposed hbe. on the other hand, total perk protein levels were unchanged in smm-treated hbe. phosphorylation of eif2α, the downstream effector of perk, and increased atf4 protein levels, which depend on the phosphorylated status of eif2α, provided additional evidence that the perk/atf4 pathway was activated by smm. these data are consistent with the hypothesis that induction of atf4 is triggered by upr activation resulting from increased synthesis of inflammatory factors. we next utilized rna microarrays to test whether atf4 target genes were induced by smm. six or 24 hr smm exposure induced atf4 target genes (e.g., ero1, an oxido-reductase that provides protection against the accumulation of endogenous peroxides during er stress; stanniocalcin 2, whose expression is associated with anti-apoptotic functions; and heme oxygenase 1). these findings suggest that 1) activation of the upr-dependent atf4 pathway is a compensatory component of the airway epithelial adaptive response to luminal infection/inflammation, and 2) activation of the atf4 pathway protects against inflammation-induced amino acid loss and oxidative stress by up-regulating genes involved in amino acid transport/metabolism and oxidative stress responses. unraveling the functions of atf4 should help determine if therapies targeted to manipulate pathway activity would be likely to improve lung function in patients with cf or other chronic inflammatory airway diseases. funded by the cff. miller, t.j.; perez, a.; qian, y.; davis, p. pediatrics, case western reserve university, cleveland, oh, usa fxyd5 is a cell surface protein originally identified in a screen for molecular markers of tumorigenesis. increased fxyd5 expression was found in tumors from stomach, thyroid, colon, pancreatic, breast and lung cancers and correlated with down-regulation of e-cadherin and poor patient prognosis. recent studies have shown that overexpression of fxyd5 promotes cell motility, decreases cell-cell attachment and increases tumor metastasis. fxyd5, also known as dysadherin, is a member of small family of proteins known to regulate the na,k-atpase. we now report that fxyd5 is upregulated in cystic fibrosis (cf) airway epithelia and modulates wound healing. we show by immunohistochemistry and immunoblot analyses that fxyd5 is increased in the lungs of s489x cf mice, and demonstrate an almost 3-fold increase in fxyd5 expression in the nasal epithelia of cf mice compared to wild-type littermates (p<0.001). furthermore, we show that fxyd5 is upregulated in nasal scrapings from human cf patients compared to controls (p<0.02). immunofluorescence data show that flag-tagged fxyd5 co-localizes with the na,k-atpase in epithelial cells, suggesting that fxyd5, similar to other members of the fxyd family, may regulate na,k-atpase function. it has previously been shown that expression and localization of the na,k-atpase is required for efficient polarization and suppression of cell motility in epithelial cells. the recurrent remodeling of pulmonary epithelium as a result of bacterial infection in cf requires that airway epithelial cells polarize and migrate to wound sites in order to maintain lung integrity. thus we hypothesized that fxyd5 may be involved in wound healing after infection. laser-capture microdissection and microarray analysis of murine lung epithelia after 3 hours treatment with p. aeruginosa indicated a significant, 5-fold increase in expression of fxyd5 that was confirmed by immunoblot analysis. others have shown that fxyd5 may mediate expression of mcp-1, a critical determinant in monocyte recruitment, through activation of the nf-kb pathway. treatment of human tracheal epithelial (hte) cells with a cftr inhibitor (172) confirmed that loss of cftr function correlated with increased fxyd5 expression by quantitative rt-pcr (p<0.001), an effect that was abrogated with treatment of pdtc, an inhibitor of nf-kb (p<0.01). we speculated that fxyd5-induced increases in cell motility may be due in part to phosphorylation at serine 163. in a murine airway epithelial cell wound healing model, serine to alanine (s163a) mutations at serine 163 inhibited wound healing compared to wild type fxyd5 overexpression, whereas aspartic acid (s163d) mutations increased wound healing (p<0.005). immunoblot and immunofluorescence analyses of these mutants suggest phosphorylation at ser163 regulates membrane localization. we conclude that fxyd5 is increased in cystic fibrosis epithelia due to increased inflammatory mediators and suggest that fxyd5 may modulate airway epithelia wound healing after infection with p. aeruginosa through phosphorylation at ser163. the inflammatory response to bacterial infection in the cf airway is exaggerated compared to normal, leading to the accumulation of millions of necrotic neutrophils. extracellular neutrophil elastase (ne) activity in the cf airway not only compromises innate defences by cleaving opsonins and reducing ciliary activity, but amplifies the inflammatory response by stimulating expression of the neutrophil chemoattractant il-8 and increases mucus production, in addition to degrading the tissue matrix leading to fatal bronchiectasis. ne therefore represents an important target for the development of new therapies. however, this strategy requires consideration of the normal physiological function of this enzyme, since previous studies in knock-out mice indicated an essential role for ne in cell migration, bacterial phagocytosis and killing. our approach was to use the intracellular neutrophil elastase inhibitor gw311616a to 'knock-out' ne activity in neutrophils in normal human blood and test the function of isolated cells in chemotaxis, bacterial phagocytosis and killing assays. whole normal human blood was incubated with gw311616a, or pbs control, for 1h at 37°c. neutrophils were isolated by sedimentation of red blood cells (rbc) on dextran 70, purification on lymphoprep and hypotonic lysis to remove contaminating rbc. chemotaxis towards il-8 was measured using a modified micro-boyden chamber. phagocytosis was assayed by the depletion of staphylococcus aureus (sa), pseudomonas aeruginosa (pa) and e. coli (ec) in supernatants following culture in a 10:1 ratio with pmn for 2h at 37°c. supernatants were diluted and remaining organisms were plated on agar and grown overnight to count viable colonies. bacterial killing was assayed by incubating bacteria and cells in a 1:1 ratio for 15 minutes to allow phagocytosis, washing off remaining organisms, and incubating for 4h at 37°c. neutrophils were lysed with water and lysates plated on agar to test for bacterial growth overnight. results; gw311616a inhibited intracellular ne dose-dependently and at 10 µm 0.168±0.062 % ne activity remained (n=5). there was no significant effect of 10 µm gw311616a on neutrophil chemotaxis, bacterial phagocytosis or killing of any organism compared to pbs-treated controls. phagocytosis data is shown in the table. thus, in the absence of ne activity, human neutrophils remain wellequipped with other defence molecules including myeloperoxidase and defensins to successfully maintain the role of the neutrophil in innate immunity. however, mouse neutrophils which lack defensins require ne activity for optimal intracellular bacterial killing, and mice are not a perfect model for studies of human infection. the development of novel inhibitors of ne to treat lung disease in cf therefore remains an important goal. supported by the cf trust of great britain. background: sphingolipid signalling may differ between individuals with cf and healthy controls. the response to bacterial inflammation is different, and uptake and inactivation of sphingosine-1-phosphate, an intracel-lular pro-inflammatory mediator, is reduced in cf cells. it may therefore continue to act on g-protein coupled receptors in the plasma membrane. (boujaoude et al, j biol chem 2001) . furthermore, ceramide originating from basolateral sphingomyelin hinders augmentation of cftr-mediated anion conductance across the apical membrane, resulting in reduction of transepithelial airway anion secretion (ito et al, bbrc 2004) . aim of study: to determine if there is a difference in the levels of alkaline, neutral or acid sphingomyelinase (smase), or in the levels of neutral or acid ceramidase, in the intestinal or bronchial mucosa and some other tissues, between wildtype, homozygous (+/+) and heterozygous (+/-) delta-f508 cftr mice. methods: enzyme activities (duan and nilsson meth enzymol 2000) were determined in intestine (and content) divided into four regions, liver, lungs, kidney and spleen from deltaf508-cftr mice (+/+) and controls (wildtype, +/-). results: there was an increased amount of neutral ceramidase in spleens from deltaf508-cftr mice (+/+) in comparison to control mice (p=0.0278). no other significant differences were seen. conclusion: delta-f508 mutation did not influence the levels of alkaline smase and neutral ceramidase acting as ectoenzymes, or the levels of intracellular smases and ceramidases, which may all generate bioactive sphingolipid metabolites in intestine and lungs. the implications of the increased level of neutral ceramidase in spleen are not known. in children with cystic fibrosis (cf) there is a clear correlation between the development of chronic p aeruginosa infection and acceleration in the decline of lung function. when chronically present, p aeruginosa takes on a mucoid phenotype and is impossible to eradicate. prior to this, when colonisation is intermittent, it is possible to eradicate it with aggressive antibiotic regimes. we sought to examine the degree of inflammation and innate defence status in the lungs of children with cystic fibrosis in various stages of colonisation by looking at a range of proteases, innate defence proteins and markers of inflammation in broncho alveolar lavage (bal). children with cf were allocated to one of three groups in relation to p aeruginosa infection; chronically colonised, intermittently colonised and non-colonised, on the basis of the leeds criteria. bal was collected as per ers guidelines as part of each patient's routine clinical care. bal was collected from control patients undergoing elective non-pulmonary surgical procedures. differential cell counts in bal were performed manually. secretory leukocyte protease inhibitor (slpi), elafin, alpha-1 antitrypsin (a1at) and lactoferrin concentrations were measured by elisa. neutrophil elastase activity and cathepsin activity were assayed by colorometric activity assays. fifty two patients were included in the study ranging in age from 3 months to 18 years (11 chronic, 10 intermittent, 17 non colonised and 14 controls). neutrophil counts, neutrophil elastase activity and cathepsin activity were markedly increased in children chronically colonised with p aeruginosa compared to those in the intermittent and non colonised groups. in contrast, levels of the antiproteases slpi and a1at and the antimicrobial peptides elafin and lactoferrin were highest in the control group and decreased as colonisation progressed, with levels in the chronically colonised group markedly lower than those with intermittent colonisation. this study demonstrates that in children with chronic p aeruginosa colonisation, there is a marked decrease in antiproteases and antimicrobial factors and a marked increase in protease activity and neutrophil influx in comparison with those who are non-colonised or intermittently colonised. these findings underline the importance of careful microbiological surveillance and early aggressive treatment of p aeruginosa infection in children with cf in order to avoid chronic colonisation. background : abnormal bronchial angiogenesis is responsible for hemoptysis in cystic fibrosis (cf). expression of vegf-a in airway epithelium induces bronchial angiogenesis in animal models. we have recently found that vegf-a and egf receptors (egfr) are increased in the airway epithelium of subjects with advanced cf lung disease. aims: to examine the effects of pa bacterial products and egfr inhibition on vegf synthesis in airway epithelium. methods: culture of non cf (nci-h292) and cf (cfte29o-) human airway epithelial cell lines. stimulation with pa lipopolysaccharide (lps). assessment of vegf mrna and protein by rt-pcr and elisa. use of chemical inhibitors, blocking antibodies and sirna. results: pa lps increased vegf gene expression and protein production time-and dose-dependently in both cells lines. using chemical inhibitors, we show that egfr and erk1/2 activation are required for lpsinduced vegf production. using blocking antibodies to egfr and its ligands, we show that tgf-alpha-dependent egfr activation mediates pa lps-induced vegf gene and protein synthesis. using pharmacological inhibitors (an ros scavenger and an nadph oxidase inhibitor) and using small interfering rna of dual oxidase (duox) 1 and tnf-alpha converting enzyme (tace),we show that lps-induced vegf upregulation is dependent on duox1-mediated ros release and tace activation. thus, pa products induce vegf synthesis in airway epithelium via a duox1-ros-tace-tgf-alpha-egfr-erk1/2 cascade. conclusions: these results describe a novel pathway by which bacterial products induce angiogenic signaling in cf and non cf airway epithelium. background: unlike bronchoalveolar lavage (bal), the airway mucosa has been under-investigated in cystic fibrosis (cf), despite the fact that irreversible airway wall changes (bronchiectasis) are a feature of endstage disease. cf is characterized by a neutrophil-dominated inflammation in bal, but little is known about the pattern of inflammation in the airway mucosa, especially in children with relatively early stage disease. we aimed to assess whether the pattern of inflammation seen in cf bal was also found in the airway mucosa in cf children. methods: to date, endobronchial biopsies and bal from 46 children (0-16 years) with cf and 8 control children (0-16 years) without lower respiratory disease have been assessed. bal cell differential was assessed on may-grünwald-stained cytospins. endobronchial biopsies were stained for neutrophils (neutrophil elastase, ne), t-(cd3) and b-(cd20) lymphocytes, eosinophils (eg2), and macrophages (cd68). area profile counts of immunopositive cells in subepithelial tissue were performed by investigators blinded to disease group. results: all cell types were increased in cf bal compared to controls. cf bal was characterized by an abundance of neutrophils (837 x 10 3 /ml vs. 3 x 10 3 /ml in controls, p<0.0001) with moderate numbers of lymphocytes (48 x 10 3 /ml vs. 3 x 10 3 /ml in controls, p<0.001). in contrast, cf subepithelial tissue was characterized by a lymphocytic infiltrate (961 cells/mm 2 vs. 575 cells/mm 2 , p<0.01) with only very few neutrophils (12 cells/mm 2 vs. 0 cell/mm 2 , p<0.05). the lymphocytic infiltrate in cf consisted mainly of t lymphocytes (91%). eosinophil counts in subepithelial tissue did not differ between cf and controls. for all cell types, there was no correlation between counts in bal and counts in subepithelial tissue. conclusions: in contrast to the neutrophil-dominated inflammation in the airway lumen, cf is characterized by a lymphocytic inflammation in the airway mucosa. the lymphocytic infiltrate consists mainly of t lymphocytes, the pathophysiological function of which may be important and is being investigated in future work. support: ers long-term fellowship and swiss national foundation grant to nr the epithelium serves as a barrier to the penetration of foreign antigens, particles, and infectious agents across the airway. the integrity of this barrier is dependent, in part, upon the apical junctional complex consisting of the tight junction (tj) and the adherens junction. alterations in tj permeability have been linked to the pathogenesis of inflammatory bowel disease and this increased intestinal permeability may actually precede the onset of chronic inflammation. in cystic fibrosis (cf), the airway lumen is filled with high concentrations of inflammatory cells, bacteria, and inflammatory mediators. since tj barrier function can be significantly reduced by inflammatory mediators, we hypothesized that measures that enhance airway tj barrier function will decrease airway responses to the continuous presence of inflammatory mediators in the lumen. to test this hypothesis, we examined the relationship between lung inflammation and epithelial permeability in vivo using a lipopolysaccharide (lps) model of lung inflammation. pseudomonas aeruginosa lps was instilled intratracheally into the lungs of c57bl/6 mice which were then euthanized at 24, 48 and 72 hrs. lung inflammation was assessed by total cell counts using a hemacytometer and differential counts by wrights staining of cytospin preparations of the bronchoalveolar lavage fluid (balf). lps increased total cell counts and neutrophil concentrations that peaked at 48 hours after lps administration, compared to saline controls. measurements of the proinflammatory murine cytokine kc in balf, by a cytokine antibody bead technique, showed an increase in murine kc at 48 hr following lps administration, which correlated with the substantial increase in neutrophil concentration. changes in lung permeability with inflammation were assessed by elisa measurements of the levels of serum protein murine albumin in balf. correlating with changes in cellular inflammation and murine kc levels, albumin concentration peaked at 48 hr after lps administration. this increase was subsequently resolved, consistent with the restoration of barrier function. an examination of frozen sections of lung from lps-treated animals showed a redistribution of the tight junction protein zo-1 consistent with the disruption of barrier function. since the p38 map kinase signaling pathway has been implicated in lps-induced airway inflammation, and an inhibitor of this kinase, sb203580 has been shown to reduce this inflammation, the effect of this inhibitor on barrier function is being investigated. in initial studies, sb203580 appears to reduce total and neutrophil cell counts by 50% in vivo. the effect of sb203580 on murine albumin concentrations in balf and on tj protein localization is currently being evaluated. a reduction in these parameters will be used as indices of improved barrier function with sb 203580. these studies will determine whether a reduction in lung inflammation correlates with a restoration of barrier function. the degree of protection provided by the p38 map kinase inhibitor sb 203580 could have important implications for inflammatory lung diseases such as cf. background: chronic pulmonary inflammation in cf is characterized by a robust neutrophil response associated with airway damage and failure to eliminate the pathogen, p. aeruginosa (pa). pa is a highly adaptable opportunist which quickly develops resistance to antimicrobials. thus, the development of specific immunotherapy targeting the neutrophil recruitment without ablating the host's immune response to infection or promoting pathogen resistance would be ideal. our group has identified il-17 as a prime target for the development of immunotherapy due to the central role that the il-23/il-17 proinflammatory axis plays in neutrophil recruitment. however il-17 does not mediate the early neutrophil recruitment seen in response to infection. hypothesis: il-23, acting synergistically with il-1, is critical to early neutrophil recruitment during pulmonary pa infection. the primary effector cells are the il-23-producing antigen presenting cells: alveolar macrophages (ams) and myeloid dendritic cells (dcs). methods: wt and il-23-deficient mice were infected with pa at 1x10 6 cfu/50ul by intratracheal (it) inoculation for 3 hours. bal inflammatory cell counts, and cytokines and chemokines were measured. am and dc cultures were infected for 3 hours in vitro and supernatant cytokines/chemokines were measured by luminex and elisa; il-23 levels were measured by taqman. these studies were designed to elucidate the role of il-23 in the early neutrophil peak and define am-and dc-mediated cytokine/chemokine production. recombinant murine il-23, il-1, and il-23 + il-1 were instilled via it into wt and il-23-deficient mice. bal inflammatory cell counts and cytokines/chemokines were measured at 3 hours. these studies were designed to elucidate the role of il-23 and il-1 in the early neutrophil peak and define am-and dc-mediated cytokine and chemokine production. results: at 3 hours post-infection, il-23 deficient mice had significantly lower percent neutrophils (p<0.02) and lower mip1α, kc, and il-6 (p<0.001) in the bal. il-17 was undetectable. there was no significant difference in bacterial load that could account for these cytokine/chemokine differences. infected wt ams elaborated significantly more mip1a, gm-csf, mcp-1, il-1, g-csf, ip-10, kc and il-6 than the il-23-deficient ams (p<0.001) and the response was inoculum-dependent (p<0.01). dcs elaborated no il-1 and exhibited il-23-dependent differences in mip-1α and mcp-1 production (p<0.05). in vivo studies of il-23 and il-1 effect demonstrated a synergistic increase in bal neutrophil recruitment (p<0.01) and cytokine and chemokine induction (p<0.01). conclusion: the first wave of neutrophil recruitment seen during pa infection is il-23-dependent and il-17-independent. ams and dcs are critical to il-23 and il-1 indcution of this neutrophil recruitment. these studies identify il-23 as a key mediator of neutrophil recruitment in the early stages of infection as well as the proximate mediator in the il-23/il-17 pro-inflammatory axis and suggest il-23 as a potential target for anti-inflammatory therapy in the treatment of pa pulmonary infection. supported by the cystic fibrosis foundation, american lung association and the nih the airways are under constant assault from air-borne pathogenic material. despite the intake of up to 100,000 bacteria per hour, the airways are sterile below the larynx in healthy individuals. the task of maintaining this sterility falls to the airway surface liquid layer (asl), the protective twophased system consisting of the viscoelastic mucus layer and the periciliary layer (pcl) through which cilia beat, sweeping the mucus layer away from the lungs. the mucus layer, which is responsible for trapping pathogenic material, is comprised of mucins (high molecular weight glycoproteins), cellular debris, dna, neurtifils, and more than 100 other proteins. this chemically heterogeneous mixture forms a viscoelastic gel that is thick enough to trap pathogens of various sizes and surface chemistries, while not sticking to the underling cellular / cilia layer, allowing the transport of trapped pathogenic materials away from the lungs. the performance of this trapping / transportation system is defined by the rheological properties of the mucus layer and the force imparted on the mucus. therefore, understanding how mucins and other chemical components of the mucus layer interact with each other to form a successful mucus gel (i.e. one that is cleared from the airways) is crucial to understanding airway defense. here we present the results of physical and chemical composition studies of sputum samples collected from patients with chronic obstructive pulmonary disease (copd) and cystic fibrosis (cf). the rheological properties of each sample was assayed using parallel plate rheolometry, probing the materials non-linear viscoelastic properties such as viscosity, elasticity, and yield stress. the physical properties of the sample are then correlated to the sample's chemical properties such as percents solids (divided between salts, proteins, and mucins), as well as the relative concentrations of the key airway mucins muc 5b and muc 5ac. our results indicate that the physical properties of sputum are not well predicted from the total amount of biosolids in a given sputum sample, but by the relative concentrations of muc 5b and muc 5ac and the interactions of these molecules with themselves and the other proteins present. further, we establish that the heterogeneous physical properties within a given sputum sample correlate to differences in the muc5b and muc 5ac concentrations. the gene modifier study (gms) was established as an effort to identify potential genetic modifiers of cystic fibrosis pulmonary disease and survival. during the course of this study over 1000 delta f508 homozygous cf patients classified as having mild lung disease, severe lung disease, or increased survival, have submitted both clinical data and blood samples for single nucleotide polymorphism (snp) analysis. the original snp analyses have shown a significant association between variants in the endothelin receptor a (ednra) gene, and cf survival, most markedly in female cf patients. sixteen additional snps within and around the ednra gene have now been genotyped, and have implicated the 5' and 3' untranslated regions of the gene as having the most significant association with pulmonary disease in females (p<0.000001), suggesting quantitative differences as a possible mechanism for the association with pulmonary phenotype. we are in the process of saturating the 5' and 3' regions of ednra with an additional 20 snps to further delineate the genetic association. ednra binds endothelin-1 (et-1) in airway smooth muscle cells, causing increased cell proliferation, smooth muscle contraction, and stimulation of inflammatory molecules. because each of these effects is known to be deleterious to the cf lung, we hypothesize that the ednra variants found more commonly in "severe" cf females ("severe" alleles) are marking increased ednra expression compared to alleles found more commonly in "mild" cf females ("mild" allelels). because the genetic association was strongest in cf females, we used the matinspector software to analyze 2kb of ednra promoter sequence and found several putative binding sites for both estrogen and progesterone. we then used a brdu assay to measure cell proliferation after stimulation with both estrogen and progesterone. these experiments showed that the asm cells with the "severe" 5' ednra genotype proliferated at levels approximately twice that of the asm cells with the "mild" 5' ednra genotype following stimulation with either estrogen or progesterone. using a single base extension protocol and quantitative pcr on human airway smooth muscle cells, we were also able to compare ednra expression from the "severe" allele, and the "mild" allele. these comparisons of ednra expression demonstrate that expression levels appear to be approximately 20% higher from alleles found more frequently in the "severe" cf females. in addition, preliminary data suggest that stimulating the asm cells with estrogen increases ednra expression by approximately 10 fold, and like the cell proliferation experiments, these increases are most pronounced in cell lines with the "severe" genotypes. these data suggest that the "severe" genotypes are marking alleles with increased expression, perhaps due to estrogen binding, that leads to increased et-1 functional effects that over time are deleterious to the cf lung. cf patients do present with variable spectra of lung disease, of which infections are most life-threatening. β-defensins have an antimicrobial activity against a broad spectrum of microorganisms and are chemotactic agents for cells of the adaptive immune system, and therefore assist in combating these infections. β-defensins 2-6 are part of a repeat region. this repeat region is polymorphic between individuals and therefore the dosage of these defensin genes/proteins varies. we developed a real time pcr assay to quantify the number of βdefensin repeats in this region. appropriate controls are needed for an accurate quantitative assay. therefore we made 6 concatemeric constructs with 1 copy of defb1 and a particular number of defb4 copies, which ranged from 1 to 6 copies. using these controls as standards, the number of defensin repeats could be accurately determined in dna samples. we then tested 146 f508del homozygous cf patients from belgian (57 patients), czech (52 patients) and south-italian (37 patients) origin. the diploid number of repeats varied between 3 and 10. for each patient group, a higher number of repeats was found in the group of patients with milder disease (fev1 >70%) compared to the group of patients with more severe disease (fev1 < 70%) (student t test, p-values of 0.0006, 0.03 and 0.019 respectively). moreover, in our cohort of 112 belgian cf patients, cf patients of 25 years or older have a significant higher number of repeats than the cf patient group below 25 (p-value 0.05). to evaluate this at the functional level, we cultured nasal epithelial cells from 3 individuals with a low number of repeats (i.e. 3 or 4 repeats) and 4 individuals with a high number of repeats (i.e. 8 repeats). the cells where grown in air liquid interface cultures. after differentiation, the cells were stimulated with 10ng tnfα. in cells with a high number of repeats, defb4 expression, as measured by the extent of transcription, was strongly upregulated by tnfα. in cells with a low number of repeats, defb4 was not upregulated (p-value = 0.015). we also tested the antimicrobial activity of epithelial cells. we challenged epithelial cells from 3 individuals (8 repeats) with a laboratory strain (pa01) of pseudomonas aeruginosa and a clinical isolate (30 -300 cfu), either in combination with tnfα or without tnfα. after 3h, surviving bacteria were counted by a plating out method. cells that were stimulated with tnfα 12h prior to the bacterial challenges were more bactericidal. the clinical strain was more vulnerable to the surface liquid than the laboratory strain. in epithelial cells from individuals having a low number of repeats, these effects were very variable from individual to individual. in summary, the β-defensin region is a modulator of cystic fibrosis lung disease. the pro-inflammatory response in cultured epithelium cells strongly correlates with the number of β-defensin repeats. cells with a higher number of repeats respond to tnfα treatment, which in turn results in a better antimicrobial activity of the surface liquid. rationale: studies of affected twins and siblings demonstrate that modifier genes are major contributors to variation in cystic fibrosis (cf) lung disease severity. we performed genome wide linkage analysis to identify regions likely to contain modifier genes affecting severity of cf lung disease. methods: 683 individuals with cf from 360 families were analyzed. pulmonary function data were collected from patient chart review and were supplemented with data from the us cystic fibrosis foundation patient registry. to minimize environmental variation, only data obtained while subjects were living with an affected twin or sibling were analyzed. the pulmonary phenotypes were defined using the best cf-specific percentile for fev1 (kulich, et al) within the last year of available pft data as a crosssectional measure (maxfev1cf%) and using two longitudinal measures: the lifetime average cf-specific % for fev1 (avgfev1cf%) and the estimated percent-predicted fev1 at age 20 (estfev1%pred@20yrs, schluchter, et al). longitudinal measures were derived from a minimum of 4 years of pft data. short tandem repeat markers (strs) were typed in all affected individuals and their parents (marshfield genotyping center: 402 markers or decode genotyping center: 1030 markers). two-point and multipoint linkage analyses were performed using sequential oligogenic linkage analysis routines (solar). results: patients represented the spectrum of lung disease severity, with maxfev1cf%'s ranging from 0 to 1, mean 0.70 ± 0.26 and avgfev1cf% ranging from 0.01 to 0.99, mean 0.59 ± 0.24. the maxfev1cf% was predictive of avgfev1cf% (r=0.89, p<0.0001) for the 486 individuals for whom both measures were available. the two longitudinal measures were also highly correlated (r=0.80, p<0.0001). linkage was found at chromosome 5 for all three phenotype definitions. peak multipoint lod scores on chromosome 5 occurred at 196 cm for maxfev1cf% and avgfev1cf% (lod 3.0 and lod 3.4, respectively) and at 191 cm for estfev1%pred@20yrs (lod 2.8). single point lod scores on chromosome 5 peaked at marker aaat072 (3.3 for maxfev1cf%, 3.4 for avgfev1cf%, and 1.88 for est-fev1%pred@20yrs). the region of linkage encompasses approximately 6 megabases near the telomere of chromosome 5q. conclusions: chromosome 5 appears to contain one or more genetic modifiers of cf lung disease severity. supported by the nhlbi, cff and genome canada through the ogi. cystic fibrosis-related diabetes (cfrd) is the most common extrapulmonary complication of cf and is an increasingly important contributor to morbidity and mortality as cf patients live longer. while pancreatic fibrosis and loss of exocrine and endocrine tissue are common in cf, 20-30% of cf adults develop defects in insulin secretion and accumulation of islet amyloid polypeptide, features typical of type 2 diabetes (t2dm) in the general population. to test whether modifier genes play a role in cfrd, we compared concordance rates for cfrd in 68 pairs of monozygous (mz) twins, 22 sets of dizygous (dz) twins, and 470 sets of 2 or more siblings (1176 individuals with cf). criteria for defining cfrd included physician diagnosis, treatment with insulin/oral agent, and 2 episodes of glucose ≥200 mg/dl. mz twins were highly concordant for cfrd (9 of 12 pairs, 75%). the young age of dz twin recruits precluded analysis of this group in isolation (0 of 4 pairs were concordant). twelve of 71 (14%) sibling pairs were concordant for cfrd. with heritability defined as: h 2 =2*(mz concordance -dz concordance), and including siblings as a proxy for dz twins, heritability is estimated as ~1.0. the same results were obtained considering only same-sex dz twins and siblings, correcting for differences in age and duration of clinical follow-up, or restricting analysis to ∆f508 homozygotes. these data support a significant role for one or more modifier genes in development of cfrd. we then tested whether cfrd correlated with a strong family history of adult-onset diabetes (at least 1 first-degree or 2 second-degree relatives on the same side of the family). of those reporting family history of diabetes, 20 of 69 had cfrd, compared to 34 of 271 with no family history (or=2.84 [1.5-5.4 ]; p=0.001). this correlation persisted after adjusting for age, sex, and pancreatic insufficiency (or=2.6; p=0.028). thus, family history of diabetes correlated with increased risk of cfrd. we then tested whether variants in tcf7l2, a transcription factor in the wnt signaling pathway, that have been reproducibly associated with t2dm in the general population were associated with cfrd in our study subjects. genotyping of four single nucleotide polymorphisms associated with t2dm (rs4506565, rs7903146, rs12243326, rs12255372, here termed snpa-d) and transmission disequilibrium testing (tdt) of 53 parent-parent-child trios revealed significant overtransmission for snpb (31:16, p=0.03) and snpc (28:15, p=0.047), and possible overtransmission for snpa (33:22, p=0.1) and snpd (31:18, p=0.06). in every case, the tcf7l2 allele overtransmitted to patients with cfrd is the same allele that confers increased risk for t2dm. furthermore, individuals with cfrd who were homozygous for risk alleles were diagnosed at a significantly earlier age (average 14.6 vs. 20.1; p=0.03). these data support a key role for modifier genes in development of cf-related diabetes, and demonstrate that cfrd and type 2 diabetes may share disease mechanisms such as alterations in wnt signaling. supported by nih dk076446, hd27799, dk44003 and hl68927, and cf foundation grant cuttin06p0. cystic fibrosis (cf) phenotypes and survival are highly variable among df508 homozygous patients, pointing to the existence of modifier genes and/or environmental factors that contribute to this disease. studies to identify genetic modifiers of cf are being carried out using dna from homozygous df508 cf patients. important clinical features, such as severity of lung disease, liver disease and meconium ileus (mi) status, are well defined. tgfβ1 has been previously identified as a modifier of cf lung disease (drumm et al., nejm, 353(14) : [1443] [1444] [1445] [1446] [1447] [1448] [1449] [1450] [1451] [1452] [1453] 2005) , but it does not explain all of the genetic heterogeneity in this population. current evidence suggests that mucus is involved in the progression of cf, making the muc genes prime candidates as modifiers of lung disease and/or other phenotypes. methods: our approach to evaluate muc genes utilizes both variable number tandem repeat (vntr) polymorphisms and single nucleotide polymorphisms (snps). vntr polymorphisms in the muc2 and muc5ac genes (n=405 and n=366 patients, respectively) were detected by southern blotting under conditions fully optimized to maximize the allele size resolution. to minimize gel to gel variation, a genomic dna mixture with the most common muc2 and muc5ac alleles was used as internal markers. accuracy and reproducibility were evaluated by duplicating the southern on the critical dna samples for muc2 and muc5ac genes (n=85 and n=63 patients, respectively). fifty snps in muc5ac, muc5b, muc2, muc1 and muc4 genes are being tested using illumina technology in 808 cf patients. results: preliminary analysis suggests there are significant differences between cf patients with "severe" and "mild" lung disease for both muc2 and muc5ac allele distribution, which is mainly driven by the male population; exhaustive statistical data analysis still is underway. the vntr data also suggest significant association between the larger muc2 allele size and cf patients with mi. the ongoing vntr analysis will be complemented by muc gene snps being genotyped. conclusions: initial results indicate that we can reproducibly characterize muc2 and muc5ac vntr alleles. additional characterization of muc2 and muc5ac vntr alleles, coupled to snp data, will allow us to better define the significance of muc gene variations as modifiers of different cf phenotypes. supported by cff perezv06g0 (jpv), cff knowle00a0 (mk), cff r026-cr02 (wko), nih rr00046, r01 hl68890, and cff drumm00a0. reporting for the gene modifier study group (mrk). we have previously reported that βenac transgenic mice, which overexpress the beta subunit of the amiloride sensitive sodium channel (scnn1b) specifically in the airways, share common features with cf, including increased enac activity, reduced airway surface liquid, mucus accumulation and obstruction, inflammation, and death. interestingly, analysis of this model also suggested the existence of potential genetic modifiers of phenotype severity, and we speculated that identification of these modifiers would provide novel insights into disease phenotype. to establish a set of reagents that could be used to uncover genetic modifiers, we have bred βenac transgenic mice from two independent founder lines (6608 and 6047, b6:c3 background) onto several strains of inbred mice, including c57bl/6n, c3h/hen, balb/cj, fvb/j, and 129/svj. these studies revealed dramatic phenotypic differences as measured by survival (0 to 80%) among strains and between lines. all lines thus far tested show ~2-3 fold increases in amiloride sensitive short-circuit current as measured by ussing chambers in the trachea. complete phenotypic characterization of c57bl/6n line 6608 at backcross generation 12 reveals high survival (66±5% in comparison to 54±4% of the mixed b6:c3 background), yet the mice maintain the pulmonary features of the originally reported mice, including increased mucus plugging, mucous cell hyperplasia, neutrophilic and eosinophilic inflammation peaking at early timepoints (5 days -2 weeks). lymphocytic nodules, which are not commonly seen in 4-6 weeks-old animals with mixed strain background, are a common feature in the c57bl/6n congenic line. emphysema and early airway epithelial cell necrosis, two phenotypes initially not strongly associated with transgene expression, are also observed. line 6608 on balb/cj and c3h/hen backgrounds has reduced survival compared to the c57bl/6n background, and generation 8 backcross lines are now being evaluated in these two strains for other phenotypic characteristics. analysis of backcross data from line 6047, which has low survival on all genetic backgrounds tested to date, including c57bl/6n, suggested that the transgene may have integrated onto a c3h locus with a dominant negative effect on survival. genetic analysis using genome-wide snp genotyping revealed a region of chromosome 4 linked to the transgene in line 6047 (lod score > 3.0). further analysis of this region is underway. in summary, backcrossing onto different genetic backgrounds is revealing genetic modifiers for phenotypes in the βenac overexpressing model. characterization of these phenotypic and genetic differences should provide clues about the mechanisms relevant to disease development. furthermore, inbred lines with variable phenotype will likely be an important reagent for the cf community as the utility of this model is evaluated in future studies. supported by nih (scor p50 hl60280) and cff (mall04go, oneal07go). introduction: new york state screens newborns with immunoreactive trypsinogen levels within the top 5% of all infants, for 32 common cf gene mutations. infants found to be heterozygote carriers are referred to a cf center to determine sweat chloride concentration. the proposed abnormal sweat chloride value for this group of newborns is ≥ 40 mmol/l, ≥ 3 standard deviation (sd) above the mean (farrell, 1996) . this retrospective study reports on the mean sweat chloride value + 3 sd in cf heterozygote newborns who have been referred for evaluation to suny upstate medical university cystic fibrosis center, and regarding the genotype of these infants with abnormal sweat chloride levels. method: from october, 2002 to december, 2006, 404 infants were referred for positive cf screening, and 300 of these (75%) were identified as heterozygote carriers by the screening program. at our center, these patients underwent pilocarpine iontophoresis, followed by collection of sweat (≥ 20 µl) in macroduct ® coils. sweat testing was performed successfully in 279 of the 300 (93%) patients. the patients' age (mean ± sd) at the time of sweat testing was 45.8 ± 19.0 days. for infants with an initial sweat chloride level ≥ 24 mmol/l, the sweat test was repeated within a week and a complete gene sequencing was requested (quest laboratory, ca). results: the sweat chloride level (mean ± sd) in newborns who were heterozygous for a cf mutation (excluding those who were found to have an additional mutation or deletion) was 10.4 ± 4.5 mmol/l (n = 273). the mean + 3 sd was 24 mmol/l, which defined our minimal value for an abnormal test. eleven infants had sweat chloride values of 27-91 mmol/l (table) ; 6 of them (55%) were subsequently diagnosed with cf by complete gene sequencing. four of the 5 remaining patients (80%) had ∆f508 mutation coupled to the 5t variant on the opposite chromosome 7 (sweat chloride levels, 24-41 mmol/l). conclusions: the reference range for sweat chloride in cf heterozygote infants appears to be significantly lower for some centers than previously reported. thus, each cf center should consider evaluating the cutoff values for the test at their site. moreover, the 5t polymorphism may account for sweat chloride elevations in heterozygote infants. method: we reviewed the charts of all 28 patients with cf who were referred as a result of the new york state newborn screening program to the suny upstate medical university cf center from october, 2002 through april, 2007. we included the patients who were identified by the state as heterozygote carriers of one of 32 common cf gene mutations, and whose second mutation was identified only after complete cf gene sequencing. results: seven of the 28 patients (25%) met inclusion criteria. six of the 7 patients (86%) were compound heterozygous for a novel or a rare cf gene mutation. one patient was compound heterozygous for a large deletion in the cf gene. the genotype and clinical status of the 7 patients are shown in table. all patients have been pancreatic sufficient to date. conclusions:the clinical effect of compound heterozygosity as a result of novel or rare cf gene mutations appears to be mild in early childhood. prior to newborn screening (with the exception of the patient with cftr deletion), these patients may not have been diagnosed with cf in the first few years of life. these patients may have subclinical airway inflammation and thus benefit from early treatment. patients with cf manifest symptoms in the pancreas, respiratory tract, male reproductive tract and sweat gland due to mutations in cftr. patients with non-classic cf have disease in a subset of these organ systems. most non-classic cf patients have two disease-causing mutations in cftr and at least one mutation permits residual cftr function. a subset of non-classic cf patients have only one cf-causing mutation after screening for a panel of common cf-causing mutations or following mutation scanning of the coding region of cftr. these patients present a diagnostic dilemma and a challenge for genetic counseling. we evaluated 10 cf patients with only one cf-causing mutation identified after a screen of 97 cftr mutations (3 patients) or scanning of the coding region of cftr (7 patients). nine of these patients, including one set of siblings, have non-classic cf with borderline or elevated sweat [cl -] plus lung disease (table) . one patient is pancreatic insufficient and has classic cf. many of these patients have features which are consistent with cftr dysfunction including a cf-like nasal potential difference (npd),p. aeruginosa infection, or congenital bilateral absence of the vas deferens (cbavd), suggesting that they have a second cftr mutation. mutations that are not detected by screening methods include insertions or deletions, mutations outside of the cftr coding region that affect rna splicing or expression, or mutations in the coding region of cftr that were missed by screening methods. to exclude the third possiblity, dna sequencing of the 27 exons and flanking introns of cftr was performed. a second mutation was identified in the coding region of cftr in 6 of the 10 patients; 3 had screening for 97 known cftr mutations (genzyme), while the remaining 3 had comprehensive scanning of the coding region of cftr by modified tgge (ambry). each of the mutations identified by sequencing has been previously described in patients with cf and is predicted to cause cftr dysfunction. these results reaffirm that patients with one cftr mutation who have biochemical and clinical features of cf are likely to have a second mutation in the coding region of cftr. thus, we suggest sequencing cftr in patients with only one mutation after mutation screening before employing other more complex genotyping methods (insertion/deletion or rna analysis). diagnostic criteria for confirming cf in symptomatic individuals includes two positive sweat tests or two known disease-causing cftr mutations. accurate sweat testing is performed at accredited cf centers, while cftr testing is available through national and specialty genetic labs. most labs offer analysis of a basic panel of cftr mutations as recommended by the american college of medical genetics (acmg), while specialty laboratories may offer an expanded panel or full sequencing/scanning. we report on one center's use of these methodologies to confirm the diagnosis of cf presenting in adulthood. the charts of 20 patients diagnosed with cf at 〉18 years of age were reviewed. 19 were sweat tested. all were genotyped. results: twelve (63%) pts had two positive sweat tests (>60 mmol/l). an additional five (25%) had at least one borderline result (40-59 mmol/l). two pts had negative results (35-39 mmol/l). one refused sweat testing because dna analysis through the acmg panel had confirmed the diagnosis prior to initial consultation. genotyping results for the 20 pts are summarized in the table below. rare mutations identifiable only through gene sequencing accounted for 18/40 alleles (45%) in our population. importantly, among this group, only one pt had a negative sweat test, a suggestive 39 mmol/l. two pts with positive sweat tests and clinical symptoms failed to reveal any cf mutations after sequencing. we continue to follow pts without genotypic confirmation, based on their clinical presentation and sweat chloride levels, and have recommended additional evaluation, including nasal potential difference studies in our series of adult-diagnosed patients, sweat test results were positive, borderline, or suggestive in all cases tested. sweat testing costs $100-$250 and results are ready in a day. genotyping costs $400-$2500 and takes several weeks. we acknowledge that circumstances may arise where reliable sweat testing is not conveniently available; but in our series, genotyping with the acmg panel would have diagnosed 10% of pts; using an expanded panel would have diagnosed 35%. genotyping is an important tool for genetic counseling, determination of eligibility for research studies, furthering knowledge of cftr dysfunction and cf pathophysiology, and for confirming a cf diagnosis after borderline or suggestive sweat test results. based on our findings and the dramatic difference in cost, we conclude that sweat testing should remain the first approach in the diagnostic workup of adult patients with a clinical presentation suggestive of cf. in colorado, 318 infants with cf (non-meconium ileus) have been diagnosed with cf by a two tiered immunoreactive trypsinogen (irt/irt) based newborn screening approach. the irt/irt algorithm has been recently adopted by other screening programs with two mandatory screening tests. while most infants in colorado have been successfully identified, the program has had a missed case rate of approximately 5%. the more common approach to cf newborn screening is the irt/dna method in which the blood spot of infants with an initial elevated irt is tested for the most common cf mutations. the initial irt cutoff is lower in the irt/dna programs than in the irt/irt programs, resulting in a lower missed case rate. the considerable number of carriers identified through the irt/dna approach puts a significant burden on the genetic counseling community, as carriers are identified at a rate of 1/20-1/25 of positive irts we propose an irt/irt/dna newborn screening algorithm that will maximize sensitivity and specificity while minimizing the number of identified carriers. using new database technologies in the newborn screening lab we will be able to identify those infants with an elevated first irt (>60ng/ml, approximately 97th percentile). all infants with an irt >60ng/ml will have a repeat irt on their second state mandated blood-spot. if the second screen is also elevated (>60ng/ml), the blood spot will be tested using a panel of 43 mutations, including mutations specific to the hispanic community. infants with one or two cftr mutations will have a sweat test to confirm the diagnosis, or rule out cf. we compared the projected statistics of our current method irt/irt to the new irt/irt/dna method, and to irt/dna is presented in the table, based on 70,000 births per year in colorado. four infants (1.5%) identified under the current irt/irt protocol would not have been identified by the mutation panel proposed in the new algorithm, out of 288 genotyped, non-meconium ileus infants. three of these missed cases are hispanic. two would be identified using an extreme irt cutoff of the 99.9th percentile (150ng/ml). the projected missed case rate would be <0.7% (0.2 -2.5%, 95% ci), using the irt/irt/dna algorithm, with 23 carriers identified, maximizing both sensitivity and specificity. this algorithm may provide a better alternative to the irt/irt screening methods in states with two mandatory screening tests, and has advantages over both the irt/irt and irt/dna methods. newborn screening for cystic fibrosis (cf) is rapidly expanding and has been implemented in at least 30 states. although most newborn screening assays are done using biochemical testing, many laboratories screening for cf include both biochemical and molecular testing of multiple alleles in the cystic fibrosis transmembrane conductance regulator gene. in response to the growing need for proficiency testing (pt) materials for molecular testing, the centers for disease control's newborn screening quality assurance program (nsqap) in collaboration with the university of wisconsin school of medicine and public health, the johns hopkins hospital, and case western reserve university, created a repository of dried-blood spot specimens with known mutations in the cftr gene to be used in a pt program. twenty milliliters of blood was collected voluntarily from adult donors with cf and sent to the nsqap laboratory. each specimen was adjusted to a hematocrit of 55% before being spotted onto whatman 903 paper (75 µl per spot), dried, and stored at -20°c with desiccant. proficiency testing (pt) panels consisted of 5 to 7 blind-coded specimens from adult donors. the panels were sent quarterly to laboratories worldwide that test specimens for cf using molecular methods. laboratories were asked to report the genotype, method used, and the presumptive clinical assessment of each specimen. twenty-two laboratories participated during both quarters 1 and 2, 2007. the laboratories used 16 different methods ranging from in-house assays to commercially available kits. most reporting laboratories tested the following 3 alleles -∆f508, g542x, and g551d. another twelve alleles were detected by most participants. nine more alleles were common among commercially available kits. laboratories were evaluated based on the clinical assessments. mutations that were not detected by a particular method were not evaluated. overall, the laboratories performed well. data compiled from both quarters demonstrated that there was 1 incorrect clinical assessment and 2 amplification failures. developing a pt program for dna-based testing is complicated by the number of methods and different alleles each laboratory chooses to test. though molecular testing for cf may be complex, pt monitors the laboratory's ability to test multiple alleles, including uncommon alleles, the limitations of various assays, and the different algorithms used for screening. the repository will also allow storage and access to rare specimens that may be useful for future research but are not readily available. who did could not reproduce. genetic counseling focused on a patient's parents, who were counseled about their recurrence risk at the time of the child's diagnosis. today, cf is a disease of adulthood. in 2002, >40% of cf patients in the us were >18; by 2010 it will be >50%. together with advances in assisted reproductive technology (art), reproduction and recurrence risk are now important issues for adolescent and young adult cf patients. methods: a 19 item questionnaire was developed from the results of prior semi-structured interviews with 18 cf patients age 16-25 years. knowledge based questions (medical issues, inheritance, and reproductive options/risks) as well as communication patterns (preferred resources for learning about cf and preferred people with whom to talk about reproductive issues) were addressed. recruited from the uab cf clinic population, 51 patients age 15-29 (mean 21), 24 male (47%), 27 female (53%), completed the questionnaire. results: regarding autosomal recessive inheritance of cf, only 33% knew that two carriers have a 25% chance of having a child with cf, and 25% knew that two carriers have a 50% chance of having a child who is a carrier. however, 82% knew that two carriers could have a child who did not have cf, and 52% knew that two carriers could have a child who did not carry cf. on their own reproductive risks, 59% knew that a cf patient had a 0% chance of having a child with cf if their partner was not a carrier, but only 26% knew that all their children would be carriers even if their partner was not a carrier. in the scenario of a cf patient with a cf carrier partner, 44% knew that a child had a 50% chance of having cf, and 24% knew that a child had a 50% chance of being a cf carrier. most patients knew about their reproductive potential, as 96% responded that cf patients are able to have children. however, when asked about whether the chance for having children was different for males and females with cf, 65% answered that it was more difficult for males, 8% that it was more difficult for females, and 27% answered "not sure." while 62% reported that they knew that there were options for male cf patients who wanted to have children, only 26% knew of art. conclusions: despite widespread availability, the lack of knowledge of adolescents and young adults with cf about the genetics of their disease continues. furthermore, these patients are unaware of both modern technologies that could enable them to have biological children and the risk of those children having cf. this study illustrates the changing needs of patient education as medical knowledge progresses. cf patients would benefit from further genetic knowledge and counseling to enable them to make informed decisions about reproduction as they mature into adulthood. center at the university of minnesota is one of three sites in the state providing confirmatory testing and follow up services for newborns identified by screening. while cf nbs identifies children with cf, most of the infants with positive screening results are carriers. our goal is to provide genetic counseling to every cf nbs patient seen at our center, and we believe that a protocol incorporating genetic counseling in the initial care plan for infants both with and without cf is imperative. the literature has shown that families who obtain genetic counseling through the cf nbs process recall genetic information more easily and accurately and are more likely to have testing to determine parental carrier status. for parents of a child that is determined to be a cf carrier, it is especially important to find the optimal method and timing of genetic counseling as many of these families are from several hours away and therefore less likely to return to clinic. to provide genetic information and emotional support for the families of infants screening positive on cf nbs, our center has a genetic counselor who serves as the cf nbs coordinator and clinical contact for the family. this allows many opportunities to speak with the genetic counselor and ask questions, as well as learn about their child's diagnosis or carrier status and the subsequent carrier testing recommendations for the infant's parents and families. to assess the success and impact of the minnesota cystic fibrosis center's nbs follow-up program and the incorporation of genetic counseling, an anonymous questionnaire was developed for parents of infants who were seen at our center due to a positive cf nbs result. questionnaires were mailed to parents of all infants seen at our center for a sweat test and genetic counseling due to a positive cystic fibrosis newborn screening result. as of the last mailing, this totals 54 families. two questionnaires were returned due to incorrect address and 28 questionnaires were returned answered, indicating a response rate of 54% (28/52). responses overwhelmingly indicated that parents were satisfied with our center's algorithm for the cf nbs follow-up program and found the information and support provided through genetic counseling to be a useful and recommended portion of the program. as cf nbs continues, it is critical that we learn about the patient's experience with genetic counseling and the nbs program, as well as identify areas needing improvement. genetic counseling is vital to the comprehensive success of our center's program, and we will report on the responses gathered from the families identified through cf nbs this first year, as well as discuss the lessons learned from setting up such projects on a state-wide basis. background & aims: aberrant splicing and nonsense mediated decay (nmd) lead to dysfunctional mrnas by skipping exons and to a reduced number of functional mrna respectively. both mechanisms have a strong quantitative aspect and may determine whether a cf patient develops a classic or atypical disease phenotype. in order to approach these highly important questions we wanted to establish a new quantitative real-time pcr based assay which allows allele specific quantification on cdna level. using this assay we like to determine the exact proportions of the f508del and non-f508del cftr mrna in cf patients compound heterozygous for the f508del mutation (for example in cf patients carrying the f508del and a nonsense mutation such as the r553x). material & methods: materials: we used genomic dna (gdna) and total rna (extracted from white blood cells and nasal epithelial cells respectively) from cf patients with compound heterozygosity for the f508del mutation, homozygosity for f508del mutation and from healthy individuals (controls). methods: the lasq (ligation dependent allele specific quantification) assay comprises 4 reactions: 1. reverse transcription of cftr mrna into cdna using gene specific primers (all rna specific). 2. overnight hybridization (12-16h) of the cftr cdna with either the f508del specific or the wt specific oligo probe pair provided by jan schouten (mrc holland). 3. ligation of the hybridized oligo pairs using the ligase 65 enzyme from mrc holland. 4. quantitative real-time pcr of the allele specific ligation products on the lightcycler (roche). results: in order to establish the lasq assay we first validated it using gdna instead of cdna as template. mixing experiments were performed to verify the accuracy of the assay. in brief, gdna (c=20mg/l) of a f508del homozygous and a f508del compound heterozygous cf patient were mixed in such a manner that 0.1, 0. amplification products of the f508del and the wt allele (both 328bp long) were analyzed by gel electrophoresis (page) and direct sequencing (abi 3100) to control specificity. our results using gdna and cdna showed that there occurs unspecific hybridization/ ligation for both probe pairs. the proportion of unspecific amplification products varies between 0.01 and 0.005 and increases the lower the initial number of templates is. however, the specificity of this assay can be significantly improved by increasing the hybridization temperature and/or decreasing the ligation time. conclusion: although some minor limitations concerning allele specificity the lasq assay has been proven to be an accurate, reliable and reproducible method for allele specific quantification and may be applied for several important questions in cystic fibrosis such as the exact determination of the amount of nmd of cftr mrna containing a premature termination codon (ptc) or the allele specific determination of aberrant splicing of cftr mrna . background & aims: as clinical presentation varies significantly among cf patients with the same genotype, e.g. in the f508del homozygous, it is evident that factors in addition to the cftr genotype such as modifier genes, are involved in determining disease severity. however, only one of several previously postulated modifier genes, the tgfβ1 gene, could recently be confirmed in a large association study. hence the identification of new modifier genes is a very important task in order to find new explanations for the heterogeneity of pulmonary disease in cf patients. we decided to search for new potential modifier genes applying a quantitative proteomic approach comparing the proteomes of a wild type (16hbe14o-) and a f508del homozygous bronchial epithelial cell line (cfbe41o-). the main goal of this study is the identification of up or down regulated proteins in the cfbe cell line which may act as modifiers of cf disease. material & methods: materials: we used two bronchial epithelial cell lines, e.g. a wild type (16hbe 41o-) and a f508del homozygous (cfbe 14o-) cell line which we obtained from dr. gruenert (california, usa). additionally, we also used nasal cells from f508del homozygous cf patients obtained either from nasal brushings or nasal polyps. methods: proteome analysis was performed by making 2d-gels using high sensitive staining protocols (ruthenium and deep purple). quantitative analysis was accomplished applying the powerful dige (difference in gel electrophoresis) method. for each dige experiment we made 4 gels whereby each cell line was twice labelled with cy3 and cy5 (=4 technical replicates). finally, identification of the protein spots was done by the use of a maldi-tof mass spectrometer. results: in a first step we established the proteomes of the two bronchial epithelial cell lines. we were able to optimize protein extraction and 2d gel electrophoresis in such a manner that the proteomes of the two cell-lines looked very similarly and the assignment of spots could easily be done. protein spots from both cell lines were analyzed using our mass spectrometry (maldi-tof ms) and allowed the identification of more than 60 different proteins so far. in a next step we quantitatively compared the proteome of the two cell lines using the 2d-dige method leading to the identification of 8 proteins which are down regulated and 5 proteins which are up regulated at least twofold in the cfbe cell line. out of the aforementioned 13 differently expressed proteins 3 could already be identified because they were among the 60 previously determined proteins. while glutathione s-transferase p and protein s100-a11 (s100 calcium binding protein) were down regulated (3.13 and 2.08-fold respectively), superoxide dismutase was up regulated (2.62-fold) in the cfbe cell line. conclusion: comparative quantitative proteomics using the dige method is a promising tool in search of potential new modifier genes which may unravel one of the key problems in cf: the large heterogeneity of pulmonary disease in f508del homozygous patients. background: cystic fibrosis is one of the most common autosomal recessive disorders among caucasians, and manifests a wide range of disease severity. although this range of disease expression can be attributed, in part, to specific mutations within the cftr gene, much of this variability has not been adequately explained. from the gene modifier study (gms-a multicenter study of 1,306 cf patients), ten genes were tested as potential modifiers of cf, and the tgfβ1 codon 10 cc genotype was associated with lung disease severity. objective: to test whether the adverse codon 10 cc genotype is associated with higher circulating levels of tgfβ1, compared to the tt genotype in cf patients and healthy controls. if true, then a link will be established between genotype, disease severity, and circulating levels of tgfβ1, and have implications for novel treatment of cf patients. methods: the study includes 60 clinically stable cf patients and 60 healthy control subjects equally distributed between the cc and tt genotypes. the cf patients enrolled are age 8 and older, of both genders, and all ethnicities that fulfilled the standard diagnostic criteria for cf, using the genetic information from the gms. healthy controls are age 18 and older, caucasian males and females, obtained from the environmental polymorphism registry (epr), a dna registry of 20,000 self-reported normal volunteers. we genotyped 485 blood samples from the epr to define 30 healthy control subjects for each of the cc and tt cohorts. subjects have a blood draw of 35 ml. blood is divided into 4 tubes and each tube is used to measure a different parameter: cbc with differential; tgfβ1 levels in platelet poor plasma by quantikine human tgfβ1 elisa kit; tgfβ1 levels in the buffy coatwhich includes platelets; and tgfβ1 mrna levels in lymphocytes from the buffy coat, using real-time pcr roche light cycler. cbc with differential is performed to quantitate lymphocytes and platelets in order to reference tgfβ1 protein and rna levels to the number of circulating blood cells. analyses will include graphical comparisons between the two groups, chi square analysis, student's two-sample t test, and one way analysis of variance (anova). results: pilot studies show that elisa tgfβ1 measurements are reproducible and mrna levels can be quantified. we have currently enrolled 14 cc genotype and 26 tt genotype of the 60 cf subjects and 11 cc genotype and 19 tt genotype of the 60 healthy controls. blood samples have been collected and processed. conclusion: genetic variants that predispose to more severe cf disease are potential targets for new therapies. we are testing the hypothesis that the adverse (codon 10) cc genotype is likely to reflect increased transcription and/ or tgfβ1 protein synthesis/ secretion. if true, "anti-tgfβ1" therapies could provide a novel therapy in cf. *reporting for the gene modifier study group. supported by cff knowle00a0, cff drumm00a0, gcrc rr00046, nih 5r01 hl68890. aim: centralized periodic evaluations of data from the screening laboratories and cf centers by afdphe (french association for screening and prevention of infant handicaps), were analysed to optimize the efficiency of the program. methods: the strategy combined d3 irt assay/dna analysis (kit elucigen cf-30 arms) /d21-fail-safe irt. revised irt-cut off levels were decided in order to maintain the percentage of positive screens around a 0.5% target. a questionnaire yearly sent to the cf centers collected the cf false negative cases. results: from 2002 to december, 31 2005, 625 cf cases were detected through nbs (2 717 905 screened infants). the i period (p) (n=553 248); d3-irt:60 µg/l and d21-irt: 30 µg/l showed a) 0.82% infant above the d3 cut-off, generating an excess of costly dna tests b) 20% had a d21-irt above the cut-off leading to a very high number of st (n=808) with an extremely low rate of cf (n=1). by increasing slightly both d3/d21 irt cutoff levels (65 µg/l, 40 µg/l) during the ii p (n=1 172 868) a) the number of positive screens decreased to 0.64% b) 11.5% of infants with elevated d21-irt had to be referred for st (n=768) with 9 cf diagnosis. since the risk to had true cf remained very low among infants with no detected mutations, during the iii p (n= 991 789), d21-irt concerned only the ones with d3-irt>100 µg/l and the percentage of infants requiring a st was reduced to 2.5% (n=146, 5 cf). the incidence of cf detected during these 3 p did not vary significantly (1/4289-1/4635-1/4081). another point of concern was the false negative cohort; with a follow-up period over 18 months, 23 cf were detected on clinical symptoms (3.4%) at a mean age of 10 months. only 3 were directly related to the modifications of the strategy. conclusion: centralisation of data made possible changes in the flow charts of the screening strategy to limit the number of false positive cases without significant alteration of the global performance of the program. there were three infants of mixed aa/caucasian origin diagnosed with cf, all had ∆508/genotype on screening. mutation data on the 53 additional aa cf patients followed at the 11 cf centers in new york was collected. six patients have not been genotyped. there were 6 patients who were homozygous for ∆508, 14 patients had only one mutation identified and 5 were not found to have any cf mutations. ∆508(27%) and 3120+1g>a(11%) were the most common mutations. there are five cf patients (including 3 infants diagnosed by nbs) with mixed racial origin who are not included in this analysis. mutations results: the median (range) mbl plasma level was 2.46 (0.04-11.14) µg/ml in cf patients, compared to 1.43 (0.02-11.30) µg/ml in controls. mbl2 genotype frequencies were similar in patients and controls. lung function level was not correlated with mbl genotype or plasma level. the frequency of colonization with pseudomonas aeruginosa was 41% in mbldeficient (xa/o and o/o genotype) children with cf and 15% in mbl-sufficient (a/a and ya/o genotypes) children (p=0.04). we found a trend of a decreased age of first onset of colonization with staphylococcus aureus, haemophilus influenza and pseudomonas aeruginosa in mbl-deficient cf patients (p=0.19, p=0.10, and p=0.14, respectively). conclusions: mbl-deficiency was associated with an increased frequency of pseudomonas aeruginosa colonization in children. mbl deficiency was not associated with lung function deterioration. in a larger cohort we hope to confirm that mbl deficiency influences age at onset of bacterial colonization in cf patients. acknowledgments although there is some evidence that cftr gene mutations may be associated with respiratory diseases, little is known about the relationship between cftr gene mutations and idiopathic bronchiectasis (ngiam et al. 2006) . we have recently showed that a rare allele (-33g>a) in the minimal cftr promoter, previously reported in patients with idiopathic bronchiectasis ( using supershift assays, we demonstrated that these transcription factors bind cftr promoter in vitro. a functional analysis, by using co-transfection assays with expression vectors of each transcription factor in pulmonary epithelial cells, showed that nrf2, irf1 significantly decrease cftr expression, whereas irf2 and sp1 increase it. in an attempt to further elucidate the mechanisms involving these factors in the cftr transcriptional regulation, for instance, to determine whether these factors could interact together in order to regulate cftr transcription, we started several experiments such as co-immnoprecipitation, multiple co-transfection and rnai. taken together, these data evidence that the variant -33a in cftr promoter should be considered as an important risk factor in bronchiectasis pathogenesis. furthermore, we have identified a novel regulatory complex on the minimal cftr promoter, which will enlighten the understanding of the transcriptional regulation of the cftr gene. a better knowledge of cftr cis-and trans-acting elements will allow to consider new approaches to modulate and/or control more specifically cftr expression. this work is supported in part by the association vaincre la mucoviscidose. (nat genet, 1999) . a recent study reports strong genetic influences for mi (blackman et al., gastroenterology, 2006) , but it did not replicate the modifier locus on chr. 19q13. inconsistency of results may reflect the variability of data reporting and different classifications of mi (i.e. surgically or medically treated). we tested the accuracy of reporting of mi on case report forms (crfs), as compared to primary source documents. methods: the crf for the gms has a checklist for past medical history. for example, we requested "yes" or "no" for mi (an obstruction of the terminal ileum at birth), but did not require source documents or identification of the type of treatment. to evaluate crf reporting of mi, we requested source documents for patients with reported mi, including a surgical or medical treatment report. if a written report from the time of birth was unavailable, a clinic note, detailing mi at birth, treatment, and evidence of a surgical, abdominal scar (if applicable), was required. verbal confirmation by the patient and evidence of a surgical scar was also accepted, if no written documentation was available. results: on crfs, 233 of 1371 patients (17%) were initially reported to have mi. to date, source documentation has been obtained for 140 patients, and 112 of those (80%) have been confirmed to have mi (92% by written report, 8% by verbal report). of the 112 with confirmed mi, 92 (82%) had surgery, and 20 (18%) had medical treatment. there were 10 false reports of mi (7.1%). mi could not be confirmed or refuted for 18 patients (12.9%) because of insufficient information (n=16) or confounding circumstances (n=2). to date, documentation for 243 patients who were reported to have no mi has been obtained, and no false negatives have been found. additional documentation for mi from other sites is expected. conclusion: at least 7%, and perhaps as many as 18%, of the reports of mi on crfs were inaccurate. gene modifier studies must include rigorous documentation of mi to ensure an accurate correlation between phenotype and genotype. studies should also characterize different classifications (i.e. surgical vs. medical treatment) to assist the detection of gene modifiers of mi. reporting for the gene modifier study group (mrk); supported by cff knowle00a0, cff drumm00a0, nih 5r01 dk66368, nih 5r01 hl68890, and gcrc rr00046. any mutation that disrupts or diminishes the efficiency of the splicing process will have an impact on disease manifestation. in the cystic fibrosis (cf) transmembrane conductance regulator (cftr) gene more than 1,500 mutations were identified, most of them being disease-causing [1] . among these,~42% are classified as missense and about 13% are classified as splicing mutations, given that they disrupt the consensus splice sites [1] . however, the splicing mutation concept is evolving, as it nowadays also includes mutations other than just those within or close to the consensus splice sites, rendering prediction of mutation consequences a hard task. nevertheless, it is still very important for the clinical settings to determine the functional effect of gene mutations. our aim here was to study the effect of i1234v, a rare cftr missense mutation in nbd2 (exon 19), directly in native tissues of a cf patient bearing f508del in the other allele, so as to gain insight on how it influence the disease outcome. to look for a possible effect at the rna level, total rna was extracted from native nasal cells and colonic tissue, and cdna producd using random primers. rt-pcr amplification was performed in the region spanning exons 18-20, being one of the primers fluorescently labelled. the products were analysed as described before [2] . the i1234v(3832a>g) mutation which creates both a novel acceptor and a novel donor, was found here to cause alternative spliced cftr transcripts lacking the last 18 nucleotides of exon 19, thus showing that only the novel donor is used in vivo. moreover, our data show that no normal (only alternatively spliced) transcripts result from this allele. we have also analysed the i1234v mutation at the protein level by producing a stable bhk cell line expressing the i1234v-cftr, after generating the respective mutant cdna construct by site-direct mutagenesis. protein expression and function was determined by immunoblot and iodide efflux assay, respectively. results show that i1234v-cftr protein is processed and functional. however, given the above-described absence of normally spliced mrna coding for this cftr variant, we have to conclude that this protein is not produced in vivo. indeed, since only the mrna coding for cftr lacking the last six aminoacids of exon 19 was detected, we are currently characterizing in vitro the proprerties of this truncated protein. altogether, our data clearly demonstrate that the functional effect of this mutation is not due to the amino acid change but to abnormal splicing. in conclusion, characterization of the consequences of mutations in native affected tissues is important, not just because this provides unexpected information about the mechanisms underlying the basic defect but also for disease diagnosis and prognosis. (darrah et al, nacfc 2006) . the purpose of this study was to examine the influence of common genetic variation in the endothelin pathway on the cf phenotype. methods: patients were recruited from the cf clinics in dublin, belfast and seattle. serial clinical data were abstracted from medical charts and local clinical databases. studentized residuals of maximum fev1, after adjusting for age, gender and height, was the phenotype of interest. twentyone maximally informative tagsnps were identified in the edn1, edn3, ednra and ednrb genes using the niehs environmental genome project and were genotyped using the illumina beadarray system. genotype and haplotype analysis were carried out using helixtree genetic analysis software. results: clinical and genetic data were available on 544 cf patients (239 from seattle and 305 from dublin/belfast). in the combined cohorts, tagsnps in the ednra gene were significantly associated with differences in cf lung disease severity (p=0.000026). this was observed in both the irish cohort (p=0.0049) and the seattle cohort (p=0.00507). the effects were independent of gender and cftr genotype. four common haplotypes (haplotype frequency>5%) were identified in the ednra gene. there was significant association between ednra haplotypes and cf lung disease severity (table 1 ). there was no association between genetic variants in ednrb, edn1 and edn3 and cf lung disease severity. conclusions: the ednra gene is a genetic modifier of the cf phenotype. our findings were seen in two independent cohorts and verify existing associations found in other populations. the endothelin pathway may be a novel therapeutic target for the treatment of cf lung disease. background: cystic fibrosis (cf) is a recessive "monogenetic" disorder, but there is heterogeneity of lung disease severity and survival reflecting environment and non-cftr genetic modifiers. the unc/cwru multisite gene modifier study (gms) identified patients who were "severe" or "mild" as teenagers or young adults and pertinent cross-sectional data was collected; however, we did not collect all pertinent information about early (age <8 years) clinical features. objective: we sought to retrospectively evaluate the early clinical features of "severe" and "young mild" patients enrolled in the gms. methods: we obtained all cff registry data available on 596 patients. there were 303 "severe" (worst 25 th percentile of birth cohort, age range 8-25) and 293 "mild" (best 25 th percentile, age range 15-28). initial analyses focused on cross-sectional plots of patient age versus multiple clinical features, including age at diagnosis, hospitalizations, cdc height and weight percentiles, presence/absence of ps. aeruginosa from respiratory cultures, and fev 1 (% pred). results: there were 15.5 and 17.6 years of cff registry data per patient for "severes" and "milds," respectively. preliminary results indicate "severe" patients were diagnosed earlier in life than "mild" patients (mean: 1.2 vs. 1.9 years, p=0.004). this difference is greater after omitting patients who had meconium ileus (diagnosis: 1.4 vs 2.3 years, "severes" and "milds" respectively, p=0.003). between 1 and 7 years of age, "severe" patients were hospitalized more frequently than "mild" patients and, from age 8 onwards, the disparity in frequency of hospitalization increased. "severes" and "milds" had similar cdc height percentiles (~35 th percentile) until age 8, at which point percentiles increased more for "mild" versus "severe" patients. by age 3, "severe" patients already had lower cdc weight percentiles than "milds" (32 nd versus 41 st percentile) and this disparity increased throughout adolescence. from age 1 to 8, "severe" patients had a 2-3 fold higher prevalence of ps. aeruginosa than "mild" patients. as early as 6 years of age, fev 1 (% pred) was ~30 points lower in "severes" as compared to "milds." however, some "severe" patients had normal lung function at ages 6-8 and overlapped with mild patients; thus phenotyping of lung severity for young patients is not optimal. all of these results were similar for males and females. summary: retrospective analysis of the cff registry data indicates that patients classified later in life as being "severe" experience a worse course of disease from early in life. understanding the early clinical course may prove helpful in defining surrogate phenotypes for modifier studies, and help define appropriate therapeutic targets. cystic fibrosis is mainly caused by small molecular defects of the cftr gene; despite the genotype is defined in the majority of patients, a number of cf cases still remain uncharacterised. the cf mutation database lists more than 35 large rearrangements that may escape detection using pcrbase techniques. the innogenetics assay, the dhplc and sequencing screening showed a mutation detection rate of 92.6% in our population. we report here the results of mlpa screening for ctfr gene rearrangements, performed on the unidentified alleles of our cf patients. our sample population consists of 691 unrelated italian cf patients (for a total of 1378 alleles), followed at cf centres of lombardia region. mlpa analysis was performed in 40 patients who still had one or two unidentified alleles after extensive analysis of cftr gene. all patients studied had classical clinical cf symptoms. subjects presented with persistent or recurrent respiratory symptoms, failure to thrive, salt loss syndrome and gastro-intestinal findings. we characterized 10 different deletions and a new duplication (dup promoter-ex.3). thus, 26.2% (21/80) tested alleles had a large gene rearrangement. the deletion of exons 22-23 (6/80) was the most frequent in our cohort. all patients had positive sweat chloride values (above 60meq/l), except the patient carrying duplication who has borderline sweat chloride value. out of 24 patients, 6 (25%) had fecal elastase levels consistent with a preserved pancreatic function: of these patients, 3 had mild mutation, 1 had severe, and 2 had unknown mutation. six patients present liver involvement. the results of the present study could indicate that compound heterozygosity for large rearrangements in cftr gene, is strongly associated with severe pancreatic disease as mutations in classes i, ii, and/or iii. l997f is a missense substitution which changes from leucine to phenylalanine at position 997, resulting from a g/c transition at position 3123 in exon 17a of the cftr gene. it has been described in patients with disseminated bronchiectasis, recurrent idiopathic pancreatitis, sarcoidosis, newborns with hypertripsinemia,. recently derichs et al. reported one healthy 3-year-old girl homozygous for l997f; therefore the pathogenic role of the variant is still unclear. in this study we present 6 subjects compound heterozygotes with l997f. no other mutations have been identified after molecular analysis performed using sequencing analysis of the whole coding region of cftr gene and mlpa technique in order to search for gene rearrangements.none of them presented ivs8 5t allele. three of them had a severe mutation in trans (r553x, 2183aa>g, and n1303k), while the other three had a mild mutation on the complementary allele (d1152h, r334q, and r334w). individuals with a severe mutation in trans presented a remarkably different clinical picture compared to those with a mild mutation in trans. the formers were diagnosed at a mean age of 2.8 years, had an average sweat chloride of 55.9 meq/l; besides, all of them have respiratory symptoms, staphilococcus aureus in sputum cultures (one had pseudomonas aeruginosa as well), and in two the chest x-rays was abnormal. the three l997f/ps mutation individuals were diagnosed at a mean age of 20.1 years, had an average sweat chloride of 39.1 meq/l; besides none of them had respiratory symptoms, abnormal chest-xrays, or positive sputum cultures, but two had a history of pancreatitis. these data seem to suggest that l997f cannot in any case be considered a neutral polymorphism. the variability of its clinical expression seems to be influenced by the mutation in trans. further studies are needed in order to support our results. expression of the cystic fibrosis transmembrane conductance regulator (cftr) is tightly regulated both spatially and temporally, yet the molecular mechanism of this regulation is not well understood. because no tissue-specific regulatory elements were recognized in the basal cftr promoter, the crucial cis-regulatory elements are likely to be located elsewhere within the cftr locus. a number of the non-coding regions of the cftr gene were found by our laboratory to contain dnase i hypersensitive sites (dhs), suggesting the presence of regulatory elements at these sites. studies presented here investigate the role of an intron 1 dhs (dhs1) in the tissue-specific regulation of cftr gene transcription. the elucidation of molecular mechanisms underlying the temporal and spatial expression of cftr may aid in developing more specific targeted therapies for cystic fibrosis (cf). footprinting analysis of cftr intron 1 revealed a protected region within the core of dhs1 at 185 + 10 kb. in silico analysis of this sequence uncovered a number of transcription factors binding motifs, including a consensus binding sequence for hepatocyte nuclear factor 1 (hnf1). we have previously identified a role for this factor in the regulation of cftr expression. results of electrophoretic mobility shift assays (emsa) demonstrate that hnf1α specifically binds to the motif in cftr intron 1 in vitro. in addition, chromatin immunoprecipitation (chip) analysis of cells expressing both cftr and hnf1α factor shows that this factor binds to the intron 1 site in vivo. when cloned as an enhancer, the dhs1 element was found to augment minimal cftr promoter activity in a luciferase reporter based assay. this increase in luciferase activity was abolished when two nucleotides within the core hnf1 binding site were mutated, suggesting a functional role for hnf1α in cftr gene transcription. further experiments are underway to determine whether additional transcription factors can be recruited to the core of the intron 1 dhs regulatory element and can interact with hnf1 and the cftr basal promoter to modulate tissue-specific cftr gene expression. the promoter of the cftr (cystic fibrosis transmembrane conductance regulator) gene is not solely responsible for its complex pattern of expression. to identify potential regulatory elements for cftr we previously mapped dnase i hypersensitive sites (dhs) across 400 kb spanning the locus. in addition to intronic dhs, a number of sites were observed that flank the cftr gene. we hypothesized that these may include insulator elements that establish the chromatin expression domain, within which the cftr gene is regulated. insulators are elements that shield against the effects of regulatory elements from adjacent genes, and they may also block silencing of integrated transgenes. using a well-established insulator assay, in which dna regions of interest are assayed for their ability to interfere with communication between a chicken β-globin enhancer and a neomycin resistance gene, we demonstrated that two dhs, at -20.9 kb from the cftr translational start site, and at +15.6 kb from the 3' end of the gene, exhibit enhancer blocking activity comparable to known insulator elements. electrophoretic mobility shift assays (emsa), demonstrated in vitro binding of the well-characterized insulator protein ctcf (ccctc-binding factor) at the -20.9 kb dhs. this was confirmed in vivo in both cftr-expressing and non-expressing cell types using chromatin immunoprecipitation (chip). in contrast, although the +15.6 kb dhs did not bind ctcf, we obtained in vitro evidence for the interaction of other factors that may be involved in insulator activity. furthermore, chip analysis of histone modifications across the cftr locus revealed striking differences between the -20.9 kb and +15.6 kb dhs, again suggesting mechanistic differences between these elements. the characterization of insulator elements flanking the cftr locus may be of direct practical relevance in the design of vectors for effective cf gene therapy. one of the problems encountered in gene therapy protocols is the relatively rapid loss of expression from the cftr cdna once it is introduced into mammalian cells. incorporation of the -20.9 kb and +15. aminoglycosides, particularly tobramycin, are primary antibiotics used to treat the airway infections in cystic fibrosis patients. lifetime, systemic exposure to these antibiotics is significant and can be associated with significant nephro-and ototoxicity. these toxicities have the potential to significantly reduce the quality of life in the aging adult cf population. we previously reported an incidence of aminoglycoside ototoxicity of greater than 50% with sensitive audiometric studies (i.e. pure tone audiometry and distortion product otoacoustic emissions). these studies indicated considerable variability in ototoxicity in patients with similar systemic aminoglycoside exposure. the literature suggests that genetic variability in mitochondrial dna can partially explain these differences. single nucleotide polymorphisms (snps) in the mitochondrial 12srrna gene are associated with aminoglycoside ototoxicity. we genotyped 90 unselected adult cf patients for four of the mitochondrial 12s-rrna polymorphisms associated with aminoglycoside ototoxicity by direct sequencing of dna harvested from peripheral blood. four patients exhibited polymorphisms in this gene. two patients possessed the a1555g transition, while another patient each revealed a polymorphism at a827g and c1494t transitions. the patient with the a827g polymorphism died prior to audiometric testing. both patients with the a1555g had audiometric studies consistent with moderate to severe ototoxicity. while the patient with the c1494t had mild aminoglycoside ototoxicity. all four patients had severe airway obstruction and at least one full course of parenteral tobramycin at 8mg/kg/d. there was no history of toxic serum levels aminoglycosides during this therapy. both patients with the a1555g polymorphism had a family history consistent with the expected maternal inheritance pattern associated with the mitochondrial 12s-rrna gene. we found no evidence of the delt961 polymorphism in this population. these studies indicate a higher than expected frequency (4%) of mitochondrial 12s rrna polymorphisms associated with aminoglycoside ototoxicity. these studies demonstrate that genetic screen-ing provides valuable susceptibility information and may change clinical decision-making. support: rising hope foundation/national cf foundation. cystic fibrosis is a genetic autosomal recessive disease that is caused by deleterious mutations in the cftr gene. in its most severe form, cf results in abnormal sweat electrolytes, sino-pulmonary disease, male infertility, and pancreatic exocrine insufficiency. in fact, cf is the most frequent cause of pancreatic insufficiency in humans. carriers of cf (i.e., heterozygotes) do not express any of these classic symptoms. recent studies indicate that significant numbers of non-cf patients diagnosed with congenital bilateral absence of the vas deferens (cbavd) or idiopathic chronic pancreatitis (icp) are compound heterozygotes and carry two cftr mutations of varying severity. in dogs, especially german shepherds and rough-coated collies, exocrine pancreatic insufficiency (epi) has been observed with symptoms similar to those seen in human patients. epi in dogs is most often caused by pancreatic acinar atrophy (paa), a degenerative disease of the exocrine pancreas that has been shown to be inherited as an autosomal recessive trait. the locus responsible for canine paa has not been identified to date. we considered the possibility that cftr mutations might be responsible for this disease. a dog that had been diagnosed with paa and one that was a known carrier of paa were screened for cftr mutations. our samples also included dogs diagnosed with idiopathic pancreatitis as well as healthy dogs. we have established protocols for detecting putative mutations in the canine cftr gene using temporal temperature gradient gel electrophoresis (ttge). in the dog with paa and in the carrier of paa, our screening methodology identified mutations in 8 of the 27 amplicons that span the 27 exons of the cftr gene. dna sequencing revealed silent mutations in exons 17, 26 and 27. in exons 17 (l888) and 26 (l1402), single nucleotide polymorphisms converted ctt codons into ctc codons, both leucine codons. in exon 27 (p1447) another single nucleotide polymorphism converted a ccc proline codon into a cct proline codon. the other identified mutations (amplicons 15, 16 and 17, 20, 22, 24, 25, 26 and 27) were found in intronic sequences and have no predicted effect on cftr expression or function. based on these findings, we conclude that cftr mutations are not responsible for paa and the epi that it causes in dogs. hypothesis: specific polymorphisms in genes of the innate immunity may modify the severity and progression of cf pulmonary disease and influence pseudomonas aeruginosa colonisation. methods: single nucleotide polymorphisms (snps) analysis of multiple genes contributing to the innate immunity ( (mbl2), masp (mbl associated serine protease) 1ç3, fcn (ficolin) 1ç2, lbp (lipopolysaccharide -binding protein), cd14, tlr (toll-like receptor 1ç10) ) was performed in 116 cf patients (age 6-44 years). spirometric data and bacterial colonisation status with pseudomonas aeruginosa were collected retrospectively. a decline in fev1 of 14 % over a 6 years period and a percent predicted fev1 value of 70 % were used to discriminate mild from more severe affected adult cf patients (≥ 16 years). the frequency of single and combined snps in well-defined subgroups of the cf population was compared. results: in adult cf patients with a mean fev1 lower than 70%, the combinations of snps of cd14 (promoter) with tlr 10 (promoter), fcn 1 with tlr 5 (exon 6), and masp 3 with tlr 9 (promoter) were more common than in adult cf patients with mean fev1 > 70 % (odds ratio (or) 6,2 (95% confidential interval (ci) : 1,7-22,2 ) , or 6, 2 (95 % ci: 1, [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] 2) and or 5,6 (95% ci: 1,7-18,4) respectively). the frequency of combination snps of masp2 with tlr2 and masp2 with tlr4 was significantly higher in cf adults with more than 14 % decline in fev1 compared to cf adults with less than 14 % decline in fev1 (or 6,5 (95% ci: 1,6-25,8) and or 5,3 (95% ci: 1,5-18,6) respectively). however, the single snps of masp2, tlr2, tlr4 were not more frequently found. in pseudomonas aeruginosa colonised cf patients, an increased frequency of combined snps of tlr1 (exon 4) with tlr6 (exon 1), lbp (exon 10) with tlr6 (exon 1) and mbl2 with tlr 6 (exon 1) (or 3,6 (95% ci: 1,3-10,1), or 3,6 (95% ci: 1,3-10,1), or 3,4 (95% ci: 1,1-10,2) respectively) was found. in contrast, for single snp tlr6 (exon 1) no statistically significant differences were found between the groups (or : 2,0 ( 95% ci: 0,9-4,6)). in general, the odds ratios for the combined snps were higher than for the single snps and remained significant when the different groups were subdivided according to cftr genotype. conclusion: certain combinations of snps of genes of the innate immunity are more frequently found in cf patients with a lower fev1, a stronger decline in pulmonary function and pseudomonas aeruginosa colonisation. however, for the single snps this trend was less pronounced or absent. congenital bilateral absence of the vas deferens (cbavd) is a rare condition associated with mutations in cftr. cbavd lacks extra-genital organ system abnormalities; it has been viewed as a mild (variant) form of cf. the disease serves as an excellent model for studying the clinical and experimental ramifications of marginal cftr activity, and the functional thresholds necessary to confer a cf-related phenotype. recently, drumm et al. (nejm, 2005) found that the codon 10 cc genotype of tgfβ-1 is associated with more severe pulmonary disease, particularly in individuals homozygous for ∆f508 cftr. tgfβ is also known to play a critical role during normal development and differentiation of the human vas deferens (han-nema et al., horm res, 2007) . because 70% of cbavd subjects carry at least one defective cftr allele (but millions of males worldwide are heterozygous for cftr mutations without developing cbavd), the present study was designed to test whether tgfβ polymorphism might serve as a genetic modifier underlying isolated vas deferens loss in the setting of cbavd. we sequenced the 5' end of the tgfβ gene in eighty cbavd individuals possessing at least one cftr mutation, and compared genotype frequency with results previously published from demographically similar controls. when cc frequency in cbavd subjects was compared to pooled estimates from earlier trials, no significant differences were seen using a non-dominant model. compared to frequencies reported by arkwright et al. and garrote et al., a greater number of homozygous cc subjects (23.8% vs. 11.4%, p<0.005) was observed. our preliminary results therefore indicate that if tgfβ-1 codon 10 polymorphism is associated with cbavd penetrance, it does not represent the major modifier of disease phenotype. in order to evaluate this possibility further, we plan analysis of a larger (matched) control set of cbavd and non-cbavd individuals, studies of tgfβ-1 activity in vas deferens epithelial cells with and without cftr, and experiments to investigate the effects of tgfβ on cftr biogenesis and function. supported by nih and cff. newborn screening (nbs) for cf has been successful in identifying cf infants, particularly those who have positive sweat tests immediately following the positive screening result. tracking by personnel at the central nbs program facilitates resolution of diagnostic status because cf nbs presents a challenge to cf center (cfc) systems and primary care providers when apparently asymptomatic infants have inadequate (quantity not sufficient (qns)), borderline or indeterminate sweat tests. short-term follow up is more problematic when the screening algorithm requests earlier sweat tests and defines a lower cutoff for indeterminate sweat test results than is used for older infants, children and adults. the massachusetts (ma) cf nbs workgroup chose a low cutoff for indeterminate results of 30 meq/l up to 6 months of age, reverting to the accepted 40 meq/l cutoff thereafter. follow up by the central nbs program identified infants whose diagnostic status remained unresolved more than 6 weeks after a qns or borderline sweat test and eliminated the need for individual cfcs to follow infants tested at more than one cfc. the program sent informational letters and a reminder recommendation to finalize the diagnosis to primary care providers of infants with unresolved diagnoses. of the 189 cf infants identified by ma cf nbs, 25 had an initial sweat result that was indeterminate and another 20 had an initial result that was qns. the central screening program has the capacity to track infant and child outcomes across cfcs; for example, we have been tracking growth outcomes of all cf infants identified by ma cf nbs, despite 31 of the 189 cf infants having moved from one cfc to another during their first 5 years of life. tracking and outcomes data will be presented. when more than one cfc operates within a screening program, centralized tracking ensures capture of quality data. despite several educational campaigns cf is often not diagnosed in early infancy in poland (the mean age of diagnosis 42 months, median is 12 months). after several years of discussions,in september 2006 a neonatal screening for cf supported by the ministery of health was started as the third obligatory neonatal screening in poland. more than 124,000 neonates were screened in the first seven months using the existing screening infrastructure and spare blood spots after pku and congenital hypothyreosis screening. the protocol was two stage irt/dna (covering the 47 most common mutations in the polish population). the final diagnosis was based on sweat tests (conventional pilocarpine-iontophoresis and conductometric nanoduct) as well as clinical examination. values of chloride in sweat >40 mmol/l and nacl >80 mmol/l were considered diagnostic for cf. 12 children were confirmed as cf. although delta f508 is the most common mutation in the polish population, only two infants were delta f508 homozygotes, six were delta f508 heterozygotes and the remaining 8 had different mutations (3 children had 3849+10kbc>). half of the diagnosed group was still pancreatic sufficient. the children had confirmed cf diagnosis at the mean age of 6 weeks. only 3 children were free from any radiological changes in lungs. the most common pathogen in this group was staphylococcus aureus. at the first visit information for parents about cf was given along with physiotherapy and dietary education. anthropometric measurements, airways bacteriology and elastase -1 in stool were performed. psychologist consultations were available. neonatal screening for cf enabled earlier diagnosis and the introduction of complex therapy in comparision with symptomatic screening. objectives: the clinical course of cystic fibrosis (cf) varies widely among patients carrying the same cftr gene mutations supporting that additional genetic modifiers could affect the cf phenotype. as inflammation is a central contributor to the pathogenesis of cf lung disease, genes involved in the inflammatory response are potential modifier candidate genes. methods: we examined the influence of 13 polymorphisms in 7 genes involved in the inflammatory response (tnf, il-1β, il-1 receptor antagonist (rn), il-6, il-8, il-10 and tgf-β1), on disease progression in a group of 329 caucasian children with cf. the genotypes were tested for an association with changes in lung function tests, pseudomonas aeruginosa colonization and nutritional status by multivariable analysis. results: we found a significant association between tgf-β1 +869t/c variants and decline in lung function measured as the forced expiratory volume in one second (fev1) and the forced vital capacity (fvc) (p=0.004 and 0.02 respectively). il-8 gene polymorphisms (-251a/t, +396g/t and +781t/c) were associated with the occurrence of pseudomonas aeruginosa colonization (p<0.02). conclusions: this study suggest that il-8 variants may influence pseudomonas aeruginosa colonization in cf, however this need further confirmation. moreover, we provide additional support to the results of other trials and strongly suggest an association between tgf-β1 variants and lung disease phenotype in cf. if such a role of tgf-β1 is confirmed by functional studies, it may have important clinical impact for the identification of patients at risk to develop more severe respiratory manifestations and for the development of new therapeutic strategies aimed at adequately balancing tgf-β1 production and action introduction: cystic fibrosis (cf) is the most common monogenic disease in caucasian population, its frequency is about one in 2500 live born. the estimated frequency in hispanic population is about one in 8500, with differences in mutation frequency especially for low frequent alleles. heterogeneity in pulmonary manifestations cannot be explained by the cftr mutation genotype. recent studies are focused in the study of modifier genes. these are genes different from the primary cftr gene that could influence the fq phenotype, acting trough mechanisms like inflammation, repair and remodeling, protease-anti protease balance, innate immune response, etc. this work shows some advances in cf diagnosis and modifier genes analysis in mexican population. methods: we tested the effectiveness of innolipa cftr36 kit (innogenetics) in 23 clinically diagnosed cf patients. thirty six frequent mutations in cftr gene were analyzed by pcr and reverse hybridization with allele specific oligonucleotides probes in dna samples extracted from blood or oral swabs. for frequency analysis of modifier genes we tested about seventy dna samples from a dna bank of patients previously tested positive for cftr mutation. the polymorphism analysis of modifier genes was made by pcr-rflp. for alpha 1 antitrypsin (aat), z and s alleles were analyzed according stanford and cols. (1999) . for tumor necrosis factor alpha (tnfα) -308 polymorphism in the promoter region was analyzed according chen and cols. (2006) . results: mutation was detected in in 20 of 23 patients (87% of effectiveness). df508 allele frequency was 52% (caucasian 60-70%) followed by 2789 +5g-a, s549n, g542x, g85e and 1078delt (20%). 28% of the mutant alleles remain undetected. preliminary results in modifier genes show low frequency for aat mutant alleles (1.4% for aat s and z alelle respectively) and 8.7% for tnf-308. conclusions: df508 alelle frequency was 52%, and we found the low fequency alleles s549n and 2789+5g-a. frequency of modifier genes mutant alleles in cf patients are low. this work is in progress of selection and recruitment of patients and controls, as well as standardization of the other genes modifiers, along with some other biomarkers that will allow to understand the physiopathology of cf and to define a possible genetic risk profile of severe pulmonary disease. over 1300 putative cystic fibrosis (cf)-causing mutations have been reported to the cf mutation database and almost half (~630) are rare missense mutations that are predicted to substitute a single amino acid. to determine if any of these rare missense mutations cause cf by altering localiza-tion of cftr, we examined mutations in the cytosolic loops of cftr since localization signals have been identified in cytosolic loops of other ion channels. amino acids alignments comparing human cftr sequence to that of 36 species revealed that cytosolic loop 4 (cl4) was the most conserved loop by identity. furthermore, cl4 had the most naturally observed mutations: 33 of the 68 amino acids were found to be mutated in cf patients and 10 sites had multiple missense mutations at the same amino acid. in addition, different substitutions of the same cl4 amino acid have been associated with different disease consequences, such as the missense mutations in the arginine at codon 1070. patients with the r1070p mutation have pancreatic insufficient (pi)-cf, those with r1070w mutation have pancreatic sufficient (ps)-cf or cbavd, while patients with r1070q primarily had pi-cf. to determine whether the r1070 mutations cause disease by affecting cftr localization, we used the flp-in system to create stable polarized mdck-gfp-cftr cells to produce isogenic clones with cftr expressed from the same integration site. confocal microscopy studies of stable mdck-frt-gfp-cftr-r1070 mutant cell lines revealed that cftr-r1070p was cytoplasmic, cftr-r1070w was apical and cytoplasmic, and cftr-r1070q was apical. to confirm these observations, quantitative biotinylation studies were performed. biotinylation assays of mdck cells stably expressing cftr-r1070p confirmed that this mutant was absent from the apical surface, cftr-r1070w had a low level of fully glycosylated protein at the apical membrane, while cftr-r1070q had fully glycosylated protein at the apical membrane comparable to wild-type cftr. thus, cftr-r0170p and cftr-r1070w displayed properties in polarized cells that were distinctly different from wild-type cftr consistent with their associated cf phenotypes. however, the profile of cftr-r1070q (apical localization and cl-channel function (seibert et al. 1996 , mickle et al. 2000 ) appears inconsistent with a pi-cf phenotype. indeed, re-analysis of cftr genes bearing r1070q revealed that 11 of 12 carried a second mutation in cis (s466x). in nine of these patients in whom cftr genotype and pancreatic status is known, presence of the nonsense mutation s466x is associated with the pi-cf phenotype. since nonsense mutations are known to cause severe gene dysfunction by promoting decay of their rna transcript, we concluded that the s466x mutation rather than r1070q is responsible for the pi-cf phenotype. the finding of s466x explained the otherwise enigmatic functional studies of r1070q and clinical observations in patients bearing this mutation. this study also demonstrates that stable expression of cftr mutants in polarized mdck cells using the flp-in system provides a useful screen for evaluating the disease potential of rare missense mutations. the clinical course of cf is characterized by recurrent pulmonary infections and chronic inflammation. in cf patients, up-regulation of toll-like receptor-2 (tlr2) gene in airway epithelial cells is believed to enhance proinflammatory responses towards bacterial tlr2 ligands. we have recently shown that decreased methylation (demethylation) of the tlr2 promoter is responsible for cf-related up-regulation of tlr2 in bronchial epithelial cells (shuto. t. et al., faseb j, 2006) . however, the molecular mechanisms responsible for dna demethylation-dependent tlr2 gene upregulation in cf cells remain unknown. here, we identified minimum region of the tlr2 promoter critical for its expression at -120/+110, which contains one putative binding site for sp1. sp1 inhibitor mithramycin a treatment reduced tlr2 promoter activity and its expression in cf bronchial epithelial cells (cfbe41o-), suggesting the importance of this sp1 site for the tlr2 gene regulation. moreover, bisulfite sequence analysis revealed the hypomethylation of adjacent this sp1 binding motif within tlr2 promoter in cfbe41o-cells, implying the cf-specific demethylation of adjacent sp1 binding motif. although mithramycin a rarely affected basal expression of tlr2 gene in 16hbe14o-cells (non-cf bronchial epithelial cells), mithramycin a inhibited tlr2 expression in 5-azacytidine (dna-demethylating agent)-treated 16hbe14o-cells. taken together, our results suggest that sp1 is crucial for dna demethylation-induced gene upregulation of tlr2 in cf bronchial epithelial cells. mucus hypersecretion due to goblet cell metaplasia is a critical feature of cf lung disease, affecting both mucociliary clearance and drug delivery. various studies in cf lungs and cf primary cultures have shown increased expression of muc5ac, the main gel-forming mucin produced by goblet cells; however, little is known about the role of muc5ac in the progression of lung disease. to determine whether overexpression of muc5ac alone is sufficient to induce lung pathology, we generated a mouse model overexpressing muc5ac. the entire muc5ac cdna (minus 26% of the predicted vntr sequence, final size ~9kb) was cloned and tagged with an internal gfp epitope at a site close to the vntr to preserve the integrity of mucin domains. gfp-muc5ac was linked to the rat ccsp promoter to drive specific airway expression, primarily in clara cells since these cells are capable of secreting mucins. blastocyst injections were used to generate transgenic founder lines in c57bl/6 background. although muc5ac-gfp mrnas were expressed in transgenic mice, lung histology did not show significant increases in inflammation, ab/pas-positive cells, or luminal secretion compared to wild-type littermates. gfp fluorescence from freshly excised lung was weak but immunohistochemistry showed that the distribution of gfp correlated with the expected expression pattern, with the majority of positive cells localized to the airways and few positive cells in the alveolar space. at higher magnification, gfp-positive granules were observed in dome-shaped cells that were also positive with cc10 antibody, suggesting proper muc5ac-gfp packaging in clara cells. bronchoalveolar lavage samples separated by agarose gel western blots and probed with gfp antibody confirmed that each line secreted muc5ac-gfp, with a migration pattern comparable to a mucin. to further characterize the secreted transgenic mucin, a pool of lavage samples was separated into supernatant and pellet. the pellet was resolubilized in 4m guanidine. both elements were passed through a s1000 size-exclusion column. analysis by light scattering and refractometry to determine molecular weight and concentration revealed that the solubilized pellet contained an enormous but compact complex (920 x 106 g/mol and 320nm radius of gyration). gfp-rich fractions were pas-positive, indicating proper glycosylation of the transgenic muc5ac-gfp. cscl density gradient studies revealed that the gfp-rich fractions had a high molecular weight but did not appear to be a prominent part of the megacomplex retrieved in lavages, unlike other tested mucins. in conclusion, we showed that the overexpressed muc5ac-gfp was a high-molecular-weight protein that was glycosylated, packaged in clara cells and formed multimers. lack of discernable phenotype in the transgenic lungs suggests that mucin overproduction alone is not sufficient to trigger goblet cell metaplasia and mucus accumulation, supporting the protective role of mucins. currently we are crossing the muc5ac-gfp with the scnn1b mouse model to study muc5ac-gfp under dehydrated conditions and we are conducting allergen-challenges in the transgenic mice. results of such studies will provide novel insights into the role of secreted muc5ac in development of lung disease in these models. supported by grants from the cf foundation. the major characteristic in cystic fibrosis (cf) is acquirement of chronic lung infections with p. aeruginosa. once established the chronic infection can only be suppressed, not eliminated. the cf patients harbour one single strain of p. aeruginosa which only rarely is replaced by other strains. during the chronic lung infection there is a year long mutual impact of the bactaria on the host, and of the host response and antibiotics on the bacteria, resulting in a continous adaptation of the bactaria in the lungs of the cf patients with selection for different clones. in contrast, mouse models of the lung infection in cf is limited to two weeks! therefore, we introduced a new strategy infecting different groups of balb/c mice with pulsed field gel electrophoresis-(pfge) identical clones isolated from one cf patient during 23-years of p. aeruginosa lung infection. 1) infecting the mice with non-mucoid isolate showed that the early isolates were significantly better in establishing an infection with reduced clearance of the bacteria from the lungs (p<0.04), increased dissimination of the macroscopic pathology (p<0.002) and a more acute type and a higher degree of inflammation (p<0.05) as compared to mice infected with a late isolate. in addition, the pulmonary cytokine response was comparable with observations in cf where gm-csf and il-10 correlated to a milder disease, whereas g-csf, mip-2 (il-8 analog), and il-1b correlated to severe disease. 2) infecting the mice with the mucoid pfge-identical isolates from the same cf patient revealed a signicantly higher mortality in mice infected with the late isolates (p<0.005). in addition, the pulmonary g-csf and mip-2 response increased in the groups of mice that were infected with the late isolates as compared to the early isolate (p<0.05). moreover, the g-csf and mip-2 increased during the first three days of infection with the late mucoid isolates (p<0.05), indicating an impairment of the host response in controlling the lung infection. 3) when tobramycin treatment was initiated 1h after infection of mice with a mucoid isolate, the number of bacteria were cleared or reduced to a significantly lower level (p<0.05), and a significant decrease of both g-csf and mip-2 at day one, two and three was observed (p<0.03). in contrast, when treatment was initiated after 24h the number of bacteria did not change significantly, and the reduction in inflammatory cytokine response became significant for both late isolates only at day three of treatment (p<0.03). moreover, cytokine levels were significantly lower in the 1h as compared to the 24h group (p<0.04) confirming the importance of early and aggressive antibiotic treatment in cf. non-mucoid isolates were reduced during the first three days of infection independent of treatment. in conclusion, we have implemented a clinical important cf-time perspective (years) in experimental p. aeruginosa lung infection, useful for patho-physiological and treatment studies. cystic fibrosis (cf) is a complex disease that affects multiple organs and results in a wide range of phenotypes in humans. mouse models of cf display many of the phenotypes observed in human patients and provide necessary tools to dissect this multifaceted disease. however, due to the involvement of cftr in various organ systems, the ability to discern the contribution of each organ system to each phenotype of cf is unknown. for example, we have found that mouse growth is dramatically reduced compared to wildtype, and mechanisms ranging from neuroendocrine disruption, intestinal malabsorption and cacchexia have been proposed. we have found that cf mice consume more calories per gram body weight than non-cf mice and intestinal absorption of dietary lipids is similar between the two. cf animals absorb 94% and non-cf 97% (p > 0.5). to identify the source of the growth deficit, it would be advantageous to restrict expression of cftr in specific tissues to determine each tissue's contribution to the phenotype. to accomplish this, we have created a conditional, null murine cftr allele. a conditional null allele is one in which the gene of interest is induced to be non-functional in either a specific tissue or cell type, or at a specific developmental time. examination of cftr absence on a tissue-by-tissue basis will greatly facilitate our understanding of the disease. the conditional cftr allele was created by "floxing" exon 10 of cftr, known to be critical for function, with flanking loxp sites that allow for the deletion of exon 10 when bacterial recombinase cre is present. to date, mice stably transmitting the floxed allele have been created. these founder mice have been crossed with mice expressing cre from the promoter of the villin gene, which restricts transcription of cre to intestinal epithelium. first generation animals show appropriate recombination and deletion of exon 10 in the gut. however, these first generation animals are heterozygous for the floxed allele. we are currently backcrossing these animals with the founders to generate animals with homozygous deletion of cftr in the intestinal epithelium. similar crosses are planned to target other tissues. this new animal model of cf will be available to the cf research community to further our understanding of cf pathophysiology. this work was supported by a grant from the cf foundation increased enac-mediated na+ absorption is a hallmark of cystic fibrosis (cf) airways. we demonstrated that mimicking na+ hyperabsorption by overexpression of βenac in mouse airways results in airway surface liquid (asl) depletion and reduced mucus clearance causing a spontaneous cflike lung disease with high pulmonary mortality, and airway mucus obstruction, mucous cell metaplasia and chronic airway inflammation in adult survivors (mall et al., nature med.10:487, 2004) . the aim of this study was to identify the initiating lesions and investigate the natural history of lung disease caused by asl depletion in βenac overexpressing (βenac-tg) mice. to achieve this goal, we performed longitudinal studies on lung morphology, airway mucus obstruction, bronchoalveolar lavage inflammatory cell counts, expression levels of mucins (muc5ac, muc5b, muc4) and proinflammatory cytokines (tnfα, kc, ifnγ, il-13, eotaxin-1), lung volume, lung mechanics, and airway na+ transport in neonatal to adult βenac-tg mice and wild-type littermate controls. we show that airway mucus obstruction in βenac-tg mice originated in the trachea in the absence of mucous cell metaplasia in the first days of life, and was associated with hypoxic degeneration and necrosis of airway epithelium, and death. in surviving βenac-tg mice, mucus obstruction extended into the lungs and was accompanied by secondary mucous cell metaplasia, increased expression of muc5ac, muc5b and muc4, and airway inflammation with transient increases in macrophages and tnfα, in eosinophils and il-13, and persistent increases in neutrophils and kc in the lung. βenac-tg mice also developed emphysema with increased lung volumes, distal airspace enlargement, and increased lung compliance. several disease surrogate markers waned in parallel with βenac transcript levels and airway na+ absorption in adult βenac-tg mice indicating that the level of enac overexpression was critical in determining disease severity. in summary, our results provide several novel insights into the in vivo pathogenesis of lung disease caused by asl depletion. we show that (i) asl depletion is sufficient to initiate severe airway mucus obstruction in the absence of mucous cell metaplasia and/or mucus hypersecretion; and (ii) airway na+ hyperabsorption and mucus plugging-induced hypoxia is associated with airway epithelial necrosis constituting a mechanism that initiates airway inflammation in the absence of infection. further, our results point to a novel link between airway na+ hyperabsorption, eosinophilic inflammation, and emphysema. the high frequency of allergic airway inflammation and emphysema in cf patients warrants further studies of the mechanisms that link asl depletion with these associated pathologies. the balance of airway surface liquid (asl) at a height compatible with efficient clearance of mucus is a process regulated by two main pathways: the secretion of clvia cftr, and absorption of na + via the epithelial na + channel (enac). to study this interplay, an immortal cell line capable of growing at an air liquid interface and having properties of human bronchiolar epithelium would be desirable for its simplicity in preparation and ease of molecular manipulation. an immortal human bronchial adenocarcinoma cell line (hbac) was infected with a lcfsn retrovirus carrying either cftr cdna (hbac cftr ) or the blank vector (hbac mock ). 8-10 day monolayers, grown on transwell clear membranes, were mounted in ussing chambers and studied under open-circuit conditions. to study regulation of clsecretion, the equivalent short circuit current was calculated in the presence of amiloride (apical, 100 µm). in hbac mock cells, forskolin (10 µm, basolateral) had no effect upon i sc . in contrast, hbac cftr cells demonstrated a robust increase in i sc ; a rapid increase to a peak (∆~20 µa cm 2 ), followed by relaxation to a stable and sustained plateau. genistein (50 µm) also stimulated i sc in hbac cftr (∆i sc 13 µa cm 2 ) but not hbac mock cells. amiloride resistant basal current was consistently higher, by 2-3 µa cm 2 , in hbac cftr versus hbac mock cells, presumably reflecting low basal cftr activity. we next examined the ability of endogenous receptors present in hbacs to regulate transfected cftr. stimulation of i sc in response to apical adenosine (100 µm) in hbac cftr was qualitatively and quantatively similar to the forskolin response. while the above experiments provided evidence to suggest that hbac cftr cells possess the bioelectric capability of sustaining anion transport via nucleoside-stimulated cftr activity, it does not guarantee a resultant vectorial flow of fluid. in the final series of experiments, we examined whether adenosine was indeed able to stimulate secretion across the hbac-cftr monolayer by assaying asl height. monolayers were loaded apically with fitc-labeled dextran (20 µl), and the majority of dye was then aspirated. monolayers were then allowed to equilibrate over a 2 hour period, and asl measured by xz scanning on a leica confocal microscope. ~100 µm adenosine and ~10 µm benzamil was then added apically in pfc and asl height measured after 10 and 30 minutes. in both hbac mock and hbac-cftr cells, the basal asl height was ~4 µm. over 30 minutes asl height of hbac mock cells did not change in response to adenosine. in contrast, adenosine stimulated hbac cftr cells demonstrated a doubling of asl height (to 8 µm) over the same period. this response is consistent with the prolonged increase in i sc observed upon stimulation with adenosine. in summary, the hbac model which displays only absorptive currents endogenously, can be transformed into a cell line in which cftr-mediated secretion can be stimulated and sustained by endogenous receptor systems present in the apical membrane. this new model will allow us to study cftr-mediated fluid transport in a defined manipulatable system. despite the identification of the cftr channel more than 15 years ago, many questions remain about its regulation and in vivo function. in order to better understand how cftr functions in vivo, numerous studies have searched for and identified potential interacting partners but to date, there is little evidence linking any of the known cftr interactors to its activity. during a forward genetic screen in zebrafish, aiming to identify genes regulating endodermal organ development, we identified a mutant, which we named baobab (bao), after the african tree that accumulates water during the wet season, which exhibits a dramatically enlarged fluid-filled gut tube. most interestingly, we have shown that various cftr inhibitors reduced the appearance of enlarged guts in bao mutant embryos, suggesting that the fluid accumulation phenotype resulted from an increase in cftr activity. to test whether increased cftr activity resulted from changes in cftr protein levels or localization we raised antibodies against zebrafish cftr. western-blot and immunolocalization studies revealed that the channel was expressed and localized as in wild type in bao mutant embryos indicating that the increased cftr function most likely resulted from the activation of the channel. these data, together with the recessive character of the mutation, suggest that the bao protein is a negative regulator of cftr activity. we have positionally cloned the bao locus and isolated the affected gene. bao encodes a cytosolic protein that has not been previously shown to regulate cftr. in agreement with the pharmacological data pointing to a gut-specific function of bao, in situ hybridization studies showed that bao is higly expressed in gut. we are currently investigating how the bao protein regulates cftr function. in addition, we are also investigating the function of cftr during zebrafish development. these studies lay the groundwork to take advantage of the zebrafish system to further investigate cftr biology. we establish a genetic model system for studying the regulation of the cftr channel that will contribute to the understanding of the pathophysiology of cystic fibrosis and several intestinal secretory conditions. acknowledegements: this work was supported by an embo long-term postdoctoral fellowship to mb and by nih grants to d.y.r.s.. we thank a. verkman for providing cftr inhibitors and k. brand for technical assistance. the trachea of the adult cf mouse (cftr tm1unc or cftr tm1kth ) expresses little cftr and exhibits neither a defect in camp-mediated clsecretion nor hyperabsorption of na + , both signature abnormalities in human cf airways. we generated a mouse exhibiting hyperabsorption of na + in the airways by over-expressing the β subunit of the epithelial na + channel (βenac, gene scnn1b). βenac mice exhibit airway pathology (decreased mucociliary transport, mucus plugging, and airway inflammation) similar to human cf airways. to determine whether inactivation of cftr in βenac mice results in a more severe phenotype, we generated a cf/βenac mouse by crossing the ∆f508 (c57bl/6n) mouse (cf mouse) with the βenac mouse (c57bl/6n). survival analysis showed a drastic reduction in survival for cf/βenac mice in comparison to cf mice or βenac mice. we studied the airway bioelectrics of 3 day-old pups of each genotype. the basal short circuit current (i sc ) was ~5 fold greater in wild-type (wt) (50 ± 13 µa . cm -2 ; mean ± sem, n=4) vs. cf (12.2 µa . cm -2 , n=2) neonatal tracheas. as previously reported, the basal i sc in βenac preps was significantly elevated (99.2 ± 14 µa . cm -2 , n=7) vs. wt tracheas. however, the basal i sc in the cf/βenac tracheas (26.6 ± 19 µa . cm -2 , n=4) was intermediate between the cf and the βenac tracheas. the amiloride-sensitive i sc was elevated similarly in the tracheas of the βenac and cf/βenac pups and was ~ 3x greater than that exhibited by the wt or cf tracheas. the post-amiloride i sc was strikingly reduced in both cf and cf/βenac tracheas compared to the wt and βenac tracheas. we suggest that the post-amiloride i sc reflects constitutive cftr-mediated clsecretion, which may help hydrate airways in the neonate. unlike the tracheas of adult cf mice, neonatal cf tracheas exhibited the classic defect in camp-mediated clsecretion, with the forskolin response in the cf and cf/βenac tracheas being reduced ~10fold compared to the response in the wt or βenac preps. the response to utp (ca ++ -mediated clsecretion) was similar in the four genotypes. absence of cftr-mediated clsecretion does not appear to cause airway pathology in neonatal cf mice, but when this defect is coupled with na + hyperabsorption, it results in a significant increase in neonatal mortality. due to lack of cftr-mediated clsecretion, cf/βenac mice appear to experience greater mucus adhesion, producing death due to asphyxiation. in addition, the tracheas of cf mice (especially congenic c57bl/6n) often appear constricted or collapsed due to a cartilaginous defect resulting in increased compliance. this anatomical defect does not appear to be detrimental to the cf mouse. however, in cf/βenac pups, that accumulate thick, sticky mucus in their tracheas, this tracheal constriction may contribute to their asphyxiation. these results demonstrate the importance of both abnormal na + absorption and decreased clsecretion in the pathogenesis of cf lung disease. supported by nih scor p50 hl060280. recently, we reported that species-specific differences in the airway biology of raav transduction exist between mice and human (am j respir cell mol biol, 2006, 34:56-64) . these differences significantly interfere with the use of mice as surrogate models for human lung gene therapy with certain raav serotypes. to survey which species might be an optimal animal model for raav preclinical testing, we utilized differentiated airway epithelia grown at an air-liquid interface (ali) from five species (human, non-human primate [nhp] , pig, ferret, and mouse) to test the efficiency of transduction with three serotypes of raav (type 1, 2, and 5). our results demonstrated significant species-specific differences in raav transduction biology in airway epithelia. the serotype preferences of raav transduction from the apical membrane in human, ferret, and pig epithelia was raav1>raav2≈raav5, different from that seen in mouse epithelia (raav1>>raav5>>raav2). surprisingly, the profile of raav transduction with these three serotypes in nhp rhesus monkey airway epithelia (raav2≈raav5>>raav1) was less similar to human than pig and ferret. additionally, raav1 lacked a bias in polarity of transduction in human airway epithelia, however, in nhp epithelia, raav1 transduced the basolateral membrane 10-fold more effectively than the apical membrane. furthermore, differences in the properties of raav1 receptors between these species was also seen. using neuraminidase treatment of the apical surface of ali cultures, we tested whether n-linked sialic acids were potential receptors for raav1 binding and/or transduction. these studies demonstrated removal of n-linked sialic acids inhibited raav1 transduction of human, nhp, and mouse airway epithelia, but not ferret and pig. however, raav1 binding in this setting did not correlate with changes in transduction; neuraminidase treatment dramatically reduced raav1 binding to nhp epithelia, but enhanced binding to human epithelia (~100 fold). these findings suggest viral binding to airway epithelial cells may not be a major barrier for raav1 transduction. evidence that proteasome-modulating agents enhancing raav transduction 2-3 logs for all serotypes and species tested, suggest that the ubiquitin/proteasome pathway represents a common intracellular block in raav transduction of airway epithelia. these findings support the notion that viral interactions at the surface of airway epithelial cells (with co-receptors and receptors) influence intracellular processing aav virions that can alter the efficiency of productive transduction. enac surface density in cortical collecting duct (ccd) epithelial cells is established by camp/pka regulated insertion and retrieval of the channel at the apical membrane, together with the recycling of internalized enac back to the apical surface (butterworth et al. (2005) journal of general physiology 125: 81-101). ubiquitination by the nedd4-2 ligase elicits endocytosis of surface enac, which may lead to its degradation. however, enac recycling to the apical surface requires ubiquitin removal by a deubiquitinating enzyme (dub). using a chemical probe approach, we identified ubiquitin carboxy-terminal hydrolase (uch-l3) as the predominant dub in vesicular endocytic and recycling compartments in mouse ccd epithelia. specific inhibition of uch-l3 (10µm of 4,5,6,7-tetrachloroindan-1,3-dione) in filter-cultured ccd cells resulted in a rapid decline in enac currents, which was accelerated by forskolin stimulation, suggesting that uch is responsible for recycling enac to the apical surface. knockdown of uch-l3 significantly reduced both basal and camp-stimulated enac currents (from 7.7±1.2µa/cm2 in control sirna treated to 1.5±0.7µa/cm2 in uch-l3 knockdown cells). to determine whether the same dub was responsible for the regulation of enac in human bronchial epithelial (hbe) airway cells, differentiated cultures derived from cf and non-cf lung tissue were treated with the uch inhibitor. to address the possibility that protease cleaved enac may be regulated differently from unprocessed channels, differentiated hbe cultures were either maintained at air/liquid interface or were submerged under liquid/liquid conditions to alter the cleavage state of enac, as we have previously described (myerburg et al. (2006) journal of biological chemistry 281: 27942-49). in all cases, no significant difference in enac-mediated na+ transport was observed between control and uch-l3 inhibited conditions. these data are in sharp contrast with the mechanisms of enac regulation observed in the ccd model system, and they support our previous findings that enac turnover and mode of regulation in hbe are distinct from the recycling mechanisms defined in the ccd epithelia (butterworth et al. primary human tracheobronchial epithelial (htbe) cells cultured at an air-liquid interface (ali) regulate ion transport, mucin secretion, and cilia beating to recapitulate directional mucus clearance, a key innate defense mechanism that fails in cystic fibrosis (cf). thus, ali htbe cell cultures are crucial for studying cf pathogenesis and for developing/validating novel therapies. different research centers use alternative protocols that enable reproducible creation of ali htbe cell cultures. however, limited primary htbe cell availability has driven the creation of cognate cell lines, accomplished mainly by the introduction of viral oncogenes. viral oncogenes disrupt normal cell physiology causing cellular hyperplasia, abundant apoptosis, genomic instability and, sometimes, poor polarization/differentiation, rendering the cells less useful than primary cells for assessing cftr function. furthermore, genetically unstable cell lines will continuously acquire changes independent of cftr genotype. bmi-1 is a polycomb group protein that controls cell cycle and cell identity via epigenetic regulation of chromatin, maintaining stem cells by suppressing expression of p16, an inhibitor of cyclin dependent kinases. bmi-1 expression has been used to create cell lines that recapitulate normal cell structure and function better than viral oncogene-immortalized cell lines. using hiv lentiviral vectors, we introduced bmi-1 and the catalytic subunit of telomerase to enhance the growth of 3 different ∆f508 homozygous cf and 3 non-cf primary human airway epithelial cell preparations. all 6 new cell lines grew for at least 40 population doublings, while their normal counterparts senesced prior to a maximum of 20 doublings. at passage 14-15, the new cell lines had a diploid karyotype compared to grossly abnormal chromosomes in cells immortalized by viral oncogenes. ussing chamber analysis of ali cultures at passage (p) 14-15 revealed variable transepithelial resistances among the cell lines ranging from 125 ->1000 ω*cm2, but short circuit current (isc) responses stimulated by forskolin and inhibited by cftr172 were true to the cell's genotype and comparable to early passage primary cells, except for one non-cf cell line with relatively low cftr isc's at p15. amiloride sensitive and utp-stimulated isc's were present but were more variable in magnitude in comparison to the currents observed in low passage primary cells. ali cultures exhibited a pseudostratified morphology with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells and occasional ciliated cells. all cf and non-cf cell lines in culture produced similar levels of il-8 at baseline and cf and non-cf cells equally increased il-8 secretion in response to il-1β, tnfα and the tolllike 2 receptor agonist, pam3cys. these novel cell lines will help fill gaps currently hindering cf research and therapeutic development. supported by cystic fibrosis foundation grant randel04g0. we recently considered that domestic dogs could represent a large natural reservoir to search for cftr mutations and, ultimately, to identify and breed animals with cf traits. the canine genome has been completely sequenced, and the canine cftr gene, found on chromosome 14, consists of 27 exons spread over 163 kb. an animal model that faithfully expresses human cf lung pathology is critical for the development of effective treatments for this most lethal aspect of cf disease. while transgenic mouse models of cf replicate numerous aspects of cf disease, they unfortunately do not develop the characteristic lung pathology including mucus plugging of airways, chronic bacterial infections, and bronchiectasis. the reason for this is not completely understood but it is likely related to significant differences in lung morphology between these species. to date, no non-murine genetic model of cf has been developed, and naturally-occurring, cf-causing mutations have not been identified in any non-human animal species. temporal temperature gradient gel electrophoresis (ttge), developed as a screening tool to identify sequence variations and mutations in the human cftr gene, was used to screen dogs for cftr mutations. dna was purified from whole blood received from veterinary clinics treating dogs for a variety of ailments. we have performed ttge analysis on dna from 179 dogs, which includes 24 dogs with pancreatitis and 4 dogs with bronchiec-tasis, and have found hundreds of potential mutations amongst the thousands of amplicons tested. ttge can detect single base pair changes within a fragment of dna. as the temperature increases, the migrating dna becomes partially melted, creating single stranded regions (internal bubbles and branched ends) that decrease its mobility through the gel. the dna fragments have distinct melting profiles that are sequence dependent; therefore, changes in sequence of even a point mutation can alter the profile and result in a change in the rate of migration through the gel that can distinguish wild type from even a homozygous mutant. thus far 65 exonic mutations have been sequenced but none have caused changes to cftr at the amino acid level (i.e., the mutations have all been silent). we conclude that ttge is a sensitive and effective method for screening large canine populations for cftr mutations. ( introduction: the search for "correctors" of defective protein processing of ∆f508 cftr has led to the development of high throughput screening assays to find compounds that promote the surface activity of the mutant protein. by necessity, primary screening assays typically use cell lines expressing recombinant ∆f508 cftr. however, "hits" identified in primary screening require scrutiny for false positives whose activity is either dependent on the ∆f508 cftr expression system or insufficiently robust to correct processing in the complex physiological setting of a native epithelium. to this end, we have developed a secondary functional assay that measures the shortcircuit current (isc) responses in organ cultures of gallbladder mucosa from ∆f508 cftr mice (cftr tm1kth ). previous studies have shown that murine ∆f508 cftr recapitulates the protein processing defect including the capability to increase processing when mucosa is incubated at reduced temperature (27°c) (j. clin. invest. 98:1304 invest. 98: , 1996 . gallbladder mucosa was chosen for this assay because the murine gallbladder can be removed aseptically for organ culture, the epithelium is essentially a monolayer, and the mucosal wall is transparent which facilitates morphological assessment. methods: using either wild-type (wt), ∆f508 cftr (df) mutant or cftr knockout (cftr ko) mice, excised gallbladders were opened longitudinally, rinsed and positioned on loose nylon mesh. the preparation was mounted between two halves of a polycarbonate culture cup with 4.5 mm aperture and placed in submersion culture for 0 -2 days at either 37°c or 27°c using supplemented ham's f12 medium mixed 50:50 with nih 3t3 fibroblast-conditioned dmem containing 2% calf serum. after incubation, the culture cups were placed in custom designed ussing chambers for measurement of isc responses to a cocktail of 10 µm forskolin and 100 µm ibmx (camp isc) at 37°c. contribution of calcium-activated clchannels to the camp isc was eliminated by addition of the distilbene dids (100 µm) to the luminal bath. results: the mean ∆camp isc responses (in µa/cm 2 ) after two days incubation were as follows: wt at 37°c = 21.1 ± 4.1 (n = 4); df at 37°c =3.9 ± 0.3 (n = 5); df at 27°c = 13.6 ± 2.7 (n = 6); and cftr ko at 27°c = 2.8 ± 0.1 (n = 3). in a pairwise comparison, the wt (37°c) was significantly greater than the df (37°c) and cftr ko (27°c) but not significantly different from df (27°c). conclusions: incubation of ∆f508 cftr mutant murine gallbladder mucosa at 27°c for two days in organ culture restored approximately 65% of the cftr-dependent isc responses to camp stimulation. the temperature-dependent effect is consistent with correction of murine ∆f508 cftr processing, indicating a physiological screening assay that is useful for preclinical testing of candidate drugs targeted at correcting the protein processing defect of ∆f508 cftr. supported by cfft and nih. the lack of a large animal model that replicates the lung disease found in humans with cystic fibrosis is a major impediment to understanding disease pathogenesis and the development of effective therapies. although cystic fibrosis is a multi-system disease, airway infection and inflammation currently cause most of the morbidity and mortality in patients. several homozygote null and missense cftr mutations have been developed in mice, but they do not exhibit the lung phenotype observed in humans. in contrast to mice, the structure and physiology of porcine lung and airways closely resemble those of humans. therefore, the goal of this project is to develop a swine model for cystic fibrosis by combining gene targeting and nuclear transfer to produce a pig with a cftr null genotype. we employed homologous recombination to disrupt exon 10 of pig cftr with a neomycin resistance cassette. we generated primary male fetal fibroblasts that were positive for cftr-targeting at one allele as detected by multiple pcr screens. genomic southern blot analysis confirmed proper gene targeting and revealed most clones were free of random integration events. these cells were used for somatic cell nuclear transfer that resulted in the birth of ten cftr null heterozygote piglets. subsequent breeding of these male pigs to wild-type females has yielded at least 9 female cftr null heterozygotes. breeding male and female heterozygotes will provide cftrnull homozygotes. our recent published and preliminary studies demonstrate that mouse slc26a9, a newly discovered member of slc26 family of anion exchangers, is expressed on the apical membrane of tracheal epithelial cells and gastric surface epithelial cells and in the tubulovesicle membranes of gastric parietal cells. the functional properties of mouse slc26a9 were examined in xenopus oocytes and in stably transfected cultured cells. in xenopus oocytes injected with the slc26a9 crna and studied with the voltage and/or ph sensitive microelectrodes, the rank order for anion selectivity at +80mv was cl->i-> no3->gluconate > sulfate, and that of the selectivity (px/pcl) was i-> cl-> no3-= gluconate > sulfate. inhibitor profile studies demonstrated that nppb at 100 µm inhibited the current at 5 min by 40% (p<0.001 compared to control period, n=6). the effect of nppb was partly reversible. dids at 500 µm had no significant effect on the slc26a9-mediated current (p>0.05 compared to control period, n=6). niflumic acid at 100 µm inhibited the current at 5 min by ~38% (p<0.001 compared to control period, n=5) which was partly reversible. cftr inh 172 at 5µm inhibited the current at 5 min by ~23% (p<0.005 compared to control period, n=5) which was reversible. clamping currents in a 85 mm k-gluconate medium and using 10 mm k-cl or k-bicarbonate revealed very low bicarbonate conductance for slc26a9 compared to that of cl-. no significant phi changes were observed in oocytes when favoring cl-exit by substituting perfusate cl-with gluconate in the presence of co2/hco3-. conversely we observed significant alkalinization in ae1-expressing oocytes in the same experimental solutions. interestingly, in hek 293 cells stably transfected with the mouse slc26a9, a significant intracellular alkalinization was observed upon switching to a chloride-free perfusate. the alkalization in slc26a9-expressing cells was inhibited by ~55% in the presence of 500 µm dids. baseline intracellular ph was significantly increased in slc26a9expressing hek293 cells vs. non transfected cells. however, baseline phi was comparable in control and slc26a9-expressing cells in the presence of 100 µm eipa, indicating the activation of endogenous nhe by slc26a9. in conclusion, slc26a9 displays characteristics of an anionic channel in xenopus oocytes, with low bicarbonate permeability. in mammalian expression systems, slc26a9 can also function in cl-/hco3-exchange mode with moderate sensitivity to inhibition by dids. we propose that slc26a9 plays an important role in anion (chloride) secretion and/or ph regulation in tracheal and gastric epithelial cells. no cell lines exist at present that robustly express endogenous wt and ∆f508 cftr on the same genetic background. zinc finger nucleases (zfns) allow the introduction of specific genetic changes at loci within the human genome (nature 435: [646] [647] [648] [649] [650] [651] 2005) . we have been using this technology in an attempt to generate human cells carrying the major cf-causing genotype -∆f508 -at the endogenous cftr locus. such lines could be expected to provide better understanding of the ∆f508 maturational processing defect, as well as new model systems for cell based assays of ∆f508 repair. we performed a bioinformatic analysis of cftr exon 10, and designed multiple distinct zfns against the ∆f508 dna sequence. all of these nucleases were assembled, cloned into mammalian expression vectors, and assayed for dna binding affinity and specificity. the zfns were then assayed for endogenous cftr locus editing in calu-3 and ht-29 cells using a novel, massively parallel methodology applied for the detection of genome editing events. the effort demonstrated that specific zfn pairs successfully bind to, and cleave, within exon 10 of the endogenous cftr locus in both cell types. we will describe experiments that yielded optimized zfns for the cftr locus, and a robust viral platform to transiently deliver genome editing reagents to the human pulmonary cell line, calu-3. supported by cff. the localization of slc26 anion transporters, as well as their function vary among members. some (slc26a2, slc26a3, slc26a6, slc26a11) are widespread, implying specific physiological function in diverse tissues. others are expressed only in a single epithelia, e.g., slc26a5 (prestin) in the inner ear. we have previously found that slc26a9 is a highly electrogenic cl --hco 3 exchanger with a large anion conductance. to determine the possible physiologic roles of slc26a9, we developed a chicken antibody (designed against sulfate transporter and anti-sigma domain, stas, of slc26a9) to localize the protein. to test the antibody specificity for functional protein, we inject xenopus oocytes with crna for slc26a6, slc26a7 or slc26a9 and documented appropriate ph and membrane potential changes. only oocytes functionally expressing slc26a9 were stained using our primary slc26a9 antibody. further, specific staining was eliminated when either pre-immune serum or blocking peptide was used. slc26a9 was only found in epithelia. in the respiratory system, slc26a9 was in the apical membrane of nasal epithelial cells and tracheal submucosal glands. membranes of calu-3 cells, derived from human tracheal submucosal glands, stained indicating that our antibody also recognizes the human slc26a9 protein. slc26a9 is found in the proximal parts of the gastrointestinal tract (esophagus, stomach), with lesser amounts in the small intestine and colon. however, slc26a9 is not present in liver. slc26a9 is present in ducts of secretory epithelia such as salivary glands and pancreas. in the urinary tract, slc26a9 is localized to the renal proximal tubule apical membrane but not in urinary bladder. in the reproductive system, we found slc26a9 in epithelia of the uterus and seminal vesicles but not the vas deference or the prostate. finally, slc26a9 is found on the eye surface, i.e., corneal epithelial cells. together, these results and the varied slc26a9 physiology suggest that several epithelial function models will need to be re-evaluated to incorporate an electrogenic cl --hco 3 exchanger and/or another apical anion channel. (support: romero-06g0, sindic-06f0). a tgfβ-1 polymorphism modifies severity of cystic fibrosis lung disease in the setting of ∆f508 cftr homozygosity (drumm et al., nejm, 2005) . the mechanism(s) underlying this observation, and whether elevated or repressed tgfβ are responsible, are not known. in addition, the pathogenic role of tgfβ in other cf organs and in murine models of the disease has not been well studied. because the tgfβ codon 10 cc genotype is associated with worsened cf pulmonary prognosis, and since high levels of tgfβ are also known to suppress cftr mrna expression and apical cftr protein expression in colonic epithelia (howe et al., exp cell res, 2004) , we developed a mouse model to test tgfβ/cftr interactions that may directly influence cf phenotype. we bred ∆f508 heterozygous mice (cftr tm1kth , ∆f508 cftr) against the tgfβ receptor-deficient murine strain (mtrii28). mtrii28 mice express a dominant-negative mutant form of the tgfβ type ii receptor which lacks the intracellular (kinase) receptor domain. the receptor binds tgfβ, but fails to transmit a tgfβ dependent signal. the tgfβ receptor transgene was inserted downstream of a metallothionein-derived promoter and expressed in numerous murine tissues following induction with 25 mm znso 4 in drinking water. an antibody raised against active tgfβ (lc-1-30-1, gift of dr. kathy flanders, national cancer institute) was used to establish tgfβ in murine tissues including lung and intestinal tract of the mtrii28 strain. rt-pcr confirmed high level transgene expression similar to gapdh internal control. nasal potential difference measurements in cftr +/+ mice expressing the tgfβ transgene indicated intact camp dependent cltransport following induction of the dominant-negative receptor. homozygous cf animals deficient for tgfβ signaling have recently been obtained and exhibit the expected litter sizes and distribution of the ∆f508 allele. studies of cf homozygous mice following induction with znso 4 are in progress, and will be used to determine whether a failure to signal through tgfβ in vivo 1) influences the severity of cf intestinal disease, 2) alters airway or intestinal bioelectric findings characteristic of cf, or 3) changes survival, histopathology or other features of the disease. these experiments are intended to provide an in vivo means of investigating tgfβ signaling and resultant cf modifier effects in vivo. supported by nih and cff. objective: to determine if the abpa phenotype observed in cftr mice was preventable by corrective gene replacement, we delivered aav5 cftr to the lung. in addition to further assess whether cftr-/-lymphocytes alone where responsible for the phenotype, we performed adoptive transfers of cftr-/-lymphocytes into cftr+/+ mice and proceeded to expose the mice to the abpa model. design/methods: cftr-/-mice underwent it administration of raav5 cftr or raav5gfp. all mice were then sensitized with 20ug of crude af antigen via intraperitoneal route. non-injected mice were sensitized without prior virus exposure. challenges were performed on all mice, using an af solution delivered by aerosol inhalation in a closed chamber on 3 consecutive days. serum ige levels were then obtained, as well as bronchoalveolar lavage fluid for differential cell counts, histologic and cytokine analysis. adoptive transfer experiments were done using spleens cells isolated from af sensitized delaf508-/-and f508+/+ mice and transfer into c57/bl6_rag-/-mice. after 7 weeks post transfer c57/bl6_rag-/-mice with f508-/-and f508+/+ lymphocytes were then challenged with af. results: following challenge with af, raav5-cftr mice had significantly lower total serum ige levels (17,251) as compared to raav5-gfp controls (26,892) (p=0.037). analysis of af-specific ige also revealed a two-fold reduction in the mice receiving the corrective gene therapy. curiously, gene transfer to the lung lowered the secretion of inflammatory cytokines from activated splenocytes of raav5-cftr when compared to raav5-gfp controls. these results suggest that correction of immune cells in the lung that traffic to the spleen may be partially responsible for the attenuation of the phenotype. in addition the transfer experiments showed that the hyper-ige syndrome can be partially transferred into b6rag-/-by f508-/-splenocytes. conclusions: correction of cftr deficiency by aav lung transfer attenuates the inflammatory phenotype associated with abpa. epithelial cells lining the vas deferens express cyclooxygenase (cox) 2 when exposed to testosterone in vivo, although no functional link to ion transport has been established. we hypothesized that testosterone modulates cftr-mediated anion secretion across vas deferens epithelia that is stimulated via cox-dependent pathways. vas deferens epithelial cells were isolated from human and porcine tissues and cultured as monolayers on permeable supports until assayed in modified ussing chambers. rna was isolated concurrently for semi-quantitative gene expression analysis. bradykinin (bk) caused an increase in anion secretion (measured as short circuit current) and this effect was inhibited by indomethacin, which indicates cox dependency. testosterone treated monolayers exhibited a bkinduced response that is 58% greater than paired vehicle treated monolayers. qrt-pcr revealed that cox-2 mrna is 19 times more abundant than cox-1 mrna in cultured vas deferens epithelial cells. the effects of bk on ion transport are inhibited by the b2 receptor antagonist hoe140 while the b1 receptor agonist des-arg9-bk produced no effect. prostaglandin e2 and selective agonists of the ep4 (1-oh-pge1) and ep2 (butaprost) receptors produced concentration dependent increases in short circuit current whereas sulprostone, an agonist of ep1 and ep3 receptors, had no effect. taken together, these results suggest that testosterone plays a key permissive role for bk-stimulated anion secretion by inducing cox-2 expression. bradykinin acts at the b2 receptor to increase cox-2 activity and the synthesis of prostaglandins that ultimately bind to ep receptors to promote cftr-dependent hco3-and cl-secretion that would be expected to modify the environment for sperm. the results further suggest that developmentally associated increases in testosterone levels during gestation and puberty will produce physiological changes in vas deferens luminal environment that are likely compromised in cystic fibrosis patients. supported by cystic fibrosis foundation schult06po and ksu cobre nih rr-17686. background: recent studies in cystic fibrosis (cf) suggest a prominent t h 2 profile; a facet that may interfere with normal bacterial clearance. this phenotype appears to increase secondary to infection with aspergillus fumigatus or pseudomonas aeruginosa and also, may interfere with macrophage activation. these same characteristics are present in cftr-deficient mice (cftr s489x -/-fabp-hcftr (+/+), hereafter designated cf mice). as a study of pathogenesis of cf related diabetes (cfrd), we previously observed increased il-10 and splenocyte proliferation in cf mice subject to streptozotocin-induced hyperglycemia. herein, we hypothesized that differences in glucose concentration would increase t h 2 profiles of splenocytes in vitro. methods: cf mice and c57bl/6j mice (b6 mice; n=3/group) were sacrificed at 14 wk of age. splenocytes were collected and cultured at the following glucose concentrations: 10, 15, 20 and 25 mm. each mouse and glucose concentration was stimulated with cona, lps and cd3/cd28. supernatants were collected at 48 hr and measured by luminex analysis. statistical analysis was performed by a one-way anova. results: stimulation by cona produced increased levels of il-10, il-4, tnf-a, gm-csf and il-2 in splenocytes of cf mice (p<0.01 for all). stimulation by lps produced decreased concentrations of il-10 in cf mice(p<0.05). stimulation of cf mice by cd3/cd28 produced decreased concentrations of il-2 (p<0.001). interestingly, no difference was observed in cytokine elaboration as a function of glucose concentration. data are presented in table as fold increase of cytokine concentration for cf mice compared to b6 mice. discussion: profound differences in cytokine production were measured in stimulated splenocyte responses of cf in comparison to b6 mice. the profiles did support the notion for a prominent t h 2 response, with the potential to interference of normal macrophage activation. in addition, differences in cytokine production profiles appear related to the nature of adaptive and innate stimuli provided (cona vs. lps). the absence of influence by glucose concentration in vitro suggests that local/immediate concentration of glucose is not relevant to prolonged expression of cytokine responses to stimuli. further studies on the impact of chronic hyperglycemia and its relation to immune response are necessary. an improved understanding of these mechanisms holds hope for a better overall outcome in patients with cf and cfrd. studies from different laboratories to characterize ∆f508 cftr clchannel activity have yielded disparate results, likely due to variations in technique, cell type, or method of analysis. in the present study, we compared ∆f508 cftr activation in two well characterized polarizing cell models. fisher rat thyroid (frt) cells have been used in high-throughput drug screening to identify ∆f508 cftr active agents, whereas cfbe41ocells have been transduced to provide robust ∆f508 cftr expression in an airway epithelial background. ∆f508 cftr biogenesis following low temperature correction was compared, indicating qualitatively similar levels of cftr transcripts and band b and c cftr. in paired studies in ussing chambers, distinct differences in isc were seen following exposure to cftr activating stimuli. in frt cell monolayers grown at 37°c, forskolin (20 µm) or genistein (50 µm) were strong stimuli of isc, producing mean isc of 13.0 +/-6.6 (forskolin) and 18.6 +/-7.2 (genistein) µa/cm 2 . regardless of exposure sequence, both agents induced equivalent changes in maximal current (59% of 42.2 µa/cm 2 by forskolin when administered prior to genistein; 54% of 44.7 µa/cm 2 by forskolin in converse order). growth at 27°c for 24 hours increased currents approximately 3.7 fold, but relative contributions of the two stimuli were similar to cells grown at 37°c (42% of total current stimulated by forskolin). in cfbe41o -∆f508 cells grown at 37°c, forskolin-dependant currents were minimal and genistein-stimulated currents small (4.2 +/-1.4 µa/cm 2 , compared to zero current in parental, nontransduced cfbe41ocells, p<0.05). in temperature-corrected ∆f508 cfbe41ocells, forskolin was a poor stimulus of cl-transport relative to genistein, contributing 15% to the total current produced by both agents (p<0.001). genistein-stimulated currents did not require pre-treatment with forskolin to produce maximal effects (28.8 µa/cm 2 with genistein alone vs. 31.1 µa/cm 2 after forskolin and genistein), and forskolin did not cause further isc when added after genistein (0 µa/cm 2 ). levels of camp did not account for these differences, since forskolin markedly increased camp in both models. to determine whether excessive phosphodiesterase (pde) or phosphatase (pp) activity limited ∆f508 cftr activation by forskolin in the cfbe41o -∆f508 cells, monolayers were pretreated with the nonspecific pde inhibitor papaverine (100 µm) or the pp2 inhibitor endothall (400 µm), each sufficient to activate isc in cfbe41oexpressing wtcftr (papverine: 59.8 vs. 1.3 µa/cm 2 with vehicle, p<0.005; endothal: 40.1 vs. 8.8 ua/cm 2 , p=0.05). neither agent restored isc in ∆f508 cfbe41oby forskolin (0.8 mv difference, p=ns). these results indicate that activity of ∆f508 cftr in polarizing monolayers is exquisitely dependant on the model chosen for study. substantial differences in ∆f508 cftr behavior in two commonly used cell models, and suggest that the failure of camp to activate ∆f508 cftr in cfbe41ocells is due to cell-specific factors that may 1) influence ∆f508 cftr folding and tertiary structure, or 2) favor interactions of ∆f508 cftr with inhibitory binding partners that limit activation by camp. adverse immune responses to viral vectors or vector-encoded proteins may hinder the practical application of gene therapy. to date, little effort has been devoted to determining the immunological consequences of topically delivered lentiviral vectors to respiratory epithelia. previously, we demonstrated that an integrating feline immunodeficiency virus-based lentiviral vector pseudotyped with a baculoviral envelope glycoprotein (gp64-fiv) readily transduced differentiated airway epithelia in vitro when applied to the apical surface. furthermore, using a luciferase reporter gene and bioluminescence imaging, we observed in vivo gene transfer to murine nasal epithelia following a single application of gp64-fiv. longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for greater than 11 months without significant decline. by histological analysis, surface epithelial cells were transduced following a single nasal application of gp64-fiv expressing beta-galactosidase. importantly, we observed that 7 consecutive doses of gp64-fiv delivered over 7 consecutive days greatly increased the number of beta-galactosidase positive epithelial cells. using quantitative bioluminescent imaging, we observed that repeated doses given over consecutive days resulted in a linear additive increase in gene expression. in addition, we performed studies investigating gene transfer efficacy and immune responses following lentiviral vector readministration with greater intervening time periods. in control mice, we observed that giving 4 nasally instilled doses (2 weeks apart) of an e1/e3 deleted ad5 vector resulted in significantly attenuated expression after the second dose. the attenuated expression correlated to the production of neutralizing antibodies. in contrast, using the same delivery protocol, 4 doses of gp64-fiv resulted in gene transfer without loss of expression of the forth dose. no significant production of neutralizing antibodies was observed following the lentiviral delivery protocol. we further report the additive increase in reporter gene expression following 7 doses of lentiviral vector delivered over 7 consecutive weeks (1 dose/week), as well as the lack of production of systemic and local neutralizing antibodies. gp64-fiv efficiently transduces and persistently expresses a transgene in respiratory epithelia and has the potential for repeat administration without eliciting adverse immune responses. these data have important implications for the application of lentiviral gene therapy to pulmonary diseases. uniform delivery of gene therapy vectors to all lung lobes is important for cf gene therapy. however, this important objective has not been achieved in large animals. to address this obstacle, we have developed an approach to specifically deliver vector into all lung lobes. in this strategy, an intracorporeal nebulizing catheter (aero-probe) is inserted into a bronchoscope to permit visual targeted aerosolization of vector specifically into each lung lobe. using this approach, 2 ml of 0.1% lpc (to transiently open tight junctions) containing 1x1012 vp of hdad-k18lacz was sequentially aerosolized into each of the six major lung lobes of a baboon. aerosolization was triggered by the ventilator upon inspiration at 0.1 ml/pulse at a rate of 8 to 10 pulses/min. halfway through the procedure a very slight, transient and fluctuating decrease in oxygen saturation of no more than 2% was noted that did not warrant supplemental oxygen. the entire procedure was otherwise uneventful and well tolerated with no clinical manifestations of toxicity. no changes in chest x-rays taken at 6 h, 24 h and at necropsy 72 h post-vector were observed compared to baseline. x-gal staining of the lungs at 72 h revealed extensive transduction of the epithelium in the large and small airways in all six major targeted lung lobes. x-gal histochemistry revealed substantial transduction that was exclusively restricted to the airway epithelial cells and submucosal glands, the target cells for cf gene therapy. assessment of the proximal major bronchi from each lung lobe revealed that~20% of the airway epithelial cells were transduced in the left upper lobe (lul), ≥ 90% were transduced in the lml, lll, rul, rml and rll, and~80% were transduced in the accessory lobe. approximately 70% of the airway epithelial cells in the tracheal sections examined were transduced. we also investigated the duration of pulmonary transgene expression. in these studies, we aerosolized a hdad expressing the baboon α-fetoprotein from the k18 promoter into the lungs of baboons. by measuring serum bafp levels, we found that pulmonary transgene expression from transduced airway epithelial cells can be detected for at least 177 days post-vector. these results demonstrate for the first time that exceedingly high levels of transduction of the airway epithelial cells and submucosal glands throughout all lung lobes in a large animal can be achieved with negligible toxicity resulting in long term trangene expression and should pave the way towards successful clinical cf gene therapy. inefficient gene transfer after repeat administration of most viral vectors has limited their suitability for cf gene therapy. however, lentiviral vectors are reported to be less immunogenic. this property combined with their capacity to integrate into the genome of transduced cells and, therefore, allowing gene expression for the life-time of the cell (approximately 3 months for airway epithelial cells and theoretically unlimited for stem cells) justify further investigation of these vectors for cf gene therapy. to ensure high transduction efficiency in the lung without the need for any pre-conditioning strategies we pseudotyped a simian immunodeficiency virus (sivagm)-based vector with the sendai virus f and hn proteins (f/hn-siv-gfp) and transduced mouse nasal epithelium (4x10e8 tu/mouse). at this titre transduction efficiency along the nasal septum was up to 5% when measured 10 and 30 days after transduction (n=4/time-point). the vast majority (approx. 80%) of gfp positive cells across all time-points were respiratory epithelial cells followed by neuronal and sustenticular cells (approx. 20%) in the olfactory epithelium. interestingly, we observed two types of duration patterns of gfp expression in mouse nose. in some mice the number of gfp positive cells decreased over time (day 30: 218±17 cells/section, n=4, day 360: 19±17 cells/section, n=6). however, in other mice strong gfp expression persisted for up to 360 days (day 360: 159±81 cells/section, n=4), significantly longer than the proposed life-span of airway epithelial cells. in order to support the idea of f/hn-siv integration into nasal respiratory stem/progenitor cells we artificially induced cell division after siv vector transduction by damaging the nasal tissue with the detergent (polidocanol), which strips of the epithelium within a few hours, while retaining the basal cell layer. the epithelium completely regenerates over the course of a few days. seven and 28 days after the vector transduction (4x10e8 tu/mouse, n=3) the nasal tissue was perfused with 2% polidocanol (10 µl/mouse) and gene expression was analyzed 4 weeks after the last detergent treatment. gfp expression was detectable in various cell types of the regenerating epithelium including ciliated and basal cells. importantly, gfp-expressing cells were more clustered after polidocanol treatment, possibly indicating origination from a common progenitor. these data suggest that, f/hn-siv integration may have occurred into differentiated airway epithelial cells as well as into stem/progenitor cells involved in the regeneration of the airway epithelium. introduction: hypersecretion of mucus is a major cause of airway obstruction in cystic fibrosis, asthma and chronic bronchitis. though mucus is a complex, non-homogeneous mixture of secretions, one group of its constituents, the mucous glycoproteins or mucins, contributes greatly to the obstruction associated with airway diseases. currently, there are a limited number of therapeutic agents available for controlling mucin overproduction and hypersecretion. the objective of this study is to develop a sensitive, cost-effective technique for reliably quantifying total mucin concentration in a robust mucin-secreting cell line for use in high throughput screening of small molecule compounds with potential to inhibit mucin secretion. methods: an enzyme-linked lectin assay (ella) was adapted using a lectin from helix pomatia (hpa), which binds specifically to n-acetylgalactosamine residues. bovine sub-maxillary mucin was used for test development and later, as a mucin standard. the test sample was "sandwiched" between hpa pre-coated onto 96-well microtiter plates and biotinylated hpa lectin. the plate was developed with a fluorescent hrp substrate, and measured at 590 nm using a standard fluorescence plate reader. using the ella, a number of carcinoma (hm3, a431, ls174, nci-h292) and immortalized lung epithelial (16hbe, cfsme) cell lines were tested to determine their responsiveness to various secretagogues. preliminary experiments indicated that the nci-h292 cells, derived from a human mucoepidermoid lung carcinoma, were most suitable for adaptation to high throughput studies. nci-h292 cells were seeded into 96-plates at a density sufficient to produce confluence in approximately 48 hours. then, individual wells were incubated for 1 hour in either control or secretagogue-containing media. supernatants were collected and mucin concentration of individual wells was determined using the ella. results: optimization of the ella provided a sensitive calibration curve from 150 ng/ml to 2.5 mg/ml. mucin secretory ratios (sr) were calculated by comparing mucin release from test and control wells. significant (p ≤ 0.05) secretory ratios were obtained following incubation with utp at 400 µm (3.17 ± 0.64), vip at 300nm (3.60± 1.41), carbachol at 2.4 mm (2.17± 1.2), interleukin-1β at 5 ng/ml (2.74± 0.47) and heparin-binding egf-like growth factor at 90 ng/ml (2.49± 0.53); all data expressed as sr ± sem, n = 6. histamine at 54 µm did not result in significant mucin secretion. conclusion: a relatively simple and economical assay has been developed for high throughput screening of small molecules for detecting their effects on mucin secretion. cystic fibrosis (cf) is the most common lethal genetic disease in north america, where it occurs with a frequency of one in ~2200 live births. it is caused by mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene, the most common being the deletion of phenylalanine 508 (delf508). the delf508 mutation causes a folding defect that inhibits maturation and trafficking to the plasma membrane, however the mutant still functions as a chloride ion channel when delivered to the plasma membrane, and partial rescue (15-20%) may be sufficient to alleviate disease symptoms. thus cftr is an attractive target for the development of pharmaceutical therapies. we recently described a novel, cell-based assay for protein trafficking which directly monitors expression of delf508 cftr on the surface of baby hamster kidney (bhk) cells and is suitable for high throughput screening of small molecule libraries. in this study we have extended the approach to natural products by studying 720 extracts derived from marine sponges that were collected from the coasts of indonesia and papua new guinea. sponges are a particularly rich source of novel bioactives and secondary metabolites. iterative use of our high throughput assay and chemical deconvolution of active fractions yielded four alkaloids jmcg1102, jmcg1103, jmcg1104 and jmcg1105. these compounds caused a detectable increase in delf508 cftr trafficking to the plasma membrane within 6 h, and after exposure to 10 pm for 24 h all four increased surface delf508 cftr expression 10-30% and enhanced camp-stimulated halide conductance. we tested a series of analogues of the alkaloids and found three others jmcg1106, jmcg1107 and jmcg1108 that also caused trafficking correction and functional ion channels in the plasma membrane. these results were confirmed in functional studies of the human bronchial epithelial cell-line cfbe stably overexpressing delf508 cftr. these compounds increased surface delf508 cftr by 15-80% within 6 h when treated with 1nm. these results identify a promising group of delf508 cftr trafficking correctors that are now being actively pursued as potential pharmacotherapeutics for cystic fibrosis along with other hits from compound library screening campaigns. work supported as part of the breathe program jointly funded by the ccff and cihr, and grants from cystic fibrosis foundation therapeutics, (cfft). kim chiaw, p. 1,2 ; bear, c.e. 2 background/rationale: biosynthetic misprocessing of the major cf mutant ∆f508-cftr has been proposed to result from the disruption in domain-domain interactions (du et al. nature struct and mol biol, 2005) that may lead to the exposure of di-arginine (rxr) retention/retrieval motifs. simultaneous site directed mutagenesis of all four rxr motifs to rxk or kxr motifs overcame retention and promoted cell surface expression of ∆f508-cftr (chang et al. mol cell, 1999) . we hypothesize that it is possible to compete inhibitory interactions with rxr motifs exposed in ∆f508-cftr by delivering synthetically derived cftr peptides bearing one or a combination of rxr motifs (cf-rxr), each conjugated to cell penetrating peptides (cpp) to permit intracellular delivery. results: confocal immunofluorescence microscopy confirms that alexa-488-tagged cpp-cf-rxr peptides can be taken up rapidly (within 10 min) by bhk cells stably expressing recombinant human ∆f508-cftr. fluorophore-conjugated peptides appear to be concentrated in vesicular compartments, consistent with the current hypothesis that cpp-peptides enter cells into endosomal vesicles via macropinocytosis from which efflux into the cytosol occurs (wadia et al. nature med, 2004) . we determined that treatment of cell cultures with cpp-cf-rxr peptide at a final concentration [1 µm] for 90 minutes is effective in inducing cell surface functional expression of human ∆f508-cftr in bhk cells as observed by a 2.3-fold increase in cyclic amp-mediated iodide efflux over control ∆f508-cftr (n=6; p < 0.01). this rescue is specific to the cf-rxr peptide as controls (cpp alone or peptides bearing a kxk motif in place of rxr) show no functional rescue of ∆f508-cftr (n=6; p > 0.05). preliminary cyclic ampmediated iodide efflux results also indicate that ∆f508-cftr functional rescue by cpp-cf-rxr peptide is as effective as temperature (27 o c) rescue. although we hypothesize that the peptide acts to facilitate trafficking of the major mutant to the cell surface, immunoblotting studies fail to show a clear conversion of the er-form (band b) to the fully processed, band c form of the protein. currently, we are developing more sensitive assays of cell surface expression as well as evaluating the possible efficacy of this peptide in potentiating function of ∆f508-cftr. discussion: future efforts include investigating the relative efficacy of peptides corresponding to the four different rxr motifs in rescuing ∆f508-cftr trafficking. the promising candidate described above is currently being evaluated with respect to functional rescue of the major mutant in primary human bronchial epithelial cells obtained from patients homozygous for ∆f508-cftr (joseph zabner and phil karp, university of iowa) and in cftr-∆f508 mice. these studies may provide the basis for future pre-clinical studies of peptide based therapies of cystic fibrosis (supported by ccff (breathe program grant) and cihr -(strategic training program in the structural biology of membrane proteins linked to disease)). several promising methods of promoting ∆f508 cftr escape from er degradation increase cftr chloride channel activity at the cell surface. however, increasing data from a number of laboratories indicate that in both epithelial and non-epithelial cell lines expressing ∆f508 cftr, the rescued chloride channel is unstable at the cell surface compared to wild-type cftr. small molecule correctors have therefore become a widely used approach to enhance ∆f508 cftr function at the cell surface. in most cases, the exact mechanism of these promising compounds has not been established. in the present study, we use stably transfected, polarized cfbe41o-cells to elucidate the effect of two of these compounds, cfcor-325 and corr-4a, on the surface stability of wild type and low temperature rescued ∆f508 cftr. specifically, we cultured cells expressing ∆f508 cftr at 27c for 48 hours to establish a large pool of surface-expressed ∆f508 cftr; we then monitored cftr internalization rates and half-life with and without small molecule treatment using surface biotinylation-based assays, performed as described previously (jurkuvenaite et al. j. biol. chem. 281:3329, 2006) . as a control, internalization and half-life of transferrin receptor was also followed. our results indicate that both cfcor-325 and corr-4a slow ∆f508 cftr endocytosis from ~30%/2.5 min to ~5%/2.5 min, and this is similar to wild type cftr internalization rates. neither wild type cftr nor transferrin receptor internalization rates were affected by corrector treatment, suggesting that the effects of the correctors were specific for ∆f508 cftr. in the surface half-life studies, both correctors doubled ∆f508 cftr halflife from 2 to 4 hours, but they did not correct the half-life to wild type levels (~8 h). again, the correctors had no effect on either wild type cftr or transferrin receptor surface half-lives. our results suggest that small molecule correctors may not only rescue cftr from erad during biogenesis, but can contribute to the stabilization of ∆f508 cftr at the cell surface, and this effect seems to be ∆f508 cftr-specific. the ∆f508 mutation in cftr causes defects in chloride channel gating and cellular processing. cell cultures models suggest the need for both a potentiator and corrector to restore ∆f508-cftr channel function and processing, respectively. previously, we identified several classes of potentiators, including 'phenylglycines', which probably bind to cftr at the cell surface and restore normal channel gating (pedemonte et al., mol. pharm. 2005; 67:1797 -1807 . we also identified several classes of correctors, including 'bisaminomethylbithiazoles', which may bind to ∆f508-cftr in the er and partially restore its folding and plasma membrane targeting (pedemonte, et al., j. clin. invest. 2005; 115:2564 -2571 . these compounds were identified from screening a diverse collection of 150,000 small molecules; the most active potentiator was phenylglycine pg01 {2-[(2-1h-indol-3-ylacetyl)-methyl-amino]-n-(4-isopropylphenyl)-2-phenyl-acetamide} and the most potent corrector was the bisaminomethylbithiazole corr-4a {n(2-[5-chloro-2-methoxyphenylamino]-4'-methyl-4,5'-bithiazol-2'yl) benzamide}. here we synthesized and characterized a hybrid molecule (corr-4a-linker-pg01) containing both corrector and potentiator fragments. based on sar data of potentiators and correctors, a hybrid molecule was designed that incorporated an enzymatically hydrolysable linker to generate and deliver the potentiator and corrector-linker fragments. using synthetic organic chemistry we synthesized a hybrid molecule containing a pg01-oh moiety and a corr-4a-linker-cooh moiety, linked with an ethylene glycol spacer through an ester bond. the potentiator and corrector fragments (after cleavage) had low micromolar potency or better. however, the intact hybrid molecule was inactive, likely because of its large size (990 daltons) and hence poor cell penetration. as proof-of-principle evidence that the hybrid molecule can be hydrolyzed to the active fragments, treatment with carbonic anhydrase or rodent stool specimens gave the appropriate potentiator and corrector-linker fragments identified by lcms. smallmolecule corrector-potentiator hybrids have potential utility as single drug therapy for cf caused by the ∆f508 mutation. supported by cff and nih. cftr is a chloride channel gated by atp binding and hydrolysis at its two nucleotide binding domains (nbd1 and nbd2). the two nbds may dimerize in a head-to-tail configuration, as observed in other abc transporters, forming two atp binding pockets (abp1 and abp2), with the atp molecules buried at the dimer interface. abp2, formed by the walker a and b motifs of nbd2 and the signature sequence of nbd1, was proposed as the site critical for the atp-dependent opening of the cftr channel, while abp1 (consisting of the walker a and b motifs of nbd1 and the signature sequence of nbd2) may contribute to the stabilization of the open channel conformation. g551d, the third most common cf-associated mutation, is characterized by a very low open probability, and patients carrying this mutation present a severe phenotype. this mutation is located in the signature sequence of nbd1 (and thus in abp2). we have recently characterized this mutant channel and found that its low activity is atp-independent. this behavior corroborates with the idea that the occupancy of abp2 by atp is crucial for catalyzing the opening of the channel. interestingly, we now found that a high affinity atp analog, n 6 -(2-phenylethyl)-atp (p-atp), increases g551d currents primarily by increasing the open time of the channel. this effect of p-atp is reduced when p-atp was applied together with atp, suggesting a competition between atp and p-atp for a common binding site. introducing the mutation y1219g (located at abp2), which reduces by more than 50-fold the atp-binding affinity in wild-type channels, does not alter the effect of p-atp in the g551d/y1219g mutant. in contrast, when we introduce the mutation w401g (located at abp1) in the g551d background, the effect of p-atp is reduced remarkably, suggesting that abp1 is the binding site where p-atp binds to increase the activity of g551d channels. these new results further confirm the idea that nucleotide binding at abp1 could stabilize the open channel conformation. since the competition experiments show that atp and p-atp are readily exchanged in the binding site, we conclude that the atp molecule is not occluded in abp1, at least under the g551d background. our observation that p-atp enhances the g551d activity by binding at abp1 implicates that abp1 can potentially be a target for drugs to bind and increase the channel activity. supported by cff grant to s. bompadre and nih grants to t.-c. hwang (hl53445r01, dk55835r01) and s. bompadre (dk75408k01). van goor, f.; hadida, s.; grootenhuis, p. vertex pharmaceuticals, san diego, ca, usa several cf-causing mutations, including ∆f508, g551d, and r117h, cause gating defects in cftr that decrease the open probability (po) of the channel, resulting in decreased cl-secretion across epithelia of multiple organs, including the lung. a therapeutic strategy for the treatment of cf is to develop pharmacological potentiators that increase po leading to increased fluid transport in affected tissues. vx-770 was identified through cell-based screening and chemistry optimization as a potent potentiator of cftr, including ∆f508-, g551d-, and r117h-cftr. in single-channel recordings of cftr expressed in fisher rat thyroid cells, vx-770 increased the po of ∆f508-cftr from 0.11 ± 0.05 to 0.45 ± 0.06. the po of g551d increased from 0.08 ± 0.03 -0.52 ± 0.19. these levels are similar to the po of wild-type cftr, indicating that vx-770 restores normal gating at the single channel level. to assess the potency and efficacy of vx-770 in native airway cells, cl-secretion was monitored in ussing chamber studies using human bronchial epithelia (hbe) cultured from the bronchi of cf and non-cf donors. in the absence of the potentiator vx-770, cftr-mediated clsecretion in ∆f508/∆f508or g551d/∆f508-hbe reached a maximum amplitude of 2.6 ± 0.5 and 2.7 ± 0.2 ua/cm2, respectively. this is ~5% of that observed in wild-type-hbe (56 ± 6 ua/cm2) and consistent with the low level of residual cftr activity reported in some individuals with severe cf disease. in ∆f508/∆f508-hbe, vx-770 caused a 2-fold increase (peak response; 5.5 ± 0.5 ua/cm2) in cftr mediated cl-secretion with an ec50 of 33 ± 8 nm, whereas in g551d/∆f508-hbe, cl-secretion increased 14fold (peak response; 36 ± 3 ua/cm2) with an ec50 of 600 ± 200 nm. the improved efficacy observed in g551d/∆f508-hbe is consistent with a higher cell surface density of g551d-cftr compared to ∆f508-cftr, which has defective trafficking to the cell membrane. vx-770 was found to be highly selective for cftr and to have good oral bioavailability in multiple species with a half-life of 6 -14 hours. the biological and pharmacokinetic data support the clinical development of vx-770 for the treatment of cystic fibrosis. we would like to acknowledge the cfft for their support and dr. joseph pilewski for providing cf hbe. the pari eflow ® rapid nebulizer is a member of the "soft mist" inhaler class for the administration of aerosolized antibiotics that eliminates the need for a compressor. objectives: to compare the pharmacokinetics and safety of tobramycin inhalation solution (tobi ® ) delivered via the pari eflow ® rapid electronic nebulizer vs the pari lc ® plus jet nebulizer with compressor. methods: we compared tobramycin levels in sputum and serum, incidence of bronchospasm and frequency of adverse events (ae) of 300 mg tobi, administered twice-daily for 2 weeks from these two delivery systems in a randomized, crossover study in 25 cystic fibrosis patients. blood and sputum samples were collected over a dosing interval after the first and last dose on each device (days 1 and 15). tobramycin serum and sputum area under the concentration-time curves (auc) were derived and the therapeutic ratio (mean sputum auc / mean serum auc) calculated. pulmonary function tests were performed before and 30 min after nebulization on days 1 and 15, and ae were recorded. results: the total nebulization time was reduced by half for the pari eflow rapid vs pari lc plus (7.3 ± 1.7 min vs 17.7 ± 3.8 min [p<0.0001] on day 1, and 8.0 ± 1.8 vs 16.3 ± 2.7 [p<0.0001] on day 15, respectively). tobramycin predose serum levels >2 µg/ml or cmax >12 µg/ml, predefined as a potential for increased risk of systemic toxicity, were not exceeded in any patient during use of either nebulizer. as tabulated below, mean systemic exposure to tobramycin from the pari eflow rapid on day 15 was slightly lower, by 14%, whereas mean sputum exposure was higher, by 80%, compared with the pari lc plus. consequently, the therapeutic ratio was nearly 2-fold higher for the pari eflow rapid due mainly to higher sputum concentrations. adverse events occurred in 16 patients using the pari lc plus nebulizer (primarily headache and abdominal pain) and in 19 patients using the pari eflow rapid nebulizer (primarily headache, cough, dyspnea). on day 15, no clinically relevant bronchospasm (defined as ≥20% decrease in fev 1 ) was observed for either treatment. the mean percentage change in fev 1 at 30 mins from predose on day 15 was -2.76% for the lc plus and -1.27% for the eflow rapid. conclusions: tobi delivered via the pari eflow rapid electronic nebulizer required shorter delivery times with similar safety and a higher therapeutic ratio (sputum/serum exposure) compared with the pari lc plus jet nebulizer. sponsored by novartis pharma ag which acknowledges support from pari gmbh introduction alpha-1 antitrypsin (aat) is a protein protecting lung tissues by inhibition of neutrophil elastase. the function of neutrophil elastase is to digest bacteria and other foreign objects in the lungs. in a person deficient of aat, the neutrophil elastase acts uncontrolled, destroying healthy tissue. the result of the damage is emphysema, which progresses if not treated. patients show a specific impaired breathing pattern of short inhalation followed by prolonged exhalation. current therapy consists of weekly i.v. infusion of aat of about 60-90 mg/kg body weight. by this method only about 2% of the administered dose is estimated to reach the lungs. since aat can currently be derived only from human blood serum by an expensive purification process, the worldwide supplies are limited and do not allow to treat all patients. an improvement of the delivery efficiency is highly desirable. inhaled treatment would offer a targeted therapy by delivering aat directly to the lungs and allow for a better use of the limited drug. the eflow ® , a novel electronic nebulizer, is more efficient than conventional nebulizers. it operates by means of a perforated vibrating membrane, applying no or only little stress to the nebulized substances. hence it is well suited for the pulmonary delivery of proteins. it has been shown previously, that a 2% aat solution can be nebulized by eflow ® without a loss of biological activity. we investigated two eflow ® configurations comparing the in-vitro delivered dose for different breathing patterns. method a highly purified 2% aat preparation (kamada ltd, rehovot, israel) was aerosolized with the eflow ® electronic nebulizer. initial studies were conducted using the 30l configuration of eflow ® . delivery performance is compared to a specially designed 30xl configuration, with increased aerosol chamber volume. aerosol delivery efficiency was determined by breath simulation using an emphysema breathing pattern (tidal volume 450 ml, 17 breaths/min, inh:exh ratio = 1:2.5) generated by a pari compas™ breath simulator. as reference, a standardized regular breathing pattern (tidal volume 500 ml, 15 breaths/min, inh:exh = 1:1) was also investigated. the respirable fraction (drug in droplets < 5 and 3.3 µm) was determined using the andersen cascade impactor at 28.3 l/min. samples were analysed for aat activity by an elastase inhibition assay. when the eflow ® 30l configuration was investigated for the standardized breathing pattern, a delivered dose (dd) of 70 ±3% of the loaded dose (92mg) was found. this value was reduced to 50 ±1% when the emphysema pattern was applied. with the new eflow ® 30xl configuration an improved dd of 81 ±3% was obtained. furthermore, when the emphysema pattern was applied, the dd did not drop strongly, but remained at values above 68%. with 83% of aerosol droplets in the respirable size range (<5µm) and 42% <3.3 µm, a high % of aat can be expected to reach both, the central and peripheral lungs of patients. conclusion inhaled aat has the potential to significantly improve the efficiency of aat replacement therapy. a customized eflow ® electronic nebulizer produced delivered doses > 60% even when breathing patterns of patients with impaired breathing capabilities were mimicked. the formulation is optimized for rapid aerosol administration through use of higher drug concentrations and is taste-masked. this study was conducted to evaluate the aerosol performance of novel higher dosed levofloxacin inhalation solutions in a customized eflow ® electronic nebulizer. methods two concentrations of mp-376 (50 mg/ml and 30 mg/ml of levofloxacin) were nebulized at fill volumes of 2 and 4 ml using the eflow ® 35l nebulizer configuration. the in-vitro delivered dose (dd) was determined by breath simulation using a standardized breathing pattern (tidal volume 500 ml, 15 breaths/min, inh:exh ratio = 1:1) generated by a pari compas™ breath simulator. the geometric droplet size distribution was determined by laser diffraction (ld). the aerodynamic droplet size distribution was determined using the andersen cascade impactor (aci). the invitro respirable dose (rd) was calculated by multiplying the respirable fraction (rf, %droplets < 5µm) derived from the cascade impaction experiment and the dd derived from breath simulation. nebulization time was determined by the electronic shut-off of the eflow ® control unit. all tests were conducted for three devices in duplicate, each (n=6). the eflow ® electronic nebulizer delivered mp-376 at both concentrations and fill volumes with equal efficiency, obtaining in-vitro dds of around 60±5 %. the average nebulization times were between 3 and 4 minutes for the 2 ml fill volume and between 7 and 8 minutes for the 4 ml fill volume and were also independent of the concentration in the investigated range. determination of the mass median diameter (mmd) by ld resulted in values between 3.5 and 4.1 µm. values of the mass median aerodynamic diameter (mmad) obtained by cascade impaction were similar (between 3.7 and 4.3 µm). the average respirable fraction was in the range between 70% and 75% for both experimental methods. conclusion the eflow ® electronic nebulizer can be customized to deliver high doses of mp-376. the short nebulization times achieved are patient friendly and should support high patient compliance. mp-376, with the potential for once daily administration, provides significant advantages over currently avail-able aerosol antibiotics. clinical evaluation of mp-376 delivered by the eflow ® nebulizer in cf patients is in progress. lavange, l. 1 ; engels, j. 2 ; accurso, f.j. 3 1. university of north carolina, chapel hill, nc, usa; 2. inspire pharmaceuticals, inc., durham, nc, usa; 3 . university of colorado, denver, co, usa introduction: percent change from baseline in a continuous outcome variable, such as lung function, is a useful descriptive measure in therapeutic clinical trials. while often favored by clinicians as the primary efficacy measure, the use of percent change as the basis for statistical comparisons raises a number of issues. defined as 100*(post-pre)/pre, where pre and post represent baseline and follow-up values, respectively, percent change is a ratio of random variables and as such, does not follow a normal distribution. analyzing percent change with standard methods can result in inefficiences and may be inappropriate without a suitable transformation (e.g., logarithmic). furthermore, the treatment effect estimated on the transformed scale is difficult to interpret. the purpose of this abstract is to describe an alternative method for analyzing percent change and illustrate its utility in cf clinical trials. methods: probability plots are generated to compare the distributions of percent change in fev 1 from baseline to study endpoint among treatment groups. the plots are similar to kaplan-meier curves, with percent change on the horizontal axis in lieu of survival time. the percentage of patients on the vertical axis depicts the cumulative percentage of patients who had a percent change at least as great as the corresponding value on the horizontal axis. a log-rank test statistic provides an overall test for any difference in distributions among treatment groups. the methods are illustrated using data from a 28-day, phase 2, multicenter, randomized, double-blind, placebo-controlled clinical trial of denufosol in cf patients with a screening fev 1 ≥75% predicted. results: a probability plot of denufosol (active doses combined, n=68) vs. placebo (n=21) is shown below. more denufosol patients (43%) experienced improvement compared to placebo (33%). the log rank test, adjusted for study site, showed that the overall distributions of percent change in fev 1 between denufosol and placebo were significantly different (p-value=0.029). conclusions: the proposed methods provide a convenient means for assessing differences in percent change and may be useful in cf clinical trials. comparisons of both a descriptive and inferential nature can be made with minimal assumptions, thereby avoiding the pitfalls in analyzing percent change with standard techniques. the methods are easy to implement with existing software packages (e.g., sas). trial supported by inspire pharmaceuticals, inc. and cf foundation therapeutics, inc. chen, x. 1,2 ; davis, p. 1 compacted dna nanoparticles formulated with pegylated polylysine and plasmid dna transfect airway cells in vivo at high efficiency, so they have great potential for gene therapy. the mechanism by which dna nanoparticles enter the cell and get expressed is not well understood. we previously showed that rhodamine labeled dna nanoparticles enter primary human tracheal epithelial (hte) cells within 15 min, and accumulate in the nucleus in 1 hr, where the transgene can be expressed. dna nanoparticles do not enter via clathrin-mediated endocytosis (cme), which leads to the degradation of internalized material in lysosomes. further characterization of the kinetics and trafficking pathway may allow us to improve the formulation of the nanoparticles and facilitate its gene delivery. as they do in hte cells, rhodamine labeled dna nanoparticles enter hela and 16hbeo-, an immortalized human bronchial epithelial cell line, within 15 min, and by 1-4 hr accumulate in the nucleus, especially the nucleolus, where they colocalize with nucleolin. expression of the gfp reporter gene in the nanoparticles was observed at 18 hr, which confirms the functionality of the labeled dna nanoparticles. cellular entry is energy dependent, as little or no intracellular rhodamine was observed at 4°c by 4 hr. we also applied rhodamine labeled nanoparticles and biotin-conjugated transferrin, a marker for cme, simultaneously. little colocalization was observed during a 15 min to 4 hr time course, so the nanoparticles do not follow this pathway. we then found a direct and strong relationship of cell surface nucleolin and the ability of the cells to take up and express dna nanoparticles. nucleolin directly binds to dna nanoparticles with kd=25.9 nm. manipulations of cell surface nucleolin in hela cells affect transfection of dna nanoparticles with a positive correlation. we therefore costained nucleolin in both hela and 16hbeo-cells treated with the rhodamine labeled dna nanoparticle. the rhodamine fluorescence colocalizes with nucleolin both in the cytoplasm at early time points and in the nucleus/nucleolus at 1 hr and 4 hr, which further supports that nucleolin might be associated with the nanoparticles during their trafficking. to confirm this association, we performed uptake experiments with ms-3, a mouse monoclonal antibody against nucleolin, alone or with rhodamine labeled nanoparticles over a 15 min to 4 hr time course. the ms-3 antibody enters hela cells at similar kinetics to the nanoparticles. at 30 min, we observed substantial amount of cytoplasmic staining, while nuclear staining tends to increase by time until 4 hr. it has little or no colocalization with transferrin in the cytoplasm, which suggests that this antibody also enters the cell via a pathway other than cme. in contrast, it showed extensive colocalization with dna nanoparticles in both cytoplasm and nucleus at all time points, which suggests that cell surface nucleolin and dna nanoparticles are associated or at least in close vicinity during their trafficking inside the cell. therefore, we suggest that dna nanoparticles enter the cell by binding to cell surface nucleolin and enter the cell via the same pathway as nucleolin antibody. chen, x. 1,2 ; davis, p. 1 dna nanoparticles are non-viral gene delivery vectors in clinical trial for treating genetic disorders including cystic fibrosis. we previously discovered that cell surface nucleolin serves as a receptor for the dna nanoparticles, and is important for their gene delivery efficiency. as nucleolin has no signal sequence or membrane-spanning domain, it is not clear how nucleolin are expressed on the outer surface of plasma membrane or what signals increase its surface delivery. we initially observed that the transfection efficiency of dna nanoparticles in hela cell decreases by 72.2% following 24hr serum-free medium treatment, which reduces cell surface nucleolin by 35.7% as determined by cell surface biotinylation and streptavidin bead pulldown. since serum depletion inhibits cell proliferation and affects cell cycle progression, we examined the level of cell surface nucleolin at different stages of cell cycle. hela cells were arrested at s phase by high concentration of thymidine, and allowed to progress synchronously through cell cycle after removing this block. surprisingly, we observed substantial increase of cell surface nucleolin at the onset of m phase, about 8 hours after release of thymidine block. it has been reported that nucleolin is phosphorylated by a cell cycle dependent kinase (cdk) cdc2, and has eight consecutive cdk phosphorylation sites. therefore it is appealing to speculate that phosphorylation by cdc2 might increase the targeting of nucleolin to the cell surface. we then determined the potential export signal in nucleolin by serial deletions. deletion of c-terminal 584 aa of nucleolin, including the c-terminal glycine/arginine rich (gar) domain and the four rna recognition motifs (rrm) does not affect its arrival at the cell surface. the n-terminal 123 aa of nucleolin is sufficient to target a gfp protein to the cell surface. in contrast, when we further delete the 8 cdk phosphorylation sites from aa 69 to 123, cell surface expression of nucleolin is significantly diminished. furthermore, nucleolin lacking the n-terminal 68 aa is not present on the cell surface. therefore, phosphorylation on the cdk sites may serve as a signal to enhance the cell surface expression of nucleolin. there has been recent interest in dry powder inhaled mannitol as a therapeutic agent in patients with cystic fibrosis (cf). it is has been shown to increase mucociliary clearance (mcc) by airway rehydration. whilst there has been one short term clinical trial of mannitol in adult subjects [1] , to date no studies have been conducted looking at its potential as a therapy in children with cf. it could be argued that children may have the potential to benefit most from a therapeutic agent that acts relatively proximally in the cf pathogenic pathway. the aim of this study was to determine acute tolerability of inhaled mannitol in children with cf. we recruited 39 children (aged 8 to 18 years) with cf. inclusion criteria were either rhdnase treatment or an fev 1 > 40% and < 70% of mean predicted normal value. 39 bronchial provocation challenges with incrementally increasing doses (5, 10, 20, 40, 80, 160, 160mg) of dry powder mannitol were carried out. subjects were pre-treated with bronchodilator 15 minutes prior to the challenge (400 mcg of salbutamol or 1 mg of terbutaline). fev 1 was measured following each dose increase up to a maximum cumulative dose of 475mg. oxygen saturation monitoring was carried out throughout. a positive challenge was defined by a drop in fev 1 of >15% from baseline. these subjects received bronchodilator treatment and had spirometry repeated at 15 minute intervals until fev 1 returned to within 5% of baseline. those children with a negative challenge had spirometry repeated 15 minutes post-completion of the challenge results mean baseline fev 1 was 68% predicted (45-94, sd 11.7). 9/39 subjects (23%) had a positive challenge. mean pd 15 (dose of mannitol required to cause a 15% reduction in fev 1 ) was 262mg (411-107, sd 104). the mean time to complete the challenge was 26 minutes (17-37, sd 5.0) for negative challenges and 27 minutes (15-40, sd 8.0) to pd 15 for those subjects with a positive challenge. we found no association between a positive challenge and age, sex, weight, height, baseline fev 1 , pseudomonas aeruginosa colonization, bronchodilator reversibility, previous aspergillus fumigatus sen-sitization, atopy or corticosteroid use. there was a non-significant trend for lower fef 25-75 (means 0.99 versus 1.40; p=0.099) and higher prevalence of aspergillus fumigatus cultured in sputum at baseline (5/9 versus 7/30 children; p=0.066) amongst those children who went on to have a positive challenge. there was no significant drop in oxygen saturation in either group. although cough was common during the challenge, other adverse events were infrequent. we find that 23% of children with cf could not tolerate inhaled mannitol as compared with 12% of adult subjects as reported in the previous study [1] . we could not identify factors predictive of a positive mannitol challenge in these patients. the most common cause of cystic fibrosis (cf) is the deletion of phenylalanine 508 (∆f508) in the cf transmembrane conductance regulator (cftr) chloride channel [1] . a major problem with ∆f508 cftr is that the protein is defective in folding so that little mature protein is delivered to the cell surface [2] . expression of ∆f508 cftr in the presence of small molecules known as correctors or pharmacological chaperones can increase the level of mature protein [3] [4] [5] [6] . unfortunately, the efficiency of correctorinduced maturation of ∆f508 cftr is low and other approaches are needed to increase the therapeutic value of correctors. we postulated that expression of ∆f508 cftr in the presence of multiple correctors that bound to different sites in the protein may have an additive effect on maturation. in support of this mechanism, we found that expression of p-glycoprotein processing mutants (cftr's sister protein) in the presence of two compounds that bind to different sites (rhodamine b and hoechst 33342) had an additive effect on maturation. therefore we tested whether expression of ∆f508 cftr in the presence of combinations of three different classes of corrector molecules would increase its maturation efficiency. it was found that the combination of the quinazoline vrt-325 together with the thiazole corr-2b or bisaminomethylbithiazole corr-4a doubled the steady-state maturation efficiency of ∆f508 cftr (about 40% of total cftr was mature protein) compared to expression in the presence of a single compound. the additive effect of the correctors on ∆f508 cftr maturation suggests that they may directly interact at different sites of the protein. the use of multiple correctors has the potential to increase the therapeutic value of pharmacological chaperones. pharmacol. 70, 297-302. the cftr protein is expressed at the surface of the airway epithelium, where it plays a critical role in maintaining airway hydration by secretion of chloride. for gene therapy of cf lung disease to be successful, it is critical that the endogenous transgene be expressed in the correct cell type and at levels sufficient to restore normal function. the expression of high levels of cftr has resulted in cftr mediated chloride secretion in a wide range of experimental systems. however, the cell type or types that need to be corrected and the level of cftr expression required to ameliorate the pulmonary manifestations of cf are not yet clear. because cftr has been localized to the ciliated cells of human airway epithelial cells, we have been investigating the use of ciliated cell-specific promoters to improve the efficiency and safety of gene therapy for cf and other airway diseases. in previous studies a fragment of the human foxj1 promoter was shown to target transgene expression specifically to ciliated cells. however, expression of cftr from this promoter in transgenic cf mice did not significantly improve the cf phenotype, as measured by the nasal potential difference (pd) technique (ostrowski et al, 2007) . this may be due to the inability of this promoter to correct the olfactory epithelium, which comprises approximately 50% of the mouse nasal cavity. in cf mice, the olfactory epithelium exhibits sodium hyperabsorption and an absence of cftr mediated chloride secretion, similar to the respiratory epithelium (grubb et al, 2007) . in this work, we used a fragment of the ciliated cell-specific promoter foxj1 to drive cftr expression specifically in human ciliated cells. replication deficient adenovirus expressing either egfp or cftr from the foxj1 promoter were used to transduce well-differentiated cultures of human cf cells following treatment with c10 to disrupt tight-junctions. cultures were studied 48 hours after treatment, and those treated with ad-foxj1/egfp showed strong expression of egfp that was dependant on ciliated cell differentiation. rna analysis demonstrated strong expression of cftr from the foxj1 promoter, and western blotting demonstrated levels of protein that were much greater than the level in normal airway cells (>100-fold). immuno-localization of cftr with specific antibodies resulted in a strong signal at the apical membrane of ciliated cells. cultures treated with ad-foxj1/cftr demonstrated a significant increase in forskolin-stimulated short circuit current (isc; 5.0 +/-0.8 µa/cm2, mean +/-sem, n=9), that was approximately 8-fold greater than the response in ad-foxj1/egfp treated cultures (0.6 +/-0.2 µa/cm2, n=9). the increase in isc was blocked by inh172, an inhibitor of cftr mediated secretion. under these conditions, ad-foxj1/cftr restored approximately 30% of the forskolin response of normal human airway cells (15.1 +/-0.3 µa/cm2, n=3). because patients expressing low levels of normal cftr mrna (5-20%) have mild disease symptoms, these studies demonstrate that the incorporation of the ciliated cell-specific foxj1 promoter into gene therapy vectors may be useful for treatment of cf. supported by nhlbi ro1 hl70199 and the cff. there is a compelling need for safe and effective ai therapy for cf lung disease. low-dose methotrexate (mtx) has been used to treat inflammatory diseases and its use has been reported in cf. encouraging results previously described regarding mtx in cf prompted this study. the study objective was to determine if mtx safely reduces inflammation in the airways of cf patients. methods: this was a single-center, open label study of mtx in stable cf patients with mild to moderate lung disease. baseline levels of neutrophils, free elastase, pro-inflammatory cytokines, and bacteria were determined from an induced sputum specimen. subjects received 15 mg/m2/week of oral mtx (single dose). after 12 weeks of treatment, a second induced sputum specimen was obtained for the same inflammatory indices. within subject comparisons (end of treatment vs baseline) were performed for the following primary endpoints: total white cell and neutrophil counts, percent neutrophils, and concentrations of free elastase, il-8, il-6, tnf-α, and il-1β. sputum quantitative microbiology was obtained at baseline and end of therapy. routine laboratories and spirometry were performed monthly. pharmacokinetic testing was performed on the final day of treatment. results: thirteen subjects were screened with 8 started on mtx. six completed the protocol and 2 withdrew early secondary to adverse events (gastrointestinal [gi] complaints and pulmonary exacerbation). five of 6 subjects completing the protocol had declines in pulmonary function: mean change in fvc was -0.257 l (range -0.47 to -0.09); mean change in fev1 was -0.15 l (range -0.29 to -0.06); change in fef25-75 was 0.01 l (range -0.25 to 0.22). mean change in weight was -2.7 pounds (range -12 to 7.5). mean change in free elastase was 62.4 µg/ml (range -68.8 to 406.4; sd 177.0). mean change in il-8 was 121511 pg/ml (range -122248 to 238400; sd 121511). similar results were obtained for the other cytokines. analysis of cytology specimens is pending. esr and crp did not show significant changes. two subjects completing the protocol had significant gi side effects, including 1 requiring admission for severe abdominal discomfort. quantitative microbiology specimens revealed an increase in colony counts in 2 subjects, decreases in 3, and mixed results in the 6th. other safety laboratories were not remarkable. pharmacokinetic studies are pending. complete statistical analysis and safety assessment are also underway. conclusions: the small sample size of this study precludes definitive conclusions regarding the safety or efficacy of mtx. however, the data suggest that 1) induced sputum inflammatory indices can be used to assess an ai in clinically stable patients, and 2) mtx therapy may not be beneficial, and may be difficult to tolerate as along-term ai therapy for cf lung disease. additional analyses of cell counts and microbiology from this study are ongoing, and may provide additional insight into the effect of mtx in the cf lung. sponsored by the cf foundation , a cellular component of many gram negative bacteria (e.g. pseudomonas aeruginosa), is a common airway stimulus. dampening the inflammatory response in cf reduces the progressive decline in lung function, so anti-inflammatory agents have become both a cornerstone of cf clinical care and a focus of therapeutics research development. reactive oxygen species [ros] can play a role in proinflammatory signaling, including the activation of nuclear factor κb [nfκb] . research has demonstrated increased oxidative stress in cf epithelial cells, potentially highlighting one of the mechanisms responsible for the exuberant inflammation observed. we have previously shown that cddo, a novel anti-inflammatory agent, significantly limits nfκb activation in cftr deficient cell culture models at nanomolar concentrations. using cf mice, we have also shown that intra-tracheal cddo limits neutrophil accumulation and the concentrations of proinflammatory cytokines and chemokines in bronchoalveolar lavage [bal] fluid in response to lps. the synthetic triterpenoids have been shown to activate the nrf2 transcription factor, thereby inducing several genes involved in redox balance. we now present data demonstrating that cddo may inhibit inflammation in models of cf pulmonary disease by reducing the oxidative stress within cells. methods: b6.129s6-cftr tm2mrc cf mice received 25µl of 10µm cddo in pbs with 3%dmso or vehicle (control) daily. drug was administered intratracheally with an atomizer (penncentury) for three days before stimulation. in separate experiments, all mice received 1µg lps or free pseudomonas aeruginosa intratracheally. mice were sacrificed by co2 asphyxiation and cardiac puncture 20 hours after stimulation and bal, serum and lung tissue were obtained. the lungs were incubated in lysis buffer containing protease inhibitors and immediately frozen. upon thawing, the tissue was homogenized and whole lung protein extracted. one mg of lung protein from each experimental animal was separated by two dimensional gel electrophoresis. gels were then imaged and analyzed with pdquest to identify protein spots that were differentially expressed in drugtreated mice compared with controls. spots of interest were excised from the gels, trypsin digested and analyzed by liquid chromatography/tandem mass spectroscopy. results: over 120 proteins with greater than 2-fold differences in expression were identified. we categorized proteins appearing in repeated analyses and identified several affecting intracellular redox regulation; including glutathione s-transferase mu1 and 2, peroxiredoxin 5 and 6, enolase 1 (alpha non-neuron), contrapsin, alpha-1 antiproteinase and cu/zn superoxide dismutase. these proteins were expressed at significantly greater concentration in the lungs of mice treated with cddo rather than vehicle. conclusion: cddo exhibits anti-inflammatory effects in mouse models of cf pulmonary disease and one potential mechanism for this effect may be the upregulation of reducing proteins to combat oxidative stress. methods: lpc (15 ul of 0.1%, 0.3% or 1.0% (w/v) in pbs) was administered (trans-orally via a cannula) to trachea of c57l/b6 mice. in sheep (1 of 5 completed) we targeted delivery of 4.5 ml of 0.3% lpc dissolved in pbs to the main bronchus of the right lung at branch 9 in a 3 month old sheep (bronchoscope, via tracheostomy). one hour later 30 ul (mice) or 0.45 ml (sheep) of a lv-lacz vector (3.12x109 tu/ml) was administered in the same manner. one week post-exposure lungs were inflation-fixed in situ, removed, stained using x-gal, sectioned, and counterstained with saf-o or h/e. results: no lacz gene expression (blue cells) was found in lungs of control (pbs-treated) mice or in the left (untreated side) of sheep lung. blue cells were found in scattered punctuate groups, or in lines of stained cells in mice and sheep. mice given the highest lpc dose (1.0%) had extensive gene transfer in larynx, trachea, carina, and in large, middle and small airways of most lobes of the lung, reaching 79% airway perimeter cell transduction in one animal. with decreasing lpc dose the distribution and number of blues cells was reduced, but transduced epithelial cells including nonciliated columnar cells, ciliated cells, and basal cells were seen in all cases. in sheep, blue cells appeared in the right main bronchus between branch 6 and branch 15, and in the branch 9 airway, and with highest expression found near branch 8 and 9. cross-sections revealed that primarily ciliated columnar cells and basal cells (and no goblet cells or macrophages) were transduced. conclusions: lv gene transfer into mouse or sheep lung can be enhanced by pretreatment with lpc. in mice this early data suggests an lpc dose-dependency. for sheep, we await further results to confirm the encouraging finding in this first study; however, it does confirm that gene transfer into the airways of large animals with a lung size similar to humans can also be achieved using lpc pre-treatment and a vsv-g pseudotyped lentivirus. the transduction of both ciliated and basal lung epithelial cells in-vivo in both models is an encouraging funding for the development and understanding of our continuing efforts to produce life-long airway gene transfer suitable for a cf airway gene therapy. supported by: nh&mrc, usa cff, sa channel 7 crf, philanthropic donations. increased airway na+ absorption is a characteristic abnormality in cystic fibrosis (cf), and is thought to play a key role in the pathogenesis of cf lung disease. we have previously demonstrated that mimicking na+ hyperabsorption by overexpression of βenac in mouse airways results in airway surface liquid (asl) volume depletion and reduced mucus clearance causing a spontaneous cf-like lung disease with high pulmonary mortality, and airway mucus plugging, mucous cell metaplasia and chronic airway inflammation in surviving βenac-transgenic (βenac-tg) mice (mall et al., nature med.10:487, 2004 ). in the present study, we used βenac-tg mice to test if inhibition of increased airway na+ absorption by the classic enac blocker amiloride has therapeutic effects on cf-like lung disease in vivo. specifically, we determined the effects of 'early' and 'late' enac blocker intervention on mortality, airway mucus obstruction and inflammation by starting intrapulmonary amiloride therapy in βenac-tg mice either at birth, i.e. prior to the onset of lung disease, or after mucus obstruction and inflammation had established. to achieve this goal, newborn, 5 day, or 4 week old βenac-tg mice and wild-type littermate controls were treated by intranasal instillation of amiloride (10 mmol/l; 1µl/g body weight, tid) or vehicle (h2o) alone for a period of 14 days. initial deposition studies showed that this treatment protocol resulted in pulmonary amiloride concentrations sufficient for inhibition of enac. during amiloride therapy, growth and survival were monitored, and any loss in body volume due to diuretic side effects was substituted by subcutaneous injection of isotonic saline (nacl 0.9%). after the 14 day treatment period, mice were euthanized, bronchoalveolar lavage (bal) was performed to determine inflammatory cell counts, and lungs were processed for histology and morphometry to quantitate airway mucus obstruction and mucous cell metaplasia. we show that amiloride significantly reduced pulmonary mortality by~70% (p < 0.001), when therapy was started in newborn βenac-tg mice. further, early amiloride treatment significantly reduced airway mucus content (p < 0.05), mucous cell numbers (p < 0.05), and bal eosinophils (p < 0.001) compared to vehicle treated βenac-tg mice. in contrast, amiloride therapy had no benefits on airway mucus obstruction, mucous cell metaplasia or inflammation, if treatment was started in 5 day, or 4 week old βenac-tg mice with established lung disease. taken together, our results are consistent with previous human studies where amiloride lacked therapeutic benefits in cf patients with established lung disease, and demonstrate for the first time that early inhibition of na+ hyperabsorption is an effective therapy for cf-like lung disease in vivo. these results warrant an evaluation of more potent and longer acting amiloride derivatives in chronic lung disease in mice, and a clinical evaluation of preventive enac blocker therapy in human trials. supported by cff (mall04g0) and ec (mext-2004-013666 objective: hypertonic saline aerosol delivered intranasally is currently being studied to enhance mucociliary clearance. there is evidence of patients' reluctance to use concentrations above 3% due to potential discomfort. our study was performed to determine the short term tolerance of 3.5% and 7% hypertonic saline versus normal saline delivered intranasally via a nebulizer/compressor system (pari sinustartm, pari respiratory equipment, midlothian, va). methods: using the sinustartm nasal aerosol delivery system, we administered 3 concentrations of saline solution, (0.9%, 3.5%, 7%) to 18 healthy, adult volunteers for 5 minutes each. a washout period of 5 minutes between treatments allowed volunteers to wipe their nose and cleanse their mouth with water. a 6 question self-administered questionnaire was completed following each treatment using a 9 point scale (1=high tolerability; 9=low tolerability). burning sensation, cold, cough, throat irritation, runny nose, and overall comfort were measured. the order of treatments was randomized and volunteers were blinded to the concentration. data: for all measurements 3.5% and 7% were equally well tolerated compared to normal saline with burning, cough, and throat irritation measuring 2.2 or below. no variable measured more than 3.8. 17 out of 18 volunteers stated they would continue this treatment on a regular basis. conclusions: our study indicates that during the time of treatment nasal aerosol delivery of hypertonic saline up to 7% is well tolerated in healthy adults. there appears to be no issue of discomfort associated with hypertonic saline that would prevent nasal aerosol treatment compliance. our overall research goal is to discover new drugs for clinical treatment of cystic fibrosis (cf) that will correct the biochemical defect in the predominant cf mutation, the ∆f508 form of the cystic fibrosis transmembrane conductance regulator (cftr) protein, which accounts for over 67% of all cf cases. this mutation leads to a misfolded protein which is rapidly degraded as well as changes in its function and half-life at the cell surface. however, it has been proposed that only a fraction of the normal surface expression level is needed to provide a clinically significant impact on the disease. thus, from a pharmacological standpoint, strategies that can partially but effectively correct these defects would be expected to have a clear clinical benefit to these cf patients, and these strategies may also be applicable to other cftr mutations. we previously demonstrated that several members of a particular class of related drugs, the anthracyclines, anthraquinones, and anthracenediones, significantly increased cftr cell surface expression and function in cell culture. in particular, we demonstrated that a non-cytotoxic concentration of doxorubicin (dox), a model anthracycline drug, significantly increased total cellular and membrane-associated cftr protein levels and cftr-associated chloride currents in human colon cancer t84 cells, and also caused a two-fold increase in ∆f508 cftr-associated chloride current in a canine mdck-c7-derived cell line that expresses a stably transfected copy of human ∆f508 cftr. our previous studies also demonstrated that dox is able to impart structural integrity to ∆f508 cftr, increasing its half-life and decreasing its proteolytic sensitivity, which is indicative of efficient folding. additionally, two other structurally related drugs, i.e., the anthracycline, daunorubicin, and the anthraquinone, mitoxantrone, were shown to have similar effects on ∆f508 cftr expression. in the current work, we have extended these effects to two other related aza-anthracenedione molecules, bbr2778 (pixantrone) which is a cancer chemotherapy drug with lower non-target toxicity than dox and which is in phase iii clinical trials, and a structurally related analog, bbr2828, which is essentially non-cytotoxic with a 100-fold lower cytotoxic potency than bbr2778. both compounds increased cftr-associated chloride currents to a similar extent as dox in cfbe human airway epithelial cells expressing ∆f508 cftr, as measured by ussing chamber experiments. the ability of the non-cytotoxic analog bbr2828 to do so is particularly important, since it indicates that correction of the ∆f508 cftr defect is not directly tied to the toxicity of this class of compounds per se but rather is a result of other structural features. thus, it is likely that this and/or other non-toxic analogs in this chemical class can be developed that have potential as clinically useful agents for treatment of cf in patients. supported by a cystic fibrosis foundation (cff) grant to jwh and rm, and by the cff-supported dartmouth cf program. cystic fibrosis (cf) is caused by mutations in cystic fibrosis transmembrane conductance regulator (cftr). cftr is not only an epithelial chloride channel, but it also regulates other transporter and ion channels, including epithelial sodium channels (enac) and aquaporin water channels. in patients with cf, absent or dysfunctional cftr results in abnormal electrolyte and fluid content on the epithelial surface. treatments intended to normalize ion transport in cf airways through non-cftr dependent pathways represent attractive approaches to alleviate the underlying pathologic defect. calcium (ca2+)-activated cl-channels have been proposed as rescue channel for the cyclic amp-dependent cftr cl-channels, offering a target for cf pharmacotherapy. increases in cytosolic ca2+ concentration activate epithelial cl-channels, but inhibit epithelial na+ channels, which is also beneficial in correcting the hyperabsorption of na+ in cf. herein we developed a high throughput screening (hts) assay for screening compound libraries with the intention of finding compounds which can increase intracellular ca2+. in the pilot primary screening, we screened a mssp library which contains 2000 known bioactive compounds and natural products. 7 compounds were classified as the real hits after the secondary screening validation with the hit percentage 0.35%. we have found that the ec50 of these 7 compounds are less than 100um in dose-response studies and none of those compounds were toxic to the cells. we have further discovered that those compounds stimulate cl-secretion through activating ca2+ dependent cl-channels in cf and non-cf human airway epithelial cells in short-circuit current experiments. in summary, our data suggest that modulation of intracellular ca2+ is a target for cf therapy. the compounds identified by our study will provide possible therapeutic leads in the treatment of cf. non-viral gene delivery particles with various synthetic polymer coatings have been developed for cystic fibrosis (cf). intracellular trafficking and in vivo tissue distribution of these particles must be carefully monitored to guide rational design of efficient delivery system. fluorescent semiconductor quantum dots (qds) allow sensitive, long-term and multi-target imaging in cellular environment, and so are a promising tool in tracing gene delivery materials in vitro and in vivo. however, application has been limited by the lack of efficient and reproducible techniques of qd bioconjugation. here, we describe a method of labeling dna nanoparticles with tunable qds that may enable us to study gene delivery in vitro and in vivo. highly fluorescent zns-coated cdse qds were first synthesized and encapsulated in amine-containing phospholipid micelles. the non-viral vector system we used is based on a polymer backbone consisting of 30 lysines with a cysteine on the n-terminus (ck 30 ) which is conjugated to polyethylene glycol (peg). this polymer vector condenses dna plasmids into stable nanoparticles that efficiently transfect airway epithelial cells in vivo. to introduce the aminecontaining qds, we utilized a hetero-bifunctional peg with an nhs ester at one end, which was first reacted with the amine groups of the qds and a maleimide group at the other end which was subsequently reacted with the thiol group in ck 30 . excess reagents were removed from the final conjugates qd-peg-ck 30 by filtration. conjugation was first confirmed by 0.5% agarose gel electrophoresis. free qds were positively charged and migrated toward the cathode. pegylated qds migrated significantly slower than free qds, and qd-peg-ck 30 conjugates migrated at still a different rate. to further demonstrate conjugation, we biotin-labeled the ck 30 and assessed the binding of qd-peg-ck 30 -biotin to avidin coated agarose beads by monitoring the yield of fluorescent beads. qds, pegylated qds, and physical mixture of qds and ck 30 -biotin were also incubated with the agarose beads as controls. fluorescent beads were detected only when qd-peg-ck 30 -biotin was present. to directly evaluate the ability of qd-peg-ck 30 to bind double stranded dna, we also immobilized double stranded dna fragments to agarose beads. qd-peg-ck 30 , but not unconjugated qd bound to the immobilized dna fragments as determined by the yield of fluorescent beads. qd-peg-ck 30 was then used to compact luciferase reporter plasmids. electron microscopy images of the compacted dna showed that qds were integrated into some dna nanoparticles. we transfected hela cells with qd-labeled and unlabeled dna nanoparticles. luciferase assay and toxicity assay indicated that qd-labeled dna nanoparticles transfected hela cells, though with less reporter gene expression than unlabeled nanoparticles and more toxicity than uncompacted qd. in conclusion, we have developed a method to conjugate qd to dna nanoparticles. improvement of the quality and biocompatibility of the qd may be required for further in vitro and in vivo studies. this system is also versatile. other less toxic optical makers can be readily tested. this study was supported by dk58318 and the cff. in cystic fibrosis (cf) the epithelial sodium channel (enac) hyperactivity plays a role in the pathogenesis of chronic lung disease. the missing enac regulation by the cf transmembrane conductance regulator (cftr) causes increased absorption of sodium ions and fluid across airway epithelia leading to the depletion of the perciliary liquid layer and to the consequent inhibition of mucus clearance. we developed a hiv-based lentiviral (lv) vector containing a sirna cassette to efficiently knockdown the expression and activity of enac in human respiratory cells. background: sildenafil has been implicated in the relocation of cystic fibrosis transmembrane conductance regulator (cftr) protein. the effect was observed in vitro and in the presence of doses roughly 300 times larger than those commonly used for treating erectile dysfunction. aim: to evaluate in vivo therapeutic efficiency of clinical doses of sildenafil and vardenafil, two approved type v phosphodiesterase inhibitors, for correcting chloride transport defect in ∆f508 mice. methods: we measured transepithelial potential difference in vivo across the nasal mucosa as a readout for sodium and chloride conductance. the effect of a single intraperitoneal injection of sildenafil (0.7 mg/kg) or vardenafil (0.14 mg/kg) was investigated in df508/df508 and normal homozygous mice. results: in df508/df508 mice, chloride conductance, evaluated by perfusing the nasal mucosa with a chloride-free solution in the presence of amiloride and with forskolin, was corrected 1 h after sildenafil administration. a more prolonged effect, persisting at least for 24 h, was observed with vardenafil. the forskolin response was increased after sildenafil and vardenafil in both normal and df508 mutant animals. no effect on sodium conductance was detected in any group of animals. conclusion: our results provide preclinical evidence of effectiveness of both drugs for correcting chloride transport defects in the cf. acknowledgments: this work was supported by grants from the french cf association, vaincre la mucoviscidose and by an educational grant from pfizer belgium. there is a strong interest in developing small molecules able to correct the phenotypic effects of cystic fibrosis (cf) mutations. many mutations (e.g. ∆f508) impair the function of cftr protein, by altering the protein targeting to the plasma membrane and/or by causing an abnormally low open channel probability. drug-like organic compounds may restore the correct membrane localization ("correctors") or increase channel activity ("potentiators") of mutant cftr. in the last years, various research teams have identified molecules with activity as correctors (corr-4a, vrt-325, miglustat, curcumin) or as potentiators (tetrahydrobenzothiophenes, phenylglycines, sulfonamides, vrt-532, 1,4-dihydropyridines). however, these compounds have been tested using different assays and the results are sometimes controversial, with marked differences in declared efficacy and potency of compounds. we have tested a panel of correctors and potentiators to directly compare their effects under the same experimental conditions. for this purpose, we have used the functional assay based on the halide-sensitive eyfp-h148q/i152l to measure ∆f508-cftr activity in frt and a549 cells. the assay for potentiators consisted in stimulation with the test compound (0.02 -60 µm) plus forskolin (on cells previously incubated at 27°c for 24 hours). the assay for correctors consisted in 24 hours incubation of ∆f508 cells with test compounds and then determination of cftr activity in the presence of forskolin plus genistein (50 µm). our results indicate that all potentiators are active in our assay with a comparable maximal effect but with values of potency that vary significantly among compounds. the potency order measured in frt cells was: dhp-194 = pg-01 (ka~50 nm) > sf-01 (ka = 140 nm) > dhp-229 = dhp-226 (ka~180 nm) > act-2b (ka = 250 nm) > felodipine (ka = 900 nm) > vrt-532 (ka = 2.6 µm) > genistein (ka = 18.5 µm). a similar order of potency was found also in a549 cells expressing ∆f508. the activity of correctors showed a more marked dependence on cell line. while the potency was comparable between frt and a549 cells, the maximal effects showed clear differences. in frt cells, corr-4a, corr-4b and corr-3c generated a maximal effect that was 1.5 -2-fold higher than that obtained by incubating the cells at low temperature. conversely, vrt-325 and vrt-640 were less effective (maximal correction equivalent to 50-75 % of low temperature rescue). in a549 cells, all compounds were instead less effective than low temperature, with vrt-325 being the molecule eliciting the highest activity (50-60 % of low temperature). our results indicate the 1,4-dihydropyridine dhp-194 and the phenylglycine pg-01 among the most potent activators of the mutant cftr channel. the similar behavior of potentiators in two different cell lines is consistent with the assumption that all potentiators act with a similar mechanism, by interacting with the cftr protein itself. in contrast, the cell line dependence of correctors suggests that they act with indirect mechanisms, possibly by interacting with proteins involved in cftr biosynthesis and trafficking. supported by cfft and telethon-italy. we also thank cfft and rfums for providing chemical compounds. lung damage in cystic fibrosis (cf) patients is determined by mucus accumulation, pseudomonas aeruginosa infection and chronic inflammation. extracellular gsh is a scavenger of free radicals produced by neutrophils in inflamed tissues. glutathione transferases (gst) are a superfamily of dimeric proteins which conjugate glutathione to a wide range of substrates including oxidants and are involved the synthesis of leukotriens. clinical beneficial effects have been reported in cf patients following treatment with the macrolide azythromicin (azm); anti-inflammatory properties have been proposed as possible mechanism. the aim of this study is to investigate the regulation of the gstt1 and gstm1 activity and expression by azm. reductions of about 25% and 40% on gst enzymatic activity were detected in ib3-1 and 2cfsmeo-cells respectively. gsts mrna expression in cf airway epithelial cell lines was analysed by quantitative pcr (qpcr). the level of gstt1 and gstm1 basal expression in cf cells ib3-1 was significantly higher than in isogenic non-cf cells c38. we found statistically significant decreases of gstt1 and gstm1 mrna of about 30% and 25% respectively in ib3-1 cells after treatment with azm for 24 hours, restoring the levels observed in c38 cells. in 2cfsmeo-cells after exposure to azm we observed 50% and 45% reductions in gstt1 and gstm1 mrna respectively. the macrolide jm, known to lack clinical anti-inflammatory properties, had no significant effects on gstt1 and gstm1 mrna expression in all cell lines. furthermore, azm did not alter the mrna expression levels of gstp1, a glutathione-s-transferase not differentially expressed in cf and isogenic non-cf cells. decreased expression of 50% and 85% of gstt1 protein has been detected by immunoblotting in ib3-1 and 2cfsmeo-cells, respectively, following treatment with azm. in the same conditions we found a drastic reduction of protein level of gstm1 in both cf cell lines. finally, gsts activity and the expression of gstt1 and gstm1 proteins in cf cells, were reduced approximately to the same level detected after treatment with interleukin 10 (il-10), an anti-inflammatory cytokine, shedding light on a possible correlation between gsts inhibition and antiinflammatory properties of azm. the effects of azm described in this study suggest that downregulation of gstt1 and gstm1 expression may result in increased availability of intracellular gsh making cf cells less susceptible to oxidative stress induced by chronic inflammation. inhibition of gstt1 and gstm1 might provide a therapeutic approach for limiting the effects of inflammation critical for lung damage in cf patients. this study is supported by italian cf research foundation; comitato di vicenza-associazione veneta per la lotta contro la fibrosi cistica; azienda ospedaliera verona, italy. tradtrantip, l.; padmawar, p.; yangthara, b.; verkman, a. activation of cftr chloride conductance by gpcrs involves camp elevation and pka-mediated cftr phosphorylation. we developed a 'pathway screen' in which cftr-mediated iodide influx is used as a read-out of gpcr activation. the cell-based fluorescence assay utilizes multiply transfected epithelial cells expressing wildtype cftr, yfp-h148q/i152l and a specified gpcr. we recently used this assay to identify a new class of nanomolar-affinity, 5-aryl-4-benzoyl-3hydroxy-1-(2-arylethyl)-2h-pyrrol-2-one vasopressin-2 receptor antagonists (yangthara et al., mol. pharm. 2007, in press ). additional screening of 100,000 diverse small molecules yielded 4 novel chemical classes of inhibitors of cftr activation. the potential molecular targets of pathway inhibitors include the gpcr, gs or gi proteins, adenylyl cyclase, phosphodiesterase, pka and cftr. a series of target identification studies was done to classify the new pathway inhibitors, which involved the use of agonists acting at different sites in the activation pathway, and specific site-of-action assays. the pathway screen yielded 3 new small-molecule cftr inhibitors that are unrelated to thiazolidinone and glycine hydrazide inhibitors. one interesting class of pathway inhibitors, thiophenecarboxylates, represent the first small-molecule phosphodiesterase activators, which strongly reduce camp and cgmp concentration in many cell types. the best thiophenecarboxylate greatly reduced intestinal fluid secretion in closed loop mouse models of cholera and travelers' diarrhea, and slowed cyst growth in a model of polycystic kidney disease. other pathway inhibitors, which are potential effectors of g-proteins and pka, are under evaluation. the gpcr-linked cftr pathway screen developed here is useful for high-throughput parallel identification of small-molecule inhibitors of multiple targets in the cftr activation pathway. potential uses of the inhibitors identified here include therapy of secretory diarrheas, polycystic kidney disease, and cyclic nucleotide-dependent tumor growth, as well as pharmacological creation of cf-phenotype in ex vivo human tissues and animal models. supported by cff and nih. inhibitors of intestinal caccs are predicted to have anti-secretory effects in certain diarrheas, and activators of airway and intestinal caccs are of potential use in cystic fibrosis therapy (activation of 'alternative' chloride channels). the purpose of this study was to identify small-molecule cacc inhibitors and activators that target caccs directly, rather than ubiquitous upstream processes such as calcium signaling. we screened a collection of 100,000 chemically diverse small molecules using a novel high-throughput screening assay involving lentiviral introduction of a yfp-based halide sensor in cacc-natively expressing human epithelial cells. cacc inhibitors were identified from iodide influx following cacc simulation by carbachol/atp. we identified five classes of cacc inhibitors with micromolar potency, including tetrahydro-cyclopentaquinolines, and 3-aryl-5-(trifluoromethyl)-pyrazoles each of which was unrelated to known transport modulators. two classes of compounds inhibited calcium-activated halide flux following stimulation by multiple types of agonists, including thapsigargin and calcium ionophores, and by patch-clamp analysis appear to target cacc directly. structure-activity analysis of 137 analogs of 'hits' yielded compounds with improved potency, which have been resynthesized and characterized for use in assays of antidiarrheal effiacy in rodent models of viral and drug-induced secretory diarrheas. screening for caccselective activators that act in a sustained manner (non-transiently) was accomplished using a similar cell-based fluorescence assay, but instead testing for increased halide influx. several classes of putative cacc activators with micromolar-potency were identified in screening of 100,000 small molecules, whose mechanism-of-action and specificity are under investigation. small-molecule modulators of cacc function that target caccs directly have potential clinical applications, and may be useful in defining the physiological roles and molecular identity of caccs. supported by cff and nih. introduction: inhaled hypertonic saline 〈hs〉improves lung function and decreases pulmonary exacerbations in older children and adults with cf. initiating therapeutic interventions in the youngest patients, particularly those that target the underlying cf defect of airway surface liquid volume depletion, has potential to preserve lung function and improve prognosis. subbarao et al performed baseline, post-albuterol and post-hs lung function testing in infants using the raised volume rapid thoracoabdominal compression technique 〈rvrtc〉and demonstrated no significant drop in lung function 〈 pediatric pulmonology, 2007〉. however, performing three sets of rvrtc maneuvers under the same sedation could prove difficult. before conducting a therapeutic trial of hs in this population, a simplified protocol must be possible at multiple centers. we sought to evaluate a simplified approach as well as to analyze changes in lung function and clinical findings after acute administration of hs. methods: in this ongoing study, clinically stable children with cf between the ages of 4 months and 4 years inhale 2.5 mg of albuterol prior to sedation with chloral hydrate. rvrtc and plethysmography are then performed before and after inhalation of 5 ml of 3‰ hs. fvc, forced expiratory volume in 0.5 seconds 〈fev 0.5 〉, fef 25-75 , frc, rv/tlc ratio, respiratory rate, oxygen saturation, and chest exam findings are recorded. predefined stopping criteria include a 20% drop in fev 0.5 or in oxygen saturation to below 90‰. results: six subjects 〈mean age 1.6 ± 0.9 years〉 have completed the protocol with 3‰ hs. comparison of post-albuterol lung function to that obtained 15 minutes after 3‰ hs revealed no changes in mean fvc 〈561 vs 562 ml; p=0.93〉, mean fev 0.5 〈426 vs 427 ml; p=0.89〉, mean fef 25-75 〈801 vs 817 ml/sec; p=0.59〉, frc 〈measured after each inhaled therapy in 3 of 6 subjects; 260 vs 260 ml; p=1.0〉, or rv/tlc 〈measured after each inhaled therapy in 3 of 6 subjects; 0.35 vs 0.34; p=0.77〉. respiratory rate, oxygen saturation and chest exam were unchanged. conclusions: results from this study demonstrate that a two-step protocol may used to evaluate the safety of hs. based on these findings, acute administration of 3‰ hs is safe in children ages 4 months to 4 years with cf. despite the known improvement in mucociliary clearance, preliminary findings demonstrate a lack of an immediate response in lung volume measures. given the demonstrated benefits in older children and adults, a multicenter therapeutic trial of hs is warranted. supported by the cystic fibrosis foundation. methods and results: we have characterized aav serotypes 1-9 in addition to twenty novel vectors isolated from human or macaque tissues to transduce the murine airway epithelium in vivo. vectors [1e+11 genome copies (gc)/mouse] expressing α-1-antitrypsin (aat) and βgalactosidase (β-gal) were co-instilled into the mouse lung or nose. transgene expression levels were monitored by assaying aat concentration in serum as well as the number and cell-types positive for (β-gal) expression in lung and nasal airways. of all vectors tested aav5 and aav6 were the two most efficient vectors in conducting airways. when these aav vectors (2e+11 gc) were subsequently evaluated on human ciliated airway epithelial cultures (haec), in contrast to our findings in mouse airways, aav5 failed to transduce haec, whereas aav6 resulted in~10% of the haec expressing transgene. since aav6 was the most efficient vector in mouse and human airway epithelium we performed structure-function analyses of the aav6 vector capsid and found two atypical capsid residues that were unique in otherwise conserved positions (f129, k531). to generate a potentially fitter vector, residue f129 was mutated to its conserved state. residue k531 was found to confer lung tropism and was left unchanged. the resulting vector, aav6.2, transduced mouse lung and nasal airway with greater efficiency than all aav vectors tested. the increased transduction efficiency of this vector was also observed (~20%) in haec derived from six different human subjects. to continue our preclinical studies in a more relevant model, aav2/6.2 expressing egfp was tested in ciliated cultures derived from macaque airways and showed 40-50% of cells expressing egfp one week after inoculation with 2e+11 gc. confocal microscopy revealed that aav6.2 targeted a significant number of ciliated cells: the airway cell-types that likely require cftr expression in cf patients. aav6.2 expressing rhesus α-fetoprotein (rhafp) was then inoculated in the nasal airways of a rhesus macaque and transduction evaluated by monitoring concentration of rhafp in the nasal lavage fluid. we have found that rhafp expression remained high (~300 ng/mg) and stable for at least 72 days. conclusion: the enhanced transduction efficiency of aav2/6.2 vector in human and macaque airway cultures and its ability to stably transduce the nasal airway of a rhesus macaque in vivo demonstrates that aav2/6.2 is a good candidate gene transfer agent for the efficient expression of cftr in human cf airway epithelium. submitted for presentation at the american society of gene therapy. supported by gsk, cff, cfpo1, p30, mtcc. pharmacological correction of ∆f508 cftr cellular processing is a potential therapeutic strategy for cystic fibrosis. recent high-throughput screening has identified synthetic small molecules, such as bisaminomethylbithiazoles (corr-4), which partially restore chloride permeability in ∆f508 mutant cells. the purpose of this study was to examine the utility of natural compounds (chinese medicinal herbs) as ∆f508 correctors. a herbal compound fraction library was constructed from 500 herbs most frequently used in traditional chinese medicine (tcm) that are believed to contain therapeutic compounds for a broad spectrum of human diseases including lung disease. for construction of the tcm fraction library, crude herbal extracts were first prepared by 95% ethanol extraction on soxhlet reflux apparatus followed by automated fractionation by preparative hplc. eighty fractions were collected from each of the 500 herbs. each fraction contained 1 to 17 (average 10.4) individual compounds as determined by analytical hplc. collected fractions were dried and 1 milligram of the material was dissolved in 200 µl dmso to generate 5 mg/ml solutions in 96-well plates. each 96-well plate contained 80 fractions from one herb. high-throughout screening was done using the frt cell-based fluorescence assay developed previously (j. clin. invest. 115:2564 -71, 2005 . of 16,000 fractions screened, 87 active fractions from 12 herbs were identified, with 26 positive fractions verified in secondary screening. the positive fraction did not increase halide transport in control non-transfected cells, and halide transport in ∆f508-corrected cells was fully abolished by cftrinh-172. we have fractionated some of the most active fractions by preparative hplc to identify which compound(s) conferred activity. for example, in one fraction there were 14 single compounds, 2 of which conferred corrector activity with ic50s < 5 µm and efficacy comparable to that of low temperature rescue. these results demonstrate the feasibility of ∆f508-cftr corrector discovery from natural compounds. further fractionation, characterization and structure determination are in progress. the unexpectedly high 'hit'rate for the natural compounds suggests their further exploration in cf therapy. cystic fibrosis (cf) is the most common genetic disease affecting the caucasian population, with an incidence of approximately one in three thousand births. cf transpires as a result from a mutation in the cystic fibrosis transmembrane conductance regulator protein (cftr), which regulates ion transport across epithelial membranes. subsequently, patients afflicted with cf have an abnormally excessive incidence of chronic lung infection, with organisms such as pseudomonas aeruginosa. because cystic fibrosis is characterized by chronic bacterial infections, excessive neutrophil recruitment to the lungs, and a coinciding increase in pro-inflammatory cytokine production and nuclear factor-kappa b (nf-κb) activation, we hypothesized that exogenous addition of the nf-κb inhibitor iκbα might ameliorate this phenotype. we cloned the human iκbα gene as well as a mutated iκbα gene into plasmids with chicken-beta actin hybrid promoters. we then tested the new plasmids, paav2.cb-hiκbα and paav2.cb-hiκbαm, in vitro in the presence and absence of pseudomonas aeruginosa infection in the ib3-1 and s9 cell lines. both plasmids produce iκbα at high levels as shown by enzyme linked immunosorbant assays (elisas). we also show that paav2.cb-hiκbα transfected ib3-1 cells, after infection with pseudomonas aeruginosa, express significantly reduced levels of interleukin (il)-1β (2 fold, p<0.0001), il-8 (13 fold, p<0.0001), and tnf (2 fold, p=0.0007) as well as nf-κb activation (2 fold, p<0.0001) compared to p. aeruginosa-infected ib3-1 controls as determined by human cytokine and nf-κb phosphoprotein bio-plex assays; cytokine expression and nf-κb activation levels in infected paav2.cb-hiκbα transfected ib3-1 cells were between levels found in infected ib3-1 and s-9 cells, excluding il-8 levels which were below s-9 levels of expression. flavonoids are among the most potent cftr modulators known. equol [7hydroxy-3-(4'-hydroxyphenyl)-chroman] is a product of intestinal metabolism of dietary isoflavones such as daidzein, and has been of recent interest in studies of cancer, cardiovascular risk, and neurologic disease. equol is metabolically stable, and 49% circulates in the free (non-protein bound) form, which is considerably greater than the proportion of free daidzein (19%). structural differences such as modification of ring c (e.g. saturation at c-2/3, and absence of carbonyl group at c-4) and an absent hydroxyl at position c-5 distinguish equol from compounds previously reported to modulate cftr activity. in prior work by our center, we showed that equol activates wt and ∆f508 cftr in membrane patches excised from bhk cells and in cfbe41o-monolayers studied in ussing chambers. activation by equol occurred only after r-domain phosphorylation in wt and ∆f508 cftr constructs, but was independent of rdomain phosphorylation in ∆r cftr, indicating activity may be related to dimerization of the nbds or other domain-domain interaction. because a molecule that alters nbd interactions might have effects on the aberrant processing of ∆f508 cftr, we screened equol and other flavonoids by preincubating compounds with cfbe41o-cells expressing ∆f508 cftr for 18 hours, and then tested for rescue of cftr dependent cl-channel activity after exchange of media solution. we found that preincubation with equol (50-100 µm) induced rescue of short-circuit current compared to vehicle treated cells (12 vs. 4 µa/cm2, p<0.001, n=8). we then evaluated 24 hour preincubation with equol (50um) for biochemical evidence of cftr processing correction in ∆f508 cfbe41o-cells grown in polarizing conditions. immunoprecipitation and in vitro phosphorylation demonstrated minimal formation of band c compared to vehicle treated cells, but adaptation of a more sensitive avidin label/biotinylation assay specific for surface localized cftr revealed clear evidence that equol preincubation led to cftr at the plasma membrane. next, we examined equol in hela cells stably transduced with ∆f508 cftr. preincubation of equol led to dose-dependent increases in halide transport measured by fluorescence-based halide efflux (spq) after stimulation by genistein (0.50 and 0.95 fluorescence slope (∆%/sec) with equol 5 and 50 µm respectively, compared to 0.2 ∆%/sec in cells pretreated with vehicle alone; p<0.005, n=120-180 cells/condition). immunohistochemical staining of ∆f508 hela cells for cftr with 24-1 c-terminus antibody showed rescue of surface localized protein with equol (50 µm) preincubation for 16 hours, while vehicle treated cells showed only perinuclear staining. in summary, we show functional, biochemical, and immunohistochemical evidence that the naturally occurring flavonoid equol corrects the ∆f508 processing defect in two model systems. a naturally occurring agent that both activates and corrects ∆f508 cftr deserves further exploration as a potential cf therapeutic, and may lead to new insights regarding domain-domain interactions that influence the activation and biogenesis of the mutant channel. supported by nih and cff. mote ∆f508 cftr maturation has been identified. although several small molecule agents have been described that overcome ∆f508 cftr processing defects in specific cellular models, few studies have directly compared the activity of temperature corrected and chemically corrected ∆f508 cftr in polarized cell systems. in the present study, we examined chemical and temperature corrected activity of ∆f508 cftr. correctors included all members of the cfft modulator library (c1, c2, c3, and c4; rosalind franklin university, chicago, il). in a screen using ∆f508 cfbe41omonolayers, maximal corrector activity across the two model systems exhibited a rank order of c4 >> c3 > c2 = c1, using forskolin (20 µm) and genistein (50 µm) stimulated isc as a sensitive test for ∆f508 cftr activity at the plasma membrane. no change in isc was observed in matched control (parental) cells lacking ∆f508 cftr expression. based on these results, we further defined the activity of c4 in ∆f508 cfbe41oand frt model systems, including dose/response and time dependence for peak isc rescue. in cfbe41o-cells, exposure to 2 µm c4 for 8 hrs produced maximal reproducible correction of ∆f508 cftr processing, with loss of activity following prolonged or high concentration exposure; in frt cells, peak effects were seen at 24 hours. ∆f508 cftr activity following small molecule treatment qualitatively mirrored temperature correction (27°c growth for 24-48 hrs) in both cell types. maximal currents produced by stimulation with forskolin (20 um) and genistein (50 µm) in ∆f508 frt monolayers following c4 pretreatment were 83% of that produced by temperature correction (p<0.001). forskolin was responsible for 57% of maximal current in frt ∆f508 cells following chemical correction and 43% of maximal currents following temperature correction. in ∆f508 cfbe41o-cells, maximal currents following chemical correction were 22% of that produced by temperature correction (p<0.001). forskolin was responsible for 10.8% of maximal currents in cfbe41o -∆f508 cells following chemical correction and 15.6% of maximal currents following temperature correction. these results illustrate dose and time response with a small molecule corrector in two polarizing epithelial model systems, and provide reassurance that observations based upon ∆f508 cftr following low temperature incubation are relevant to functional analysis after chemical correction. similarity in activation properties between chemical and low temperature correction suggest it is unlikely that the two maneuvers result in ∆f508 cftr with significantly different structural properties. the studies also indicate fundamental differences in ∆f508 cftr behavior in frt compared to cfbe41ocells, and emphasize the importance of identifying an agent that can restore camp dependent regulation to bronchial epithelial cell types. supported by the nih, cff and cfft. participants: n=38 patients (21 randomised to azm and 17 to placebo) who had successfully completed a course of intravenous antipseudomonal antibiotics immediately before the trial (mean age: 23.7 years; mean fev1: 62% of predicted). measurements and results: after treatment (mean dose of 21.2 mg/kg body weight once a week) pulmonary function declined in both groups compared to baseline (i.e. after cessation of iv antibiotics). the azithromycin group had signifcantly better results regarding the mean changes in serum crp (azm: +0.9 mg/l, placebo: +21.6 mg/l, p=0.019), lipopolysaccharide binding protein in serum, lbp (azm: +0.9 µg/ml, placebo: +7.0 µg/ml, p=0.015), serum interleukin-8 (azm: -3.1 pg/ml, placebo: +2.9 pg/ml, p=0.001) and alginate in sputum (azm: +85 µg/ml, placebo: +353 µg/ml, p=0.048). quality of life (german version of the cfq) showed significantly better results after azm in adolescents and adults. azithromycin was well tolerated with no increase in treatment-related adverse events. conclusion: once-weekly azithromycin ameliorated inflammatory reactions and improved quality of life. a decline of pulmonary function after cessation of intravenous antibiotics could not be prevented, however. this study has been sponsored pfizer gmbh, germany this open-label, multicenter study was conducted in the usa and australia to evaluate the clinical responsiveness of a patient-reported outcome measure, the cfq-r respiratory scale, by determining the minimal clinically important difference (mcid) following a 28-day course of tobramycin inhalation solution (tis). cf patients (n=84 [≥6 to <18 yrs, n=56; ≥18 yrs, n=28]) with pseudomonas aeruginosa and clinical symptoms predictive of a pulmonary exacerbation (increased cough, increased sputum /chest congestion, decreased exercise tolerance or decreased appetite) were enrolled. three efficacy measures were included: 1)change in forced expiratory volume in 1 second (fev1) from baseline (day 0) to end of treatment (day 28) or end of study (day 42); 2)one question about change in respiratory function (days 28 and 42; global rating of change questionnaire, respiratory domain, grcq rd;0 =no change;7 =maximal improvement or worsening);and 3)change in cfq r-respiratory scale (day 28, 42). average change from baseline fev1 (mean [standard deviation, sd]) was 5.1% (16.5) at day 28 and 4.9% (16.0) at day 42. based on grcq-rd at day 28, 22 patients (28%) reported no change in respiratory symptoms (score <1.1), 32 (41%) a minimal change (≥1.1 to <3.1), 16 (20%) a moderate change (≥3.1 to <5.1), and 9 (11%) a large change (≥5.1). mean (sd) change from baseline cfq-r respiratory was 5.7 (19.2) at day 28 and 5.9 (20.7) at day 42. at day 28, change in cfq-r was moderately correlated with change in fev1 and with grcq-rd; the correlation was stronger for patients with baseline fev1 <75% of predicted fev1 values (see table) . mean change from baseline on the cfq-r respiratory scale was 10.0 at day 28 for patients with grcq-rd scores indicating minimal change in symptoms (≥1.1 to <3.1; n=30); this provided an estimate of the mcid for the cfq-r respiratory scale for those in exacerbation. this mcid value was consistent with estimates from distribution-based methods( 1 ⁄2 sd and standard error of measurement). the cfq-r was responsive to changes in pulmonary symptoms in patients in mild exacerbation following tis treatment; the mcid in this population was 10.0 points. responses on the cfq-r-respiratory scale were moderately correlated with changes in fev1. funded by gilead sciences, inc. this was an open-label, multicenter study conducted in the usa. we determined the minimal clinically important difference (mcid) for the cfq-r, respiratory scale following a 28-day course of tobramycin inhalation solution (tis) in patients with cf and chronic pa infection (n=140, 14 children [<13 yrs]). patients had received ≥3 courses of tis (mean = 5.4) within the previous year, however their respiratory symptoms were stable at study entry, with forced expiratory volume in 1 second (fev1) between 25% to 75% of predicted values. efficacy measures included: 1) percent change in fev1 (l) from baseline (day 0) to treatment end (day 28); 2) a single question about change in respiratory function (day 28; global rating of change questionnaire; respiratory domain, grcq-rd; 0 =no change, 7 =maximal improvement or worsening); 3) change in cfq-r-respiratory scale (day 0 to 28); and 4) change in log10 pa colony-forming unit (cfu) density in sputum (day 0 to 28). the mcid for cfq-r was estimated using three methods: 1) change in cfq-r (day 0 to 28) for the patient subset with minimal change in respiratory function, as determined by the grcq-rd at day 28; 2) cfq-r standard error of measurement (sem) from a validation sample), and 3) 1 ⁄2 standard deviation (sd) of the cfq-r respiratory scale scores. at day 28, change from baseline fev1 (mean [sd]) was 1.0% (11.3), change from baseline cfq-r was -0.6 (12.6), and change in log10 pa cfus was -0.4 (1.5) . based on the grcq-rd at day 28, 37 patients (41%) reported no change in respiratory symptoms (score <1.1), 40 (44%) a minimal change (≥1.1 to <3.1), 10 (11%) a moderate change (≥3.1 to <5.1), and 4 (4%) a large change (≥5.1). pearson r-values for the correlation of change in the cfq-r-respiratory scale and change in fev1, log10 cfus and grcq-rd were 0.1, 0.08, and 0.46, respectively. estimates of the mcid for cfq-r-rd ranged from 4.6 to 5.9 for adults/adolescent patients (table) . in patients with cf who had no immediate need for antipseudomonal therapy at study entry, the cfq-r-respiratory scale appeared responsive to changes in patient disease perception following 28 days of tis treatment; the mcid for cfq-r was approximately 5 points for the adolescent/adult patient population. funded by gilead sciences, inc. ex vivo chloride secretion measurements (intestinal current measurement, icm) in cf patients have been established over the past 15 years to study the cftr-basic defect in more functional detail. modified micro-ussingchambers are used to registrate the transepithelial short-circuit current (isc) in freshly obtained human rectal suction biopsies as a measure of ion transport after stimulation with secretagogues. hereby, the cftr clchannel, its amount of residual function in cf and alternative cl-channels can be investigated by a standardised protocol.in the course of the development of cftr pharmacotherapeutics as well as agents activating alternative cl-channels, icm may function as an useful outcome parameter in preclinical and clinical trials. it is easy to perform repeatedly at all patients ages and comprises the safety advantages of an ex vivo method which is relevant especially for early study phases. aim of this study was to describe reference values and quantify the intraindividual variability of different icm parameters. methods: a total of n=574 rectal biopsies from n=212 individuals; with pancreatic insufficient (pi)-cf (n=22; mean age 14.5 years), pancreatic sufficient (ps)-cf (n=9; 10.8 years), excluded cf by icm diagnostics (n=169; 12.8 years) and healthy control (n=12; 26.3 years) were included into analysis. for calculation of intraindividual variability, 2-4 biopsies/patient were compared with respect to basal tissue resistance (rt basal), basal open circuit potential difference (pd basal), basal short circuit current (isc basal) and the isc responses to stimulation with carbachol (10-4 mol/l, serosal), 8-bromocyclic monophosphate (camp) (10-3 mol/l, mucosal+serosal) + forskoline (10-5 mol/l, serosal) and histamine (5x10-4 mol/l, serosal).results:we determined icm reference values for the groups of pi-cf (isc basal 23.4 ± 18.1 µa/cm 2 , ∆isc carbachol 0.4 ± 3.6 µa/cm 2 ,∆isc camp/forskoline 2.0 ± 2.9 µa/cm 2 ,∆isc histamine -3.1 ± 4.6 µa/cm 2 );ps-cf (isc basal 45.6 ± 31.5 µa/cm 2 ,∆isc carbachol 3.4 ± 6.8 µa/cm 2 ,∆isc camp/forskoline 8.0 ± 10.4 µa/cm 2 ,∆isc histamine 6.3 ± 10.8 µa/cm 2 ),and healthy control (isc basal 39.0 ± 26.1 µa/cm 2 , ∆isc carbachol 27.3 ± 7.1 µa/cm 2 , ∆isc camp/forskoline 15.2 ± 10.6 µa/cm 2 , ∆isc histamine 27.4 ± 15.2 µa/cm 2 ). for the total cohort, mean coefficients of variation were: rt basal 29%, pd basal 48%, isc basal 49%, ∆isc carbachol 59%, ∆isc camp/forskoline 64%, ∆isc histamine 80%. conclusion:this first comprehensive analysis of the intraindividual variability of icm basal tissue and cl-secretion parameters provides the basis for the method as an useful outcome measure for future clinical trials aiming to rescue the cftr basic defect. possible effects of pharmacological therapeutics in cf relevant human epithelia have to be adequately interpreted with respect to subject variability and laboratories reference data. ex vivo cl-secretion measurements have the potential of being an essential step in the evaluation process of cftr-correcting/potentiating agents on their way from laboratory screening to the application in human cf tissue without any risk of toxicity. center, placebo-controlled, double-blinded pilot study we assessed safety and tolerability of 2.5 mg/d moli1901 versus placebo (normal saline) administered by inhalation (pari lc plus) once daily for 28 days. patients included were ≥16 years of age in phase i and ≥12 years of age in phase ii, with a fev 1 >60% predicted and stable lung disease. overall, 12 subjects received moli1901 and 6 placebo. exclusion criteria included abpa, b. cepacia infection and severe liver disease. the study involved 7 clinic visits over a period of 8 weeks to assess adverse events, spirometry, pulse oximetry and quality of life. a total of 111 adverse events (aes) were observed in 16 subjects (101 ae in 12 subjects receiving moli1901), with only 1 (productive cough) in the moli1901 group being of severe intensity. the most frequent aes related to the study medication were (productive) cough (43x) and dry throat/throat irritation (14x), and most of these resolved within 1 hour after inhalation. in the moli1901 group no significant ae, defined as a decline of fev 1 ≥20% from baseline accompanied by symptoms, a decrease in oxygen saturation to <90% or a fall of 8% from baseline requiring therapeutic intervention, or a change in safety parameters judged to be clinically significant was observed. this trial was not primarily designed to show efficacy; however, the median change in fev 1 from day 1 to day 56 was -3% in the placebo group, and +2% in the moli1901 group, and this difference was significant (wilcoxon test, p=0.0217). similarly, there was a significant difference between the median change in fef 25-75% from day 1 to day 56 in the placebo group as compared to the moli1901 group (-10% vs. +1%, p=0.029). no significant changes were observed for the other study days or for fvc and pulse oximetry. moli was well tolerated in this trial, with the observed aes generally being mild and of short duration. these encouraging explorative results are currently being further evaluated in explorative and confirmatory trials. introduction: results from published data elucidate that microbes found in the upper respiratory tract are similar or the same as those found in the lower airways of cf patients. inhaled, aerosolized drug delivery to the lower respiratory tract is an established treatment route. however, drug delivery systems capable of depositing drug to the paranasal cavities are not yet established and require evidence of deposition and efficacy. pari developed the vibrent™ paranasal drug delivery system to enable the aerosol and drug to penetrate into the nose and sinuses. objectives: this study was conducted to demonstrate that the pari vibrent™ pulsating drug delivery system is capable of ventilating the human paranasal sinuses of 3 healthy volunteers. methods: 81mkr-gas was continuously ventilated through the nasal tract of three healthy non-smokers in front of a single-head gamma camera (diacam, siemens, germany), using the pari vibent™ pulsating drug delivery system. the nebulizer was coupled to the right nostril and a flow resistor to an output tube was inserted into the left nostril. during ventilation with the krgas (about 10 sec) the subject closed their soft palate to transmit the pulsation and to prevent penetration into the lower respiratory tract. the gas supply of the vibrent™ was directly taken from the 81mkr-gas generator output channel. kr-gas ventilation imaging was performed with and without pulsation. serial images were recorded with anterior and lateral views. additionally, mri (magnetic resonance imaging) lateral slices of the subjects' head were recorded. the gamma camera images were superimposed to the mri images by adjusting the spatial resolution. with no pulsation from the vibrent™ no ventilation of sinus cavity was visualized by gamma camera images and radioactivity was detected in the nose only. when pulsation was added the maxillary sinuses can be visualized in the gamma camera images of all volunteers. conclusions: without pulsation no ventilation was observed. gas penetration to the paranasal sinuses can be demonstrated using the pulsating action of the pari vibrent™, potentially enabling drug delivery via aerosols. this confirms results of in vitro studies using a cast model. 81mkr-gas ventilation of the nasal cavities during 10 sec breath holding in front of a planar gamma camera head (anterior) using the pari vibrent without (w/o, left image) and with (w, right image) the pulsation system. the delivery and the exhaust tubing of the kr-gas are shown together with the outline of the head, obtained from mri pictures. introduction: pediatric patients with cf were previously studied in clinical trial studies of denufosol, a novel selective p2y 2 agonist that enhances ciliary beat frequency and activates chloride secretion to hydrate the airways in the lung. pediatric patients are often discouraged from participation in clinical trials until later stages of drug development. aims: in order to evaluate the safety experience with denufosol in this population, we have retrospectively examined integrated data for pediatric cf patients with mild to moderate pulmonary disease that participated in three phase 2 studies. demographic and baseline characteristics in addition to safety and tolerability results for 115 cf patients aged 5-18 years old are reported. methods: three phase 2, multicenter, randomized, double-blind, placebo-controlled, parallel group studies were conducted. patients were randomized to receive either denufosol (20, 40 or 60 mg) or placebo (normal saline) tid for 28 days by inhalation. only study 1 included denufosol 40mg, while all 3 studies included denufosol 20mg and 60mg. the fev 1 predicted normal required to be eligible was >75% (study 1); 60%-90%, inclusive (study 2); >60% (study 3). all three studies included a one-week pre-randomization period during which reproducibility of fev 1 (l) ±12% was required in order to be randomized to double-blind study medication. patients were allowed to use bronchodilators, dornase alfa and corticosteroids in studies 1, 2 and 3. patients were allowed to use oral antibiotics including macrolides and inhaled tobramycin solution in studies 2 and 3. results: a total of 115 cf patients 5-18 years old were randomized and dosed in three 28-day studies. eighty-one received denufosol (active doses combined) and 34 received placebo. demographics were similar for all treatment groups -denufosol pediatric patients had a mean (sd) age of 11.8 (±3.70) years old compared to placebo pediatric patients who had a mean (sd) age of 11.9 (±4.05) years. denufosoltreated patients were 59% male and placebo-treated patients were 53% male. the mean (sd) percent predicted fev 1 at baseline was similar between treatment groups [89.9% (±15.13) and 92.0% (±14.28) for denufosol and placebo, respectively]. the overall incidence of treatment emergent adverse events (ae) was similar between treatment groups (84% denufosol, 82% placebo). the most common ae was cough, reported by 48% and 47% of patients that received denufosol and placebo, respectively. seven percent of denufosol and 6% of placebo patients prematurely discontinued from the study due to aes. there were no differences in compliance with administration of study drug (96% in patients given denufosol and 100% in patients given placebo). conclusion: doses of denufosol up to 60mg given tid for 28 days were well tolerated in pediatric cf patients 5-18 years old. these data demonstrate that inclusion of patients 5-18 years old is feasible regardless of administration of denufosol or placebo. a longer term phase 3 study of denufosol in cf patients >5 years old in north america is currently ongoing. acknowledgements: this research was funded by inspire and the cystic fibrosis foundation. objective: pulmonary delivery of anti-infectives provides the potential to attain pk-pd indices exceeding those which can be achieved with systemic dosing. mp-376 is a novel formulation of levofloxacin that enables delivery of high concentrations over a short period, and provides taste masking. the objective of this study was to: i) determine the safety of aerosol doses of mp-376 and, ii) determine pharmacokinetics of levofloxacin in serum, urine, and sputum following aerosol doses of mp-376 using the pari eflow nebulizer in normal healthy volunteers (nhv) and patients with cystic fibrosis (cf). methods: nhv and patients with stable cf were enrolled in a single within-subject ascending dose study of 3 dose levels (loaded doses of 78, 175, and 260 mg) of inhalational mp-376 (levofloxacin solution for inhalation) or placebo. study participants were monitored for safety and changes in pulmonary function. serum, urine, and sputum (cf patients only) samples were collected at various times following the dose and assayed for levofloxacin concentration using hplc. in addition, each participant received a single iv dose of levofloxacin to determine the systemic bioavailability following aerosol mp-376. noncompartmental and compartmental methods were used to determine serum, sputum and urinary pharmacokinetic parameters. pharmacokinetic deconvolution methods were used to determine the amount of the dose remaining in the lung over time. results: this study is ongoing. dosing in 8 nhvs (6 active mp-376, 2 placebo) has been completed. there were no serious adverse effects, and no significant changes in pulmonary function were noted. preliminary pharmacokinetic data from 4 nhvs show a proportional increase in serum levofloxacin concentrations with increasing aerosol mp-376 dose; serum aucs were 3.1, 1.9, 5.1, and 8.9 mg-h/l for the iv dose, low, mid, and high aerosol doses, respectively. deconvolution of the serum levofloxacin concentrations from aerosol mp-376 and iv dosing shows absorption of drug from lung over time. conclusion: preliminary results show that mp-376 is well tolerated following aerosol administration in normal volunteers. serum concentration data following aerosol dosing suggests that absorption of levofloxacin into the systemic circulation is a major route of elimination from the lung. studies in cf patients are in progress and will be presented. methods: a 10 mg/kg aerosol dose of mp-376 was given using a microspray aerosol generation device. when placed just above the tracheal bifurcation, and activated, it delivers a bolus aerosol dose to the lung. a 10 mg/kg intravenous (iv) dose of lvx was given as a slow bolus into the lateral tail vein. plasma lvx was determined in all rats up to sacrifice at 1, 2, 3, or 6 hours, at which time rats were humanely euthanized and bronchialalveolar lavage (bal) performed. fluids were analyzed for lvx concentration using an hplc/ms method. results: the plasma lvx versus time profiles of both iv and aerosol doses were best described by a two compartment model. the plasma auc after an iv dose of levofloxacin and an aerosol dose mp-376 were similar (3.79 mg᭹hr/l vs. 3.72 mg᭹hr/l, respectively) suggesting near 100% bioavailability from the lung. after an aerosol dose, the mean residence time (mrt) was prolonged when compared to the intravenous dose (0.88 vs. 0.79 hours). this delay in absorption was associated with an increase in bal lvx auc0-6h in bal (1.6 mg᭹hr/l vs. 8.3 mg᭹hr/l for iv vs. aerosol dosing, respectively). conclusion: these data show that levofloxacin is highly available to plasma following a single microspray aerosol dose of mp-376. the aerosol dose does produce a slightly longer mean residence time in plasma, suggesting delayed, but complete absorption from the lung; this delay in absorption was associated with increase in bal aucs. these data suggest that high concentrations of lvx can be attained in lung fluids following an aerosol dose of mp-376. auc: area under the time-concentration curve; cl: clearance; cmax: maximum concentration; f: bioavailability; mrt: mean residence time. methods: animals were assigned to one of five exposure groups for all studies. for the 28-day toxicology studies, animals were exposed for up to four hours daily for twenty-eight consecutive days, followed by a 28-day recovery period. respiratory function and cardiovascular safety were conducted on the first and last day of the 28-day study in dogs. rats were exposed to 5, 10, or 20 mg/kg/day, and dogs were exposed to 5, 10, or 15 mg/kg/day. at the conclusion of the study, necropsy was performed and all respiratory tissues were harvested, weighed, and underwent gross and microscopic examination. a separate dog respiratory safety study was conducted in which dogs were exposed to 5, 10, or 20 mg/kg. results: four week repeat dose inhalational exposure of mp-376 at target doses up to 15 mg/kg/day in dogs and 20 mg/kg/day in rats was not associated with any test article-related changes in any respiratory tissues. aerosol administration of mp-376 at doses of up to 20 mg/kg in dogs was found to have no acute effects on minute volume, tidal volume, respiratory rate, or ecg. conclusion: local effects due to the inhalational administration of levofloxacin formulated as mp-376 were not observed in either rats or dogs. these data suggest that the risk of respiratory toxicity from nebulized doses of mp-376 is low. methods: bacteria were grown overnight in mueller-hinton broth (mhb) at 37°c and then sub-cultured into fresh mhb and allowed to reach log phase (4 hours). female swiss mice were infected under anesthesia by intratracheal instillation of 0.05 ml of a 2 x 106 cfu/ml bacterial suspension. ip doses were selected to provide lvx exposures (as auc) comparable to that obtained with systemic dosing regimens. for the p. aeruginosa infection, treatment was initiated by either the ip or aerosol route 2 hours post-infection. mice were euthanized 1 and 4 hours after the start of treatment, lungs harvested, and bacterial counts in lung determined. results: lvx plasma pharmacokinetic profiles were nearly identical following intraperitoneal or aerosol dosing. the geometric mean log cfu/lung pair (sd) for the p. aeruginosa are shown in the table below: in both the k. pneumoniae and p. aeruginosa lung infection studies, aerosol administration was more effective than systemic administration; for k. pneumoniae, the extent of bacterial killing at 24 hrs was ca. 1.4 log cfu greater with aerosol mp-376 than with systemic lvx. conclusion: aerosol administration of mp-376 produces a greater extent of bacterial killing than systemic dosing of lvx in mouse models of pneumonia due to k. pneumoniae and p. aeruginosa. the majority of morbidity and mortality in cystic fibrosis patients is caused by chronic and persistent lung infections especially with pseudomonas aeruginosa. since galactosyl ceramide had been previously shown to be involved in pseudomonas internalization, ceramide levels in the plasma of cf patients were assessed and compared to healthy volunteers using hplc followed by mass spectrometry. the results demonstrated that cf patients displayed significantly lower levels of several ceramide species. also, cftr-knockout mice displayed diminished ceramide levels in cf related organs (lung, pancreas, and ileum) and plasma compared to wildtype controls. treatment with a semi-synthetic retinoid (fenretinide), which was previously reported to induce ceramide in neuroblastoma cell lines, was able to increase ceramide concentrations in cf related organs in cftr-knockout mice to the levels of wildtype mice. treatment also dramatically improved the ability of cftr-knockout mice to control pseudomonas infection. following infection with pseudomonas-impregnated agar beads, fenretinide treated cftr-knockout mice were able to clear bacterial infection as efficiently as wildtype mice. overall, these findings not only documented a novel deficiency of ceramide in cf patients but also demonstrated a pharmacological means to correct this defect in cftr-knockout mice. our data provides a strong rationale for clinical intervention that may benefit cystic fibrosis patients suffering from cf lung disease. recent reports show that adult bone marrow-derived stem cells can localize to and acquire phenotypic and functional markers of lung epithelium. these findings raise the novel possibility of stem cell therapy for multitude of lung diseases. however, only small numbers of adult marrowderived stem cells localize to lung and it is not clear whether these cells will be clinically useful. we investigated whether mesenchymal stem cells (mscs) obtained from human cord blood might have increased potential to participate in structural lung remodeling. cord blood was obtained from normal deliveries at the university of vermont. mononuclear cells were isolated and plastic adherent cells were expanded and characterized as mscs according to international society for stem cells research (isscr) criteria. following systemic (2x106 cells/mouse by tail vein) administration to sublethally irradiated (1.4 gy) immunodeficient (nod/scid) mice, lungs harvested 1 day, 2 weeks, 1 or 3 months later demonstrated small numbers of human b2-microglobulin positive cells in the airway epithelium at all time points. small number of cells was found also stain positive for pancytokeratin (pan-ck) and rarely, we identified cells of b2-microglobulin+/pan-ck+ in the airway epithelium of these mice after 2 weeks. we are currently characterizing the phenotype of these cells with ccsp and cftr but these data suggest that cb-mscs may be a potential alternative source of stem cells for use in lung remodeling. high-throughput screening (hts) and other drug development programs have identified cftr activators and potentiators that require secondary evaluation and mechanistic confirmation. we recently evaluated the cfft modulator library (rosalind franklin university, chicago, il) and found some, but not all, cftr potentiators induced potent phosphorylation of the regulatory-domain (r-d), conventionally viewed as the first step in cftr activation. to assess whether this observation has functional significance regarding ion channel activation by these agents, we evaluated cftr potentiators in cfbe41o-and fisher rat thyroid (frt) cells stabily transduced with ∆f508 cftr. cells were studied in modified ussing chambers under control conditions and after correction of ∆f508 cftr misprocessing using either low temperature (27οc x 48 hrs) or pre-incubation with the chemical corrector c4. total currents were determined following potentiator (at reported ec 50 ), forskolin (2 µm), and genistein (50 µm) stimulation. in temperature corrected ∆f508 cfbe41ocells, potentiator 1 (p1, cfpot-532), an agent that does not induce r-d phosphyorylation, caused modest activation when acutely administered (5 µm) (15.5 vs 1.1 µa/cm 2 in vehicle treated cells, p < 0.005). importantly, this agent potentiated forskolin mediated short-circuit current (isc: 9.8 vs 1.5 µa/cm 2 , p < .05). forskolin accounted for 43% (vs. 4% with vehicle) of total current, demonstrating potent rescue of camp dependent cftr activity, an effect not previously reported in this cell type (p<0.005). in contrast, two cftr potentiators that induce r-d phosphorylation, p8 (uccf-029, a benzoflavone intermediate, 2 µm) and p10 (uccf-152, an isoxazole, 50 µm) induced modest direct activation of cftr (5.3 and 6.1 µa/cm 2 respectively, p=0.10 and p<0.01), but no evidence of forskolin potentiation (p8: 2.3 µa/cm 2 , 10% of total stimulated current p=ns; p10: 0.4 µa/cm 2 , 1%, p=ns). in ∆f508 frt cells grown at low temperate, p1 elicited strong potentiation of forskolin mediated currents (121.7 vs. 46.7 µa/cm 2 with vehicle, p <0.01), and modest direct cftr activation (8.7 vs. 0.51 µa/cm 2 , p < 0.05). p8 and p10 were limited to minimal (not significant) activation and no evidence of forskolin potentiation. in cfbe41oand frt cells pretreated with c4 (to chemically correct ∆f508 cftr processing), p1 again potentiated forskolin mediated current (101.3 vs. 43.2 µa/cm 2 with vehicle, p<0.001 in the frt model) but did not directly activate i sc . findings with p8 and p10 were otherwise as seen with low temperature corrected cells. in summary, in ∆f508 cftr polarized epithelia, p1 both activates and potentiates cftr activity (with potentiation being the predominant effect, a unique observation in cfbe41ocells), while p8 and p10 confer activation of cftr without forskolin potentiation. given that p8 and p10 induce r-d phosphorylation while p1 does not, our findings suggest that agents that do not confer phosphorylation of the r-d may be better suited to rescue the endogenous camp mediated component of i sc . screening of potential therapeutic agents for effects on r-d phosphorylation may help predict utility at restoring ∆f508 chloride channel activity. supported by nih and cff. activation of cftr is conventionally viewed as a two step process: pka-regulated phosphorylation of multiple sites within the regulatory domain (r-domain), followed by atp dependent gating mediated by binding sites at the interface of the two nucleotide binding domains (nbds). cftr 'potentiators', small organic molecules that overcome mutant cftr gating defects at the cell surface, have been proposed as therapies for cf. although a number of these agents are advancing to the clinical testing phase, their mechanism(s) of action are not well understood. as a step towards better characterizing cftr potentiators available through the cfft modulator library or other resources, we are developing standardized biochemical and functional assays to evaluate the r-domain during cftr activation in living cells. we have previously described a gel-shift method by which phosphorylation of isolated r-domain (residues 635-836) can be monitored. using this method, we have confirmed that one potentiator, p1 (cfpot-532), does not induce phosphorylation of the r-domain (4% of forskolin response, n=7, p = ns). unexpectedly, two potentiators, p8 (uccf-029, a benzoflavone intermediate) and p10 (uccf-152, an isoxazole), robustly confer phosphorylation of the r-domain (p8: 32% of forskolin response, n=8, p = 0.002, p10: 37% of forskolin response, n=8, p = 0.004). we found the phosphorylation conferred by either p8 or p10 could be inhibited with the pka inhibitor h89 (10 µm). maximal stimulation of phosphorylation occurs within 2 minutes, indicating time dependence similar to forskolin. importantly, neither p8 nor p10 increased total cellular camp, a finding confirmed by a number of other laboratories. the results may therefore implicate compartmental inhibition of cftr-associated phosphotases (eg. pp2a) or phosphodiestereases (eg. pde4) as an underlying mechanism by which isoxazole or certain flavone-derivative compounds stimulate cftr. to further test this hypothesis, p1, p8 and p10 are being examined for effects on ∆r-cftr chloride channel activity. these studies provide a means by which novel cftr potentiators can be biochemically categorized based on r-domain phosphorylation, a measure of the first step of cftr activation. compounds working through distinct mechanisms may have particular relevance to certain cftr mutations, and could provide synergy in the clinical setting. supported by nih and cff. mucociliary clearance (mcc) is an innate defense mechanism that protects the lungs from bacteria and viruses. mcc requires maintenance of a thin layer of airway surface liquid (asl) to eliminate inhaled particles. the asl volume is tightly regulated by a balance of ion and water transport across the airway epithelia. in cf, the loss of cftr clsecretion coupled with unregulated na + absorption via enac results in asl volume depletion and impaired mcc. although significant progress has been made in the identification of the basic disease mechanism in cf, therapeutic approaches that address abnormal cftr biogenesis are not currently available. the extracellular nucleotides atp and utp are important mediators of asl volume and mcc. in the airways, secreted atp acts on the g-protein coupled p2y 2 receptors to fine-tune mcc via the regulation of apical ion transport, ciliary beating, and mucin secretion. we have previously demonstrated that atp controls asl volume by inhibiting absorption through enac and increasing secretion through apical membrane chloride channels. p2y 2 receptor agonists are good candidates to treat cf. however, the rapid hydrolysis of atp and utp on the airway surface of cf patients limits the effectiveness of this approach. consequently, inspire pharmaceuticals has developed di-nucleotide molecules which retain the ability to activate p2y 2 receptors, but are more resistant to hydrolysis by ectonucleotidases. denufosol, inspire's lead compound for cf treatment, potently activates p2y 2 receptors to stimulate cl -/water secretion, ciliary beating, and mucin release in epithelial tissues. however, the stability and potency of denufosol has not been determined using human bronchial epithelial (hbe) cells. in primary cultures of hbes, denufosol was significantly more stable than utp. under thin-film conditions, the initial hydrolysis rate of a therapeutic concentration of denufosol (1 mm) was 0.08 nmol × min -1 × cm -2 with a half-life of approximately 1 hour. by comparison, denufosol is more than 10 times more stable than utp on the mucosal surface of hbes. importantly, the increased stability of denufosol translated into an increase in efficacy for this compound in vitro. equipotent concentrations of denufosol and utp significantly increased asl height. however, denufosol produced maximum asl height increases that were both greater (77.6% versus 33.2% maximum increase over basal asl for denufosol and utp, respectively) and longer-lasting (72.2% versus 16.6% increase over basal asl following a 60 minute application of denufosol and utp, respectively) compared to utp. furthermore, the asl height increases are specific for denufosol as the addition of apyrase blunts the response to utp, but not denufosol. our data demonstrate that denufosol is more stable than utp on the mucosal surface of human airway epithelia, which results in larger and more sustained increases in asl volume. recent phase ii clinical trials show that administration of denufosol over 28 days was well tolerated and associated with improved lung function in mild cf patients. taken together, these data suggest that denufosol is a promising candidate molecule for cf therapeutics. mucociliary clearance (mcc) is the primary airway host defense against chronic exposure to infectious and noxious agents. mcc is dependent upon ciliary beating and the volume and composition of the airway surface liquid (asl). asl volume is regulated via isotonic fluid transport which is dominated by na + absorption in the superficial epithelium. in cf, unregulated na + absorption through enac drives asl volume depletion and a subsequent decline in mcc. the accumulation of mucus in the airways of cf patients supports persistent and life-threatening bacterial infection. increasing airway surface hydration represents a promising therapeutic approach for treating cf. in vivo, aerosolized osmotic agents such as hypertonic saline (hs) improve mcc and lung function in cf patients. however, the benefits derived from hs treatments are transient, as nacl is relatively rapidly absorbed by airway epithelia. previously, donaldson et al. (nejm 2006) tested the hypothesis that blocking na + absorption with the enac inhibitor amiloride would increase the efficacy and extend the benefit of hs. surprisingly, amiloride blunted the response to hs, which resulted from a previously unrecognized property of amiloride to inhibit aquaporin-mediated water transport. while potent inhibitors of enac would likely be of great benefit to the cf treatment milieu, the compounds then available were not adequate for this purpose. parion sciences has developed novel 2-substituted pyrazinoylguanidine compounds that selectively inhibit enac and are >100-fold more potent than amiloride. in the present study, we evaluated the ability of two compounds, 552-02 and 680, to increase asl volume under thin film conditions. in primary cultures of human bronchial epithelial cells (hbes), 552-02 alone produced a small, but significant increase in asl height. in cf cultures exposed to phasic motion that simulates the shear stress generated by normal tidal breathing, 552-02 alone increased asl heights by 46.7%. additionally, we tested the effects of 552-02 pre-addition in combination with 4% hs. as expected, hs alone produced a rapid and substantial increase in asl height (671.7% by 10 minutes) which declined to 50% bỹ 1 hour post-treatment. pre-treatment of cultures with 552-02 prior to hs was both more potent (867.8% by 10 minutes) and longer-lasting (50% initial response at ~4 hours) than hs alone. on normal hbes cultures, the 680 compound alone increased the basal asl height by 107.7% following a 2 hour exposure. similar to 552-02, 680 in combination with hs likewise extended the longevity of the hs effect (t1/2 ~4 h versus ~1 h for 680 + hs and hs, respectively). by blocking na + absorption, the parion compounds alone increased asl volume in both normal and cf hbes. strikingly, when used in combination with hs, the parion compounds enhance and sustain the increase in asl volume associated with hs alone. our data demonstrate that the parion compounds alone are sufficient to increase basal asl volume. furthermore, these data provide a proof-of-concept that combinational therapies utilizing osmotic agents and compounds that regulate ion transport will provide therapeutic benefits to cf patients. methods: a total of 24 patients (with fev 1 ≥40% of predicted) received 500 mg of arikace by inhalation for 14 days. drug was administered using the pari lc star nebulizer. laboratory parameters, adverse events and pulmonary function tests were collected for all study subjects in order to determine safety and tolerability of arikace™. sputum samples were collected to determine changes in bacterial density, and amikacin pharmacokinetic parameters were assessed at selected time points in urine, serum and sputum specimens. change in fev 1 , fev 1 % predicted, fef 25-75 % and fvc relative to baseline and change in log 10 cfu on days 7 and 14 were assessed. results: on day 7, 14 and 21, the observed change for fef 25-75 % was 0.49 (p < 0.001), 0.42 (p = 0.02) and 0.34 l/sec (p = 0.04), respectively. on day 7 and 14, the observed change for fev 1 was 0.24 (p = 0.002) and 0.13 l (p = 0.10), respectively, and was 7.49 (p <0.001) and 4.38 l/sec (p = 0.03) for fev 1 % predicted. significant relationships (p ≤ 0.05) between log 10 cfu and serum auc:mic ratio, and between changes in log 10 cfu and fev 1 , fev 1 % predicted and fvc were identified. treatment was safe and well tolerated with the most frequent adverse events being dyspnea and headache of mild to moderate severity. conclusion: inhaled arikace™ 50 mg/ml was well tolerated and in select patients improved pulmonary function. together these clinically relevant changes from baseline likely represent drug effect and warrant further development of liposomal amikacin for inhalation in patients with cf. allergic bronchopulmonary aspergillosis (abpa) is a disease caused by hypersensitivity to aspergillus fumigatus (af). the prevalence of abpa is approximately 1%-15% of patients with cf and can contribute to worsening of their pulmonary disease. the treatment of abpa consists of high-dose oral corticosteroids for many months and may include also antifungal antibiotic such as itraconazole. the clinical effectiveness of corticosteroids is usually shown by an improvement in clinical symptoms and radiological parameters, as well as a reduction in total ige. the prolonged period of systemic steroids may cause significant side effects such as cushingoid appearance, hypertension, glucose intolerance and more. over the past 5 years, we have used pulse high-dose methylprednisolone in 5 patients with cf. method:all 5 patients were diagnosed as suffering from abpa by the standard criteria as the may have mucoid impaction or central bronchiectasis on chest radiography, an elevated ige (>417 iu/ml), the presence of specific ige anti af and an elevated eosinophil count. the patient may have a positive skin prick test to af allergen. they were treated once a month with iv pulse high-dose methylprednisolone (10mg/kg/a day) for 3 days once a month for several months until the total ige decreased to normal values. three patients were treated also with itraconazole. in 2 patients itraconazole was discontinues due to side effects. results: we treated 5 patients age 10-35 years old (3m/2f). four patient are pancreatic insufficient and 1 pancreatic sufficient. ige was 622+/-371 (119-1213) at the time of diagnosis and decrease to 55+/-64 (57-89). fev1 increased from 65+/-16 to 78+/-15. the patients also gained weight and improved their chest x-ray finding. side effect were minor and mainly during the treatment days and resolved 1-2 days after each treatment and included malaise during the infusions, glucose intolerance on infusion days in one case, and hypertension in another case. conclusion: iv pulse steroid treatment should be considered in abpa as it was found effective with fewer side effects that should be expected from prolonged oral corticosteroid treatment. lung pathology in individuals with cystic fibrosis (cf) is linked to sodium hyper-absorption. the defective regulation of the epithelial sodium channel enac is thought to be a major contributor to reduced airway surface liquid (asl) volume and impaired mucociliary clearance of the airways. thus, strategies designed to inhibit enac function may result in clinical benefit. rna interference (rnai), mediated by short, double-stranded rna molecules, can be used to target complementary rna sequences for degradation via the rna induced silencing complex (risc). we investigated the possibility of using rnai to reduce expression of enac in the mouse lung, as proof of principle for a strategy to reduce sodium hyperabsorption in the cf lung. potent sirna molecules capable of efficient knockdown of enac alpha, beta and gamma subunits were identified in an optimised cell culture system utilising mouse kidney m1 cells (50,000 cells per 24-well; 20pmol sirna complexed with lipofectamine 2000; 48 hour harvest) in which enac expression was quantified using real-time rt-pcr. approximately 80% knockdown of each enac subunit was observed with the most potent sirna molecules in this system. subsequently, sirna molecules were delivered to the lungs of female balb/c mice via hydrodynamic tail vein injection (40 µg naked sirna, n=5-6). after 24 hours, lungs were harvested, rna extracted and enac mrna measured using real-time taqman pcr. whereas, enac subunit mrna levels in mice treated with a negative control sirna were not different from untreated mice (p < 0.05), delivery of enac alpha and beta-specific sirna molecules resulted in a reduction to 56.5 ± 12.3 % or 33.3 ± 4.7 % of the expression levels observed in untreated mice, respectively (p < 0.05, mann-whitney u). interestingly, despite efficient knockdown in cell culture, in vivo treatment with the most potent enac gamma-specific sirna molecule led to no reduction in enac gamma expression compared with untreated controls (p > 0.05). these data show proof of principle that enac expression can be reduced in the lung using sirna. further work is needed to assess the functional consequences of inhibiting enac in the lung. gene therapy for cystic fibrosis lung disease will likely require longterm transgene expression; however, in the lung, many gene transfer agents have resulted in only transient gene expression. previously we have shown that the choice of enhancer/promoter elements has a strong influence on the duration of reporter gene expression following delivery of non-viral vectors to the mouse lung (gill et al., 2001 , gene ther, 8, 1539 . however, it is also possible that other elements of plasmid vector design play an important role. using a clinically relevant mouse lung aerosol model, we have studied the effect of varying the plasmid cpg-dinucleotide content on the duration of expression from plasmids that had an identical enhancer/promoter sequence. firstly we constructed a cpg-free plasmid vector containing a synthetic cpg-free luciferase gene under the transcriptional control of the human cmv enhancer/ef1α promoter. secondly, we constructed a similar vector containing an identical enhancer and promoter but with a small number of cpg motifs, in the luciferase gene (97 cpgs) and in the backbone sequence (52 cpgs). we then investigated the level and duration of transgene expression following aerosol delivery of these plasmids complexed with the genzyme lipid gl67 to the lungs of balb/c mice (2.5 mg/ml pdna, 6mm gl67, 10ml, pari lc+ nebuliser). total lung extracts were assayed for luciferase activity at days 1, 2, 7, 14 & 28 (n=6 per time-point). the cpgcontaining plasmid initially directed approximately 2-fold higher levels of reporter gene expression than the cpg-free plasmid (p<0.05 both days 1 and 2 post-delivery). however, while expression from the cpg-containing plasmid subsequently fell slowly to background levels, expression from the cpg-free plasmid increased 2-fold to day 7, and remained at the peak levels observed with the cpg-containing plasmid until the end of the experiment at day 28 (cpg-free plasmid 4-fold, 16-fold and 50-fold higher expression at days 7, 14 and 28 post-delivery respectively; p<0.05 for each). these data suggest that the ability of a promoter/enhancer sequence to direct long-term expression in the mouse lung is dependent by the sequence of the plasmid backbone. one explanation for this observation is that a specific, but as yet unidentified, sequence exists in the cpg containing backbone that triggers transcriptional silencing. alternatively, the well-described host inflammatory response to cpg-containing dna, may be involved and the cpg content of the vector could be responsible for these differences. we are currently evaluating the potency and duration of effect of cftr expressing, cpgfree, plasmids in late-stage pre-clinical studies prior to the initiation of a further round of clinical studies of non-viral gene transfer in cf patients. background: novel approaches to cf therapy by improving/restoring cftr chloride channel function using gene therapeutic tools or small molecules are currently undergoing phase i and phase ii clinical trials. there remains a great need for accurate and practical methods of measuring cftr function in vivo to act as primary outcome measures of efficacy. we propose that an in vivo assay measuring cftr function in sweat glands offers several potential advantages over the nasal potential difference test. objective: to develop a reliable test of sweat gland function that is capable of measuring the range of cftr function in vivo. methods: we are performing repeated measures of sweat gland function in healthy controls (n=10), obligate heterozygotes (n=10), pancreatic sufficient (cfps, n=10) and insufficient cf patients (cfpi, n=10). sweat secretion is stimulated by iontophoresis (30µa/cm2) with 1% pilocarpine. to elucidate the most sensitive and discriminatory parameter of sweat gland function within this study group we are performing simultaneous measurements of: a) the transglandular potential difference of a stimulated skin area using an ecg electrode (espd) and b) single sweat gland potential differences in single sweat glands (spd). using a wescor® collector cup we also measure: c) sweat secretion rate and (d) sweat chloride concentration (sweat cl-). results: we report interim results of our ongoing study (table) . sweat rate was not different between the groups, but a gender difference was observed (mean±sd; 12 male: 1.6 ± 0.7µl/min/cm2; 15 female: 0.9 ± 0.5µl/min/cm2, p<0.001) as previously described. our preliminary data show that espd as well as sweat cl-allow good discrimination among healthy controls, cfps and cfpi (p<0.01 and p<0.05 respectively). spd show similar trends to espd but with greater overlap (differences not significant). conclusion: these encouraging preliminary results, particularly the espd method and sweat cl-following stimulation with a lower iontophoretic current, justify further efforts to complete enrolment of subjects and to further refine technical challenges such as minimizing the effect liquid junction potentials across the ecg electrode. results will also be compared to the classical sweat test using higher iontophoresis current and npd measurements in these patients. this study is supported by the ccff and genome canada. ino-4995, a prodrug, has been demonstrated to inhibit nasal potential difference (pd)in human cf nasal airway epithelia and cf mice and this effect is more potent with repeated dosing. as such it is being developed as potential therapeutic for cystic fibrosis. ino-4995 cell entry is facilitated by its hydrophobic propionoxy(methyl)ester protecting groups which can be hydrolyzed by intracellular carboxyesterases after the prodrug enters the cell. once the protecting groups are removed, the drug (ino-4913) with the ether-linked octyl group is expected to be more slowly metabolized. however, the precise kinetics of the uptake of ino-4995, its conversion to its active metabolite, and methods. confluent cultures of hela or t84 cells were incubated for varying periods of time ranging from 10 min to 3 hrs with medium containing [ 3 h] ino-4995 (2 x 10 6 cpm/ml) and 5µm ino-4995 cold carrier to ascertain the rate of uptake. in pulse chase experiments after cultures were incubated for 2 hours, they were washed and incubated with media without radiolabel for varying periods of time. after the indicated times, the media was removed and the cells were washed, harvested, extracted and the aqueous and organic extracts subjected to sax and reversed phase hplc respectively. results ino-4995, a prodrug, has been demonstrated to inhibit nasal pd in human cf nasal airway epithelia and cf mice and this effect is more potent with repeated dosing. ino-4995 cell entry is facilitated by its hydrophobic propionoxy(methyl)ester protecting groups which can be hydrolyzed by intracellular carboxyesterases after the prodrug enters the cell. once the protecting groups are removed, the drug (ino-4913) with the ether-linked octyl group is expected to be more slowly metabolized. however, it is not known whether the mucus present in cf airways could impede its access to airway epithelia or whether ino-4995 would cross the epithelial layer to enter the bloodstream. here, in order to address these issues we measured its uptake into human tracheal epithelial sheets in the presence and absence of mucus and compared its transepithelial permeation to mannitol. we studied the uptake and transepithelial fluxes of [ 3 h]ino-4995 in primary cultures of human tracheal epithelium with transepithelial resistance of from 500 to 1000 ohms.cm 2 . the mucosal surface of some tissues was rinsed three times with pbs to remove mucus for comparison with control cells which were not rinsed. elisa for human airway mucins performed on the rinsings confirmed that three rinses sufficed to remove the mucosal mucous blanket. the development of long-term, safe and efficacious gene therapies for lung disease will require efficient gene delivery to stem cells which produce the differentiated progeny of affected epithelial tissues. recombinant lentiviral vectors are being considered for gene therapy applications for the lung because they can efficiently transduce a wide variety of stem and progenitor cell types. however, it is of concern that strong constitutive viral promoters have produced oncogenesis in x-scid patient recipients of retroviral vectors in part due to the disregulation of proto-oncogenes by the strong viral promoter/enhancer located near the integration site. we propose that regulation of transgene expression to the tissue or cell type of interest will enhance the safety of gene therapy for respiratory disease. in this study we have investigated the potential for respiratory specific transgene expression from integrated lentiproviruses in cell lines in vitro and in mice in vivo. the lentiviral vectors constructed for this study contained regulatory elements predicted to produce lung specific transgene expression: the surfactant protein c promoter (spc) for alveolar epithelial type ii cell (aecii) expression, the clara cell 10kd protein (cc10) for clara cell expression in the airway, and the jaagskiete sheep retrovirus (jsrv) promoter for expression in both cell types. transgene expression from the spc and cc10 vectors was restricted to aecii and clara cell lines respectively, while expression from the jsrv vector was observed in multiple respiratory and non-respiratory cell types. following intra-tracheal delivery of lentivector supernatant to mice, transgene expression was observed in aecii from the spc lentivector (n=8 mice), and in clara cells from the cc10 promoted lentivector (n=6 mice). transgene expression was not detected in non-respiratory tissues following intravenous delivery of cc10 and spc lentiviral vectors to neonatal murine recipients (n=4 and n=7 respectively). in summary, incorporation of genomic regulatory elements from the spc and cc10 genes resulted in respiratory specific transgene expression in vitro and in vivo. these vectors will provide a useful tool for the study of lung biology and the development of gene therapies for lung disorders such as cf. cystic fibrosis (cf) is associated with chronic pulmonary inflammation and progressive lung dysfunction, associated with the formation of oxidants derived from neutrophil myeloperoxidase (mpo). indeed, respiratory tract levels of mpo are extensive, and have been found to correlate with decreases in respiratory parameters or disease severity in cf. mpo-derived oxidants thus may play a role in perpetuating the progressive lung dysfunction associated with cf. based upon these premises, we reasoned that mpo could represent a novel therapeutic target for the treatment of inflammatory diseases such as cf. herein we report that substituted urea and thiourea compounds are potent inhibitors of mpo. utilizing tetramethylbenzidine (tmb) as a classical heme peroxidase substrate, we screened a large number of structurally varied urea and thiourea derivatives. alkyl, cycloalkyl, and aromatic ureas and thioureas all proved to be effective mpo inhibitors, however, with a high degree of variability in potency. of those compounds examined, substituted phenylthioureas proved to be the most potent mpo inhibitors. various ortho-, meta-, and para-substituted phenylthioureas (ptus) were then assessed for inhibitor potency. the most potent inhibitor of mpo was 2-chlorophenylthiourea (ic50 = 700 nm, ki = 320 nm). the potency of ortho-ptus as mpo inhibitors showed a linear relationship with ortho sigma substituent constants (reflecting resonance, steric and inductive effects). moreover, cyclic voltametry experiments with ortho-substituted ptu compounds revealed a linear relationship between inhibitor potency and thiourea oxidation potential. this correlation is indicative of a substituent effect on the relative ease of sulfur oxidation within the thiourea moiety. this finding suggests that the redox-active sulfur plays a crucial role in mpo inhibition, perhaps via a direct interaction with the heme of mpo. thioureas proved to also inhibit the chlorinating activity of mpo, whereas sustituted ureas were not effective in this regard. both ureas and thioureas were also effective inhibitors of dityrosine formation by mpo. these findings will help facilitate the rational design and optimization of mpo inhibitors with even greater potency, which could serve as novel therapeutic agents for the treatment of cf. gene therapy is the transfer of a normal copy of the cftr gene and should, in theory, provide a long-term cure for cf. the development of prenatal screening programs for cystic fibrosis has provided an opportunity to identify cf patients in utero, and may provide an opportunity to treat the disease before birth, prior to the onset of disease symptoms. the long-term goal of our study is to investigate the therapeutic potential of delivering the normal cftr gene to epithelial stem cells in fetal cf mice by in utero administration of recombinant lentiviral vectors carrying the normal cftr gene. this would provide us with the unique opportunity to "correct" the chloride channel defect in the epithelial stem cells, which give rise to the epithelial cells in the tissues affected by cf at a very early stage. the aim of the current study is to optimize the in utero gene delivery of recombinant lentiviral vectors, carrying an egfp reporter gene to epithelial stem cells in fetal mice. mice have been used for these studies, as mouse strains with a variety of cf mutations are available. in all studies, the uterus was exteriorized following a mid-line laparotomy, and the injections were made directly into the amniotic fluid of individual fetuses. in the first studies, the survival of pregnant females and pups following saline injection at e16.5 (n=2) and e17.5 (n=3) was evaluated. all five females survived the procedure and an average of 54% (26/48) of the pups present at the time of injection were born alive. this experiment demonstrated that the surgical procedures were reasonably safe. in the next experiments, we injected 5.0ul lentiviral supernatant (~4.5x107iu/pup) into the amniotic fluid of each pup and evaluated the effect of the gestational age pups on transduction efficiency. the pregnant mice were allowed to carry the fetuses to term. at p0, p7 and p28, 2-3 pups of each litter were sacrificed and skin, trachea, lung, stomach and intestines were harvested for analysis of gene transfer and egfp transgene expression. all but one pup (11/12) from the e12.5 time point aborted prior to parturition. from the e14.4 and e16.5 injections, 6/12 and 7/7 pups were born naturally, respectively. at e14.5 and e16.5, proviral sequences were observed in the skin, trachea and stomach, although the level of transduction varied among individual fetuses and tissues. immunofluorescence analysis on the skin of one mouse with a high proviral copy number demonstrated that some of the egfp positive cells were also positive for vimentin or f4/80, markers of mesenchymal cells and macrophages, respectively but negative for pancytokeratin. our preliminary studies suggest that in utero gene therapy with lentiviral gene delivery may be safe, however ongoing studies will provide more in depth analysis on safety and feasibility of delivering the normal cftr cdna to epithelial stem cells in the tissues affected by cf in utero. future studies will investigate the therapeutic potential of delivering a lentiviral vector containing the cftr gene to fetal pups in a murine model of cf. supported background & objectives: cystic fibrosis is characterized by progressive loss of lung function resulting from chronic bacterial infection and concomitant airway inflammation. while a number of bacteria are known pathogens in cf, mounting evidence suggests that the lungs of cf patients harbor numerous bacterial species, and that interactions within this ecosystem may affect outcomes. however, the composition of this community is not well characterized. we therefore initiated work to profile the bacterial diversity of the lungs of cf patients with end-stage disease by 16s rrna gene sequencing. methods: genomic dna was purified from snap-frozen explanted lung tissue of 21 randomly selected cf patients undergoing transplantation in toronto between 1998 and 2006. bacterial 16s rrna genes were amplified from each sample using broad-spectrum pcr primers, and pcr products used to generate 16s rdna libraries for each patient. 16s rdna profiles for each patient were generated by automated dna sequencing of up to 96 randomly selected clones per library, and tentative species assignments made by comparison to known 16s rrna gene sequences using phylogenetically-based analyses. results: sequence analysis of 1514 16s rdna sequences from this preliminary set of 21 patients (avg. = 72 sequences/patient; range = 36-96) demonstrates a range of complexity of the microflora in cf lungs, with some patients showing a single dominant organism and others showing multiple co-existing species. in total, over 60 species have been identified to date in this set of patients. these include species identified previously by clinical microbiology, including pseudomonas aeruginosa (detected in 20/21 patients), and burkholderia species (detected in 4/5 patients known clinically to be infected). numerous organisms not previously reported in the lung were also identified, including several from the taxonomic order burkholderiales. interestingly, while organisms commonly associated with cf were detected in patients transplanted throughout the year, a cluster of four plant-associated species demonstrated striking seasonal variation in rates of detection. conclusions: the lungs of cf lung transplant patients harbor a diverse bacterial community. sequencing of 16s rrna genes is a powerful method to profile this diversity. correlation of these profiles with clinical information may help identify new patterns of infection and important interactions between species in this community. introduction and aims: pulmonary infection, primarily with pseudomonas aeruginosa, is the leading cause of morbidity and mortality in cystic fibrosis (cf) patients. we have demonstrated that anaerobic bacteria, belonging primarily to the genera prevotella, veillonella, propionibacterium and actinomyces, can be cultured from the sputum of cf patients. the aim of this study was to determine the antibiotic susceptibility of these anaerobic isolates grown planktonically and as biofilms. methods: the planktonic susceptibility of anaerobic isolates to antibiotics used in the treatment of cf (meropenem, piperacillin/tazobactam) as well as to antibiotics traditionally used to treat anaerobic infection (clindamycin, metronidazole and ampicillin) was examined using e-test® strips. selected anaerobic isolates were grown as biofilms for 4 or 24 hours in 96 well trays prior to challenge with antibiotic. bacterial biofilm formation was determined using crystal violet. results: the planktonic susceptibility of 39 anaerobic isolates (14 prevotella, 5 veillonella, 5 propionibacterium, 8 actinomyces and 7 additional isolates) comprising 9 different genera was determined. although all of the anaerobic isolates examined were sensitive to meropenem when grown planktonically, high levels of resistance to the anti-anaerobic drugs, metronidazole and clindamycin were observed. examining susceptibility by genera, the veillonella were the most resistant to piperacillin/tazobactam and the propionibacterium the most resistant to metronidazole. furthermore, several isolates, including 4 of the 14 prevotella isolates examined, were found to be resistant to multiple antibiotics. the susceptibility of anaerobic biofilms to antibiotics was found to be isolate and antibiotic dependent. generally antibiotic treatment of 4 hour old biofilms was often able to eradicate biomass and prevent biofilm formation; however, antibiotic treatment after 24 hours of biofilm formation with up to 100x the mic of an antibiotic often failed to eradicate the biofilm. conclusion: these results suggest that alternative antibiotic treatment regimes may be necessary to treat cf pulmonary infection if anaerobes are present. these results also suggest, based on in vitro susceptibility, that meropenem and not metronidazole or clindamycin would be the most effective antibiotic in treating any anaerobes present in cf pulmonary infection. early eradication protocols for the first appearance of pseudomonas aeruginosa (psa) have become standard practice in many cystic fibrosis (cf) clinics. the details of such protocols often vary significantly between clinics, making it important to compare efficacy from one approach to another. however, comparison of reported results is currently very difficult. this is not only because of variations in completeness of reporting, but especially because calculation of average time to regrowth cannot by definition include patients who remain free of psa regrowth at the time the "average" is calculated (nor those in whom a re-appearance of psa is found to be due to a different strain). such results would be better expressed using "time to event" (tte) statistics (analogous to "life tables") and different protocols can be better compared using this methodology. we report for the first time, results from our psa early aggressive eradication protocol for the period 1995-2007, expressed using tte statistical analysis. children are seen in clinic on average 4 times yearly for full assessment, and at each clinic visit sputum or pharyngeal cultures obtained by the physiotherapist. standard treatment for "first growth" pseudomonas aeruginosa consists of 2 weeks of intravenous antibiotics (piperacillin 600 mg/kg/day plus tobramycin 12 mg/kg/day) followed by 3 weeks of oral ciprofloxacin (20-30 mg/kg/day) and inhaled colymycin (100 mg bid) for 6 months. a total of 145 treatment courses have been completed for the period 1995-2007 with a 90% clearance rate, defined as 3 or more consecutive negative cultures for pseudomonas aeruginosa over 6 months. since the initiation of this protocol, the percentage of patients colonized with psa in our cf pediatric clinic has declined from 44% in 1995, down to 15% in 2007. subsequent regrowth of psa occurred in 1/3 of patients. treatment courses may then be repeated a second, third, or fourth time. to determine whether these repeat psa isolates are newly acquired or identical to the previous isolate, rapd typing of these isolates is routinely assessed. a total of 44 isolates have been rapd tested and have identified the same strain as the previous isolate in 16 (36%) and a different strain in 28 (64%)treatment courses. the average time to regrowth for those who had a recurrence of psa following this treatment protocol was 24 months, but this does not include those patients who have had no further growth of psa for periods up to 10 years. to reflect the results for all patients, including those who have not as yet had further regrowth of psa, we have constructed tte curves. these measures can be used to compare results using our protocol with results from other approaches to clearing pseudomonas aeruginosa in cf. retsch-bogart, g.z. 1 inhaled antibiotics have been part of the therapeutic armamentarium for patients with cf for decades. we studied the effect of inhaled aztreonam on respiratory symptoms in cf in a phase 3, double blind, placebo (pl) controlled trial of azli, a novel formulation of aztreonam, which enrolled 164 patients with cf from 67 centers in the us, canada, australia, and new zealand. all patients have completed study participation. inclusion criteria included age ≥ 6 years, pa in sputum or throat swab, fev1 ≥ 25% to ≤ 75% predicted, and no use of anti-pseudomonal antibiotics in the previous 14 days. following a 14 day screening period, patients were treated for 28 days with either azli 75 mg or pl. study drug was administered tid using the pari eflow® electronic nebulizer after pre-treatment with bronchodilator. concomitant standard cf therapies were allowed, with the exception of anti-pseudomonal antibiotics, azithromycin and hypertonic saline. the primary endpoint was change from baseline to day 28 in scores from the cystic fibrosis questionnaire-revised (cfq-r) respiratory domain, a validated patient-reported measure of respiratory symptoms. other efficacy measures included change in pulmonary function (fev1, fvc, and fef25-75), number of hospital days and number of courses of iv, oral or inhaled anti-pseudomonal antibiotics. microbiological endpoints included change in pa bacterial density in sputum, change in susceptibility of pa to aztreonam, and emergence or disappearance of other pathogens. safety evaluations included adverse events, airway reactivity (defined as acute decrease in fev1 ≥ 15% at 30 minutes after dosing) and clinical chemistry and hematology. complete efficacy and safety results from this trial will be presented. salam, a.p.; orchard, c.; wee, a.; hodson, m. introduction: oral ciprofloxacin is often used for pseudomonas aeruginosa (psa) infections in patients with cystic fibrosis (cf) not requiring intravenous antibiotics. oral chloramphenicol is commonly used at the royal brompton hospital (rbh) as an alternative to ciprofloxacin, but less so in other cf centres due to concerns about aplastic anaemia and uncertainty regarding effectiveness. this study aimed to address three questions: 1) what proportion of psa is sensitive to ciprofloxacin and/or chloramphenicol? 2) were there any adverse haematological effects with the use of oral chloramphenicol at rbh? 3) how effective is chloramphenicol in comparison to ciprofloxacin? study design: we carried out a retrospective review from the rbh cf database (1985) (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) together with analysis of patients' notes. methods: 1) psa sensitivities to ciprofloxacin and chloramphenicol were recorded from sputum samples taken in late 2004. 2) all blood dyscrasias in patients who had received chloramphenicol from 1985 to 2004 were reviewed. 3) the database was searched for patients who had received a course of either oral chloramphenicol or ciprofloxacin from 2004 to 2005. 25 patients from each group were randomly selected. fev 1 , fvc and oxygen saturations pre and post treatment completion were analysed, as were markers of symptom improvement. results: 1) 216 sputum samples from 216 patients grew psa. some were sensitive to only one antibiotic, and there were some cross-sensitivities, but in total 35.46% were sensitive to chloramphenicol and 54.25% to ciprofloxacin. 2) 648 patients received oral chloramphenicol and 755 oral ciprofloxacin from 1985-2004. multiple courses were often given, but usually no more frequent than 3 monthly with chloramphenicol. over 20 years, 4 cases of blood dyscrasia were recorded, none due to chloramphenicol, and all recovered. 3) in the ciprofloxacin group: the mean oxygen saturations improved by 0.25 % (p 0.672), the mean fev 1 improved by 5.3% (p 0.126), and the mean fvc by 3.35% (p 0.202). in the chloramphenicol group: the mean oxygen saturations improved by 0.48% (p 0.118). ), the mean fev 1 improved by 4% (p 0.438) and the mean fvc improved by 5.9% (p 0.047). 56% of patients in the chloramphenicol group and 56% of patients in the ciprofloxacin group reported symptom improvement. a significant proportion of psa is sensitive to chloramphenicol and in over 20 years there have been no documented cases of related aplastic anaemia. clinical improvements with choramphenicol comparable to ciprofloxacin were also recorded. our data shows chloramphenicol is safe and may be the oral antibiotic of choice in ciprofloxacin allergy or psa resistance to ciprofloxacin. a randomised prospective study is needed to further evaluate the efficacy of chloramphenicol in comparison to ciprofloxacin. pseudomonas aeruginosa lung infections are one of the main factors causing disease in cf patients. at present the generally recommended treatment is repeated courses of antibiotics. this has resulted in fewer infections and a markedly improved prognosis for the patients. but it has also severe drawbacks such as antibiotic resistance, disturbed normal flora and allergenicity. antibiotic resistance is an emerging problem world wide and alternatives are urgently needed. specific chicken antibodies, anti-pseudomonas igy, have potential both as an alternative and a complement to antibiotics (pediatr pulmonol 2003; 35:433) . anti-pseudomonas igy prevents p. aeruginosa from adhesion to epithelial cells and has affinity for flagellin in vitro. humans do not produce anti-igy antibodies and oral administration of igy is generally regarded as safe. the risk that bacteria should develop resistance to igy is extremely low. in total, 21 cf patients have received antipseudomonas igy for up to 12 years. since november, 2003 the drug is given to a group of cf patients (at present 14 patients) on individual permissions granted by the swedish mpa and reimbursed by the swedish government. in our first study about the effect of igy there were 2.5 positive cultures/100 months, compared to 13.7/100 months in a control group. up to december 31, 2006, nearly five years later, the effect is maintained with 2.4/100 months for the whole study period. we are at present compiling the data from the control group during the same period. only two siblings in the igy-treated group have been chronically colonized (with an identical p. aeruginosa strain), which still is non-mucoid after 4.5 and 1.5 years. during the whole treatment period there have been no cultures positive for mucoid p. aeruginosa or b. cepacia and other pathogens (s. maltophilia, a. xyloxidans, atypical mycobacteria and a. fumigatus) have only appeared sporadically -possibly due to the relatively low use of antibiotics. all patients have preserved pulmonary functions and nutritional status. there have been no adverse events. thus treatment with anti-pseudomonas igy has diminished the number of positive cultures and delayed the onset of chronic infection. the need for antibiotics is reduced as well as their adverse side effects. in conclusion, igy is an important complement to antibiotics for prevention of p. aeruginosa infections in cf. introduction and aims: we have previously shown by culture that the lungs of clinically stable cystic fibrosis (cf) patients are not only colonised by commonly recognised aerobic bacteria, such as pseudomonas aeruginosa, but also by a range of potentially pathogenic anaerobic species. the aim of this study was to determine whether anaerobes are also present in the sputum of cf patients with an acute exacerbation of pulmonary infection. methods: sputum samples were collected and processed using strict anaerobic bacteriological techniques, prior to commencing and at the end of antibiotic therapy, from 32 adult cf patients admitted for treatment of an acute exacerbation of pulmonary infection. bacteria within the samples were detected by plating on selective agars, quantified by total viable count and identified by pcr and sequencing of 16s ribosomal rna genes. as a control induced sputum samples were collected, using hypertonic saline, from 20 healthy volunteers who did not have cf and processed using similar methods. results: anaerobes from a range of species including prevotella, veillonella, propionibacterium, actinomyces and gemella were detected in high numbers (up to 6 x 10 7 cfu/g of sputum) from all patients prior to commencing antibiotic therapy with the predominant primary pathogens (p. aeruginosa or b. cepacia complex) detected in similar numbers. anaerobic bacteria were also detected in sputum samples at the end of antibiotic treatment and for 22/32 (69%) patients they were present in lower numbers than detected before antibiotic treatment. moreover, in 11/32 (34%) patients there was greater than a one log reduction in the total viable count of anaerobes. similar data for detection of the predominant aerobic pathogens was also collected for 27 of these patients. p. aeruginosa or b. cepacia complex was present in lower numbers following antibiotic treatment in 22/27 (81%) patients and in 13/27 (48%) patients there was more than a one log reduction in the total viable count. anaerobes were not detected in the induced sputum samples from 4 volunteers and in the remaining samples they were present in much smaller numbers, ranging from 10 2 to 10 5 cfu/g of induced sputum, than detected in cf sputum. the most commonly isolated bacteria from the induced sputum samples were the actinomyces and streptococcus, with prevotella cultured from only 2 samples. no veillonella and propionibacterium species were isolated from the induced sputum samples and combined with the low frequency of isolation of prevotella species, these results strongly suggests that the prevotella, veillonella and propionibacterium species recovered from cf sputum samples are not oral contaminants. conclusion: these results indicate that anaerobes are present in large numbers within the cf lung during an acute exacerbation of pulmonary infection. their presence could be of important clinical relevance to cf patients as they may contribute significantly to the inflammatory process. airway infection and inflammation cause the majority of morbidity and mortality in cystic fibrosis (cf). the microbiology of cf is complex. not only are the routinely identified pathogenic bacteria present, increasingly a number of other bacteria are associated with pulmonary disease. in addition, the normal microbiota likely contribute to airway disease in cf via intracellular signaling, but remains largely unstudied. the application of culture independent methods for bacterial identification in cf airway secretions provides for the first time the ability to characterize the bacterial communities in the cf airway comprehensively and efficiently. here we present the results of a two-year study of cf airway samples to describe the bacterial communities present by ribosomal rna sequences. we have examined 231 airway specimens from 117 subjects (82 cf, 35 controls). these results provide a unique perspective on cf airway microbiology. bacterial communities identified in sputum were more complex than communities identified in bronchoalveolar lavage fluid (balf). however, pulmonary status determined the level of complexity, with higher complexity observed during stable pulmonary function. we identified a collection of anaerobic bacteria that were present at high levels during pulmonary exacerbation in a subset of cf subjects. detection rates ranged from 8-50% of cf subjects examined depending on the specific organism (table 1 ). these organisms of interest are suspected to be involved in pulmonary exacerbation especially when cultures for standard cf pathogens are negative (~20% in our center). preliminary data will be presented from quantitative real-time pcr experiments to track these organisms. we also will present the application of new meth-ods for community comparisons. this analysis provides the ability to look at how clinical information from individuals with cf corresponds to the microbial communities that occur in the airway. overall, the results point to a much richer bacterial community associated with the cf airway than previously thought. the results also point to new opportunities to understand the differences within the cf population, and thus potentially improve individual patient outcomes. cystic fibrosis (cf) patients are characterized by persistent microbial colonization of the airways and recurrent pulmonary infection. however, due to the domination of bacterial communities present in cf sputum by a small number of species (typically pseudomonas aeruginosa), culture-independent approaches to characterize the depth of bacterial diversity in cf sputum have yielded limited information. here we describe application of a novel microarray, the 16s rrna phylochip to comprehensively describe the bacterial diversity present in temporal sputum samples of 3 cf patients who experienced periods of exacerbation (defined as hospitalization) and remission. in addition, these samples permitted assessment of bacterial community dynamism during periods of antimicrobial administration in these patients. phylochip analysis identified 980 bacterial taxa present across these patient samples, including a large number of known human pathogens. antimicrobial administration caused a profound decrease in bacterial diversity in all patients examined. despite this, a sizeable fraction of each community remained stable or proliferated during the period of antimicrobial administration, suggesting many of the bacterial taxa present were resistant to the antimicrobials administered. taxa exhibiting this behavior included p. aeruginosa, arcobacter cryaerophilus, streptococcus constellatus and burkholderia mallei amongst other pathogens. these results suggest that complex polymicrobial communities exist in cf sputum that exhibit significant antimicrobial driven dynamism and consist of multiple antimicrobial resistant pathogens. future studies will focus on whole community gene expression to determine which virulence systems are activated in response to bacterial community perturbations and contribute to pathogenic processes in cf airways. the lungs of chronically infected cystic fibrosis (cf) patients have been intensively studied. it is well documented that pseudomonas aeruginosa is the predominant pathogen in cf (høiby 1974 , baurnfeind et al.1987 , koch 2002 , and that p. aeruginosa accumulates in heaps (aggregates), detected in cf sputum (høiby 1977) and intraluminal in the lung (baltimore et al. 1989) . the bacterial density is highest in the bronchi (potts et al 1995) and these p. aeruginosa microcolonies are embedded in a matrix (lam et al. 1980) . the morbidity of the infection is due to an extensive and ongoing pmn response which apparently does not eradicate the bacteria, instead leading to slow degradation of the lung. in addition, even highest deliverable doses of antibiotics fail to clear the bacteria completely, though survival has significantly increased the last 30 years due to aggressive anti p. aeruginosa therapy (koch and høiby 2000) the present study was performed to investigate the significance of the aggressive therapy for the distribution of p. aeruginosa and how p. aeruginosa is organized and persists in the cf lung. the material used was: autopsies from dead short term colonized cf patients (n=12) obtained before today's aggressive antibiotic treatment , and explanted lungs from long term p. aeruginosa infected cf patients (n=3) (i.v. antibiotics every 3rd month since 1976 + inhalation of colistin daily since 1987. histological sections of the lungs were investigated using he stain, gram stain, alcian blue, antibodies against alginate and p. aeruginosa specific pna-fish combined with bacuni pna-fish and with dapi as counter stains. due to the high specificity of the pna-fish probes employed in this study, our results provide strong evidence for that before the aggressive antibiotic therapy, p. aeruginosa infected and destroyed the cf lung due to fast spreading into the respiratory zone. today antibiotics suppress but can not eradicate the bacteria from the conductive zone, whereas the remaining respiratory zone is protected from massive biofilm infection for prolonged time. the conductive zone serves as a reservoir; here the bacteria are organized in microcolonies embedded in puss, a trait which is independent on the time course of the infection and amounts of antibiotics. these microcolonies consisted solely of p. aeruginosa. in addition, we found no bacteria adhering to the epithelial tissue. the pus consisted mainly of leukocytes, surrounding the microcolonies. a smear of dna and dead leukocytes were detected just around the microcolonies, possible due to the quorum sensing dependent rhamnolipid killing recently described by us (jensen et al. 2007) . we conclude that p. aeruginosa persists in the cf lung due to its ability to create microcolonies i.e. biofilms. within these biofilms the bacteria are protected against antibiotics and the host defence. respiratory viral infections, including influenza (flu) and respiratory syncytial virus (rsv) contribute to the morbidity of children with cf. additional human respiratory viruses have been identified in children such as human metapneumovirus (hmpv) and coronavirus (cov), but these have not been examined in cf patients. in our laboratory, pcr assays have been developed to identify both traditional and newer respiratory viruses in healthy and immunocompromised children. these assays are usually performed on nasal swabs or nasal wash samples. however, oropharyngeal (op) and sputum samples are routinely obtained in cf for bacterial culture. the goals of this trial are to determine whether these available samples can be used for viral pcr studies and to examine the epidemiology of respiratory viruses in cf. for this single center observational trial, 44 cf patients, ages 6.1 to 17.7 yrs (mean 12.8 yrs), have been recruited. subjects provide op and nasopharyngeal (np) samples at quarterly clinic visits and with acute pulmonary exacerbations requiring hospitalization; sputum is also collected if patients can expectorate. np and op samples are pooled when both are available. viruses assayed are flu a and b, rsv, parainfluenza (piv) 1, 2, 3 and 4, hmpv, cov, adenovirus (adv), and rhinovirus (rhv). subjects are being followed for two respiratory viral seasons. we report here the methodology of viral pcr detection in cf sputum, as well as the results of viral detection during the first respiratory viral season in the seattle community. the mean length of time subjects were followed was 0.84 yrs (median: 0.92 yrs, range: 0.18-1.18 yrs). overall, 47 samples (20% of the 235 samples analyzed) were positive. twenty-four subjects (69.1%) had at least one viral infection detected and 8 (18.2%) had two viruses at different visits. both sputum and pooled op/np samples were available at 55 visits and were concordant in 80% of cases. among the discordant cases, it was more common for sputum to be positive and op/np to be negative (9.1%) than the converse (3.6%). the most frequently identified viruses were rhv (32 isolations); piv3 and cov were the next most prevalent with 4 and 3 isolations, respectively. in conclusion, sputum viral pcr shows promise for the diagnosis of both traditional and novel viral infections in cf. as part of ongoing clinical trials in cf, non-fermenting gram negative bacilli (nfgnr) identified at site laboratories are often sent to the tdn core microbiology laboratory for confirmation of identification. among nfgnr from cf patients, p. aeruginosa are the most frequently isolated and also are acknowledged to be the easiest to identify. however, in a survey of 428 "p. aeruginosa" isolates sent to the laboratory in the past year, pcr identified 30 (7%) as alternate species. these included: non-aeruginosa pseudomonads, stenotrophomonas maltophilia, achromobacter xylosoxidans, ralstonia spp., alcaligenes spp., brevidimonas spp., and bordetella bronchiseptica. identification of these nfgnr can be difficult; however, p. aeruginosa is an important isolate for the clinical microbiology laboratories in all cf centers to be able to identify. many methods are available including conventional biochemical testing and rapid identification kits. in the tdn core laboratory we previously demonstrated the importance of combining phenotypic identification (morphology plus a short set of biochemical tests) and genotypic identification (16s rdna sequencing of v3 and/or pcr using three primer sets) and developed an algorithm for testing. in this study, we examined all of the cf cultures performed in the clinical microbiology laboratory at chrmc over the past two years to determine the number of isolates for which it was necessary to perform genotypic identification in nfgnr. among this collection of 182 nfgnr from clinical cultures sent to the microbiology laboratory from the seattle children's hospital cf center, pcr identified 43 isolates as burkholderia cepacia complex. next most commonly identified were a. xylosoxidans (28), p. aeruginosa (23) and non-aeruginosa pseudomonas spp. (23). three isolates remained unidentified, even with pcr sequencing of the v3 determinant. the clinical microbiology laboratory at chrmc has served as the core microbiology laboratory for tdn clinical trials for 8 years and thousands of cf samples are processed annually. the result is that there is increased experience with cf isolates among the laboratory personnel. however, even with experienced technologists, a significant number of nfgnr required genotypic identification in the past two years. thus, it is important for clinical microbiology laboratories with less experience in the identification of cf nfgnr to utilize additional molecular techniques or send problematic isolates to a reference laboratory. based on these results we propose an algorithm for the most efficient means of identifying nfgnr from cf samples using a combination of tests including phenotypic (colony morphology, biochemical testing) and genotypic identification (pcr, sequencing). background: the epidemiology of s. aureus has been changing in both cf and non-cf patients. in non-cf patients, methicillin-resistant s. aureus (mrsa) has traditionally caused infections in patients with risk factors such as hospitalization, surgery, and anterior nares colonization. recently, community-acquired (ca-) mrsa infections have increased among patients without known risks. in cf, colonization of the anterior nares with methicillin-susceptible s. aureus (mssa) is associated with transmission between patients and household members. the prevalence of respiratory tract colonization/ infection with mrsa has increased in cf, but little is known about risk factors for mrsa, including possible transmission among families, and if strains are ca-or healthcare-acquired. methods: this is a case-control, multicenter study to investigate potential risk factors for mrsa among children with cf 1-17 years of age. subjects are enrolled from 3 cf centers in the new york metropolitan area that have a mrsa prevalence ranging from 11% to 21%. cases are children with a positive respiratory tract culture for mrsa within the past 5 years. controls (2 age and center matched per case) are negative for mrsa. the aims of the study are: [1] to determine the point prevalence of anterior nares colonization with s. aureus in children with cf and their household members; [2] to assess potential risk factors for mrsa by administering a survey to the parents of subjects inquiring about factors such as crowding, pets, and participation in contact sports and by reviewing the medical record for antibiotic use, hospitalization, and surgery; [3] to determine potential transmission of s. aureus within families and centers by assessing the molecular epidemiology of strains using pulsed field gel electrophoresis and meca type of mrsa isolates. results: to date, 42 cf subjects (12 cases, 30 controls), mean age 8.9 years, and 100 household members (1 to 5 per subject) have been enrolled. preliminary data demonstrate positive anterior nares cultures among cases, controls, and household members to be 7/12 (58%), 7/30 (23%), and 24/100 (24%), respectively. of the 38 s. aureus isolates, 27 were mssa and 11 were mrsa (5 cases, 3 household members of cases, and 3 household members of controls). during the past 6 months, 4 household members have been hospitalized and one has stayed overnight in a nursing home; 4 had staphylococcal infections and 2 had skin infections. data collection and molecular analysis are ongoing. conclusions: we expect this study to provide insight into risk factors for respiratory tract colonization/ infection with mrsa, the role of ca-mrsa in cf, and potential strategies to prevent mrsa. aims: 1. revise infection control guidelines for the institution 2. establish outpatient infection control policies consistent with those used for hospital inpatients 3. cohort young and recently diagnosed cf patients without pseudomonas or mrsa to clinic days separate from colonized patients 4. establish a protocol for eradication of mrsa in newly colonized patients. methods: working with representatives from hospital epidemiology, the cf center, inpatient units, outpatient clinics, respiratory therapy, and cf families, a uniform infection control policy for cf patients in all settings (inpatient, outpatient, home care) was drafted and revised. arrangements were made to cohort newly diagnosed patients and children <5 years free of pseudomonas and mrsa to separate clinic days from colonized patients. review of the 41 mrsa positive patients in our pediatric population, for whom data was available, showed median age of acquisition of 8 years (range 1 mo to 18 yrs). for 3 patients the first culture obtained at diagnosis or transfer from another center was mrsa positive (1 mo, 1 yr, 4 yr). an eradication protocol for newly acquired mrsa has been developed involving treatment with oral and topical antibiotics and phisohex or clorox washes for 5 days, plus changing disposable respiratory care equipment at the beginning and end of the treatment period. surveillance cultures of nose, groin, and airway or sputum are done after completion of the treatment protocol and re-treatment initiated if still positive for mrsa. conclusion: using a three-tiered approach of infection control guidelines, cohorting, and mrsa eradication protocol we expect to decrease our overall rate of mrsa and protect our newly diagnosed and young cf patients from acquisition. objective: studies have shown that respiratory pathogens can be transmitted within cf centers. in a recent cross-sectional study at 7 cf centers, we assessed the rate of bacterial shedding by cf patients during office visits and noted that 6% of patients carried respiratory flora on their hands. the current study was performed to assess the effectiveness of alcohol-based rubs on hand carriage of respiratory pathogens by patients during the course of office visits. methods: four bacterial organisms were chosen for study: pseudomonas aeruginosa (pa), staphylococcus aureus (sa) [including methicillin-resistant (mrsa) strains], stenotrophomonas maltophilia (sm) and burkholderia cepacia complex (bcc). at the beginning of clinic visits, hands of study patients were cultured using the "glove-juice" technique (gjt) [1] . hand hygiene was then performed using an alcohol-based rub. at the end of the visit, hands were again sampled using the gjt. samples were sent to the microbiology laboratory at dartmouth-hitchcock medical center for culture and identification of study organisms. hand and respiratory tract isolates were compared using pulsed field gel electrophoresis (pfge). recovery experiments were also performed to assess the sensitivity of the gjt. results: samples were collected from 100 encounters (50 adult; 50 pediatric). median encounter time was 72 min (range 60.5-170.0 min). recovery experiments determined that the level of detection for each study organism using the gjt was ~10 2 colony forming units. confirmation of matches between hand and respiratory isolates was determined by pfge. the number of patients with study organisms recovered from the respiratory tract was: 38 pa, 31 sa, 21 mrsa, 8 sm, and 2 bcc. the overall hand contamination rate with a matched respiratory tract isolate at either culturing time was 14% (95% ci, 8.6-22.2%). before use of the alcohol rub, hand contamination was 9.0% (95% ci, 4.9-16.2%). in those patients with hand contamination by respiratory pathogens prior to alcohol rub, 50.0% (95% ci, 23-77%) had negative cultures at the end of the clinic visit. however, there was strong evidence of hand contamination during the encounter, despite hand hygiene, as the overall rate at the end of visits was 8.0% (95% ci, 4.2-15.0%). there was a trend toward increased hand contamination in those suffering from pulmonary exacerbations versus clinically stable patients (28.6% [95% ci, 11.8-55%] vs. 11.6% [95% ci, 6.5-20%], p=0.10). conclusions: contamination of patient hands by respiratory tract pathogens is observed in the outpatient setting in approximately 14% of patients. a single application of an alcohol-based hand rub, while safe and inexpensive, has limited antiseptic effectiveness during the full course of a typical cf clinic visit. repeated use of alcohol-based hand rubs during outpatient encounters would likely be more effective. these data support recent recommendations in the cf foundation consensus guidelines for infection control [2] (nthi) is the most common initial bacterium infecting the airways in cystic fibrosis (cf). nthi infection usually precedes pseudomonas aeruginosa infection in many chronic lung diseases, including cf, emphysema, diffuse panbronchiolitis, and immotile cilia syndrome. it is hypothesized that nthi acts as a gateway organism paving the way for subsequent infection with p. aeruginosa, however, the mechanism by which this occurs remains poorly understood. we used a novel co-culture model of persistent nthi biofilm infection on airway epithelial cultures to study epithelial immune responses following nthi biofilm infections. objective: we hypothesized that prolonged exposure to nthi biofilms causes airway epithelia to become tolerant to inflammatory stimuli and show subsequent decreased innate immune responses. methods: nthi were inoculated onto the apical surface of calu3 air-liquid interface epithelia and grown in co-culture. rna from 3 sets of paired airway epithelial cultures at 4, 24, 48 and 96 hours both with and without nthi inoculation was isolated and hybridized on custom affymetrix chips. data were normalized with rma and data biases were removed using the mixed model anova method. differentially expressed gene analyses were performed using anova, and pathways analyses were performed using gene set enrichment analysis (gsea). to test the hypothesis that prolonged nthi infection induces tolerance, epithelial cultures were pre-treated with either 4 days of nthi or 4 days of pbs before stimulation with pbs, il1β (100 ng/ml) or 10 6th cfus of live p. aeruginosa before subsequent measurement of epithelial responses. results: both rma and gsea analyses of microarray data showed many innate immune and pro-inflammatory responses at early timepoints that largely returned to baseline after 4 days of co-culture, despite an increasing apical nthi biofilm infection. stimulation with il1β or p. aeruginosa after 4 days of nthi infection resulted in significantly decreased il8 production compared to uninfected controls. nthi treated cultures subsequently infected with p. aeruginosa also displayed increased tight junction integrity and resistance to cell culture death. conclusions: persistent nthi infections on airway epithelia resulted in increased tolerance to bacteria and inflammatory stimuli and increased resistance to bacterial toxicity. airway epithelial tolerance may contribute to a failure to clear subsequent bacterial infections. these phenomena would help to explain how early infections with nthi may promote chronic bacterial infections and p. aeruginosa acquisition in cf and other nthi related airway diseases. the colonization of cf airways by p. aeruginosa leads to intractable and persistent lung infections that resist permanent eradication by antibiotics. the lack of efficacy of current therapies is believed to be due to the formation of p. aeruginosa antibiotic resistant biofilms in the cf airways. however, little is known about biofilm formation on human airway epithelial cells. thus, we designed a continuous flow, live cell imaging system to grow p. aeruginosa biofilms on glass or on the apical surface of polarized human cells. cfbe41ohuman airway epithelial cells homozygous for the ∆f508 mutation (cfbe), and cfbe41o-cells complemented with wt-cftr (cfbe+wt-cftr) were grown as polarized monolayers at an air-liquid interface and gfp-labeled p. aeruginosa were applied to the apical surface of the cells. after 6 hours biofilms did not form on glass but large bacterial macrocolonies developed on cfbe cells. surprisingly, biofilms also developed on cfbe+wt-cftr cells: however, p. aeruginosa biomass on cfbe+wt-cftr cells was significantly reduced compared to cfbe cells (0.60±0.12 µm 3 /µm 2 vs 1.58±0.20 µm 3 /µm 2 ). the minimal bactericidal concentration for tobramycin was >10,000 µg/ml for p. aeruginosa grown on cfbe and cfbe+wt-cftr cells. these results explain why tobramycin given clinically, which is~1,000 µg/ml in bronchoalveolar fluid collected from cf patients, fails to eradicate the persistent infection by p. aeruginosa. p. aeruginosa also formed biofilms on human bronchial epithelial cells, which produce mucus. indeed, biomass was similar on cfbe+wt-cftr cells, which do not produce mucus, and human bronchial epithelial cells. to determine if human airway epithelial cells secrete factors that facilitate biofilm formation, conditioned medium was collected from cfbe and cfbe+wt-cftr cells and applied to p. aeruginosa grown on glass. under these conditions, biofilm formation was dramatically facilitated, with a biomass of 0.02 ± 0.01 µm 3 /µm 2 when p. aeruginosa was grown in conditioned medium collected from cfbe+wt-cftr cells and 0.11 ± 0.02 µm 3 /µm 2 when grown in conditioned medium collected from cfbe cells. the secreted factor(s) were greater than 5 kda, as assessed by size exclusion. our results suggest that human airway epithelial cells secrete factor(s) that facilitate biofilm formation by p. aeruginosa, in the presence and absence of mucus, and that secretions from cf cells are more effective in promoting biofilms than secretions from non-cf cells. the ability of non-cf cells to facilitate biofilm formation appears to contradict the observation that non-cf lungs do not harbor p. aeruginosa. we propose that the innate immune response in the non-cf lung effectively eliminates p. aeruginosa from the lung, thus, biofilms do not form even thought the airway epithelial cells secrete factors that have the potential to facilitate biofilm formation. however, in cf when mucociliary clearance is compromised, p. aeruginosa accumulates in the lung and secretions by cf airway epithelial cells greatly enhances the formation of drug resistant biofilms (supported by the nih (ro1 ai51360, ro1-hl-074175, p20-rr018787, t32-dk-07301), and the cff (stanto97ro, anders06fo). ma, l. 1 ; parsek, m.r. 2 ; wozniak, d.j. 1 1. microbiology and immunology, wake forest university health science, winstonsalem, nc, usa; 2. university of washington, seattle, nc, usa bacteria in natural, industrial and clinical settings live in surface-associated communities termed biofilms, which are the source of persistent infection and resistance to antimicrobial treatment. biofilm development is initiated by the attachment of planktonic cells to a surface, followed by formation of microcolnies, and finally disperses swimming cells from microcolnies to occupy a new surface. to maintain the community structure, bacteria in a biofilm are usually enmeshed in an extracellular polymeric matrix, which consists of nucleic acids, proteins, and polysaccharides. exopolysaccharides (eps) have been known as a component of the biofilm matrix for years. however, little is know about how the eps matrix forms and develops and whether an eps can form a matrix independently. in the present report, we use lectins that specifically detect the mannose or galactose structure in pseudomonas aeruginosa psl eps. this technique allows us to visualize psl eps on the bacteria cell surface and in the biofilm matrix. our results indicate that psl eps is likely anchored on the cell surface in a helical pattern and clearly forms a matrix, which holds bacteria in the biofilm and on the surface. in a flat multiple-layer biofilm, psl eps is equally distributed in the entire biofilm. however, microcolonies reveal peripheral staining of psl eps with minimal staining of matrix in the center of the microcolonies. instead, this region has swimming cells indicating a biofilm development stage prior to dispersion. more strikingly, this area also has concentrated dead cells and/or extracellular dna, which fills up the viod spaces in the lower center of microcolonies at the stage prior to dispersion. these data provides a plausible mechanism for how p. aeruginosa sacrifices a portion of cells to make void spaces and free another portion of cells for future dispersion. in addition, our data also show that psl eps matrix and dna matrix are not overlapping, which suggests that these two components of the matrix work coordinately to encase bacteria in the biofilm. finally we show that the psl eps matrix is present in biofilms formed by mucoid p. aeruginosa, and formation of the psl eps matrix is independent of the production of alginate and pel, two eps that also contribute to p. aeruginosa biofilm formation. overall, our data indicates that psl eps functions as a primary scaffold, holding biofilm cells together in the matrix. moreover, our data along with published literature suggest a possible model for how p. aeruginosa biofilms persist in cystic fibrosis patients. 1 1. clinical microbiology, rigshospitalet, copenhagen, denmark; 2. biocentrum, technical university of denmark, kgs. lyngby, denmark; 3. department of paediatrics, copenhagen cystic fibrosis centre, rigshospitalet, copenhagen, denmark pseudomonas aeruginosa predominates chronic lung infections in patients with cystic fibrosis (cf). a hallmark of chronic p. aeruginosa lung infections in cf patients is the presence of mucoid p. aeruginosa biofilms surrounded by numerous polymorphonuclear leukocytes (pmns) in the lower airways. how p. aeruginosa escapes the bactericidal pmns is not fully understood. several virulence factors of p. aeruginosa is controlled by quorum sensing (qs); a density dependent mode of inter-bacterial communication based on signal transmitter molecules. active qs is present during chronic p. aeruginosa lung infections in cf patients and we have previously demonstrated a qs-regulated tolerance of biofilm bacteria to the antimicrobial properties of the pmns. the precise qs-regulated effect on the pmns is, however, unknown. in the present study, further elaboration using flow cytometry and microscopy revealed that qs-competent p. aeruginosa (pao1 and ∆rhli lasi complemented with c4-hsl and 3-oxo-c12-hsl) induces rapid necrosis of the pmns. this mechanism was also observed in in vitro biofilms and in mouse lungs infected with p. aeruginosa embedded in alginate. by using hplc fractionation the toxicity could be ascribed to a single substance. using lc-ms and 2d nmr this substance was identified as rhamnolipid b. in accordance, concentrations of rhamnolipid exceeding 200 µg/ml were found in the toxic supernatants from wild-type p. aeruginosa (pao1) whereas non-toxic supernatants from qs-deficient knock-out mutants (∆rhlr lasr and ∆rhli lasi) and from ∆rhla mutants contained low amounts of rhamnolipids approaching 0 µg/ml. our results demonstrate the potential of the qs system to facilitate infections with p. aeruginosa by utilizing rhamnolipid to disable a major first line of defense of the host -the pmns. furthermore, our study emphasizes the inhibition of qs as a target for the treatment of infections with p. aeruginosa. carlsson, m. 1,2 ; pettersson, a. 1 ; andersson, c. 1 ; wieslander, j. 4 ; eriksson, l. 3 ; segelmark, m. 2 ; hellmark, t. 2 1. dept of microbiology, immunology, and glycobiology, university of lund, lund, sweden; 2. department of nephrology, lund university, lund, sweden; 3. heart lung division, the cystic fibrosis center, lund university hospital, lund, sweden; 4. wieslab analys ab, lund, sweden the clinical consequence of chronic pseudomonas (p) aeruginosa colonization in cystic fibrosis (cf) varies between individuals for unknown reasons. auto-antibodies against bactericidal/ permeability increasing protein (bpi-anca) are associated with poor prognosis in cf. we hypothesize that there is a correlation between the presence of bpi-anca, the biological properties of the colonizing bacteria and the clinical conditions of the hosts. we have compared isolates of p aeruginosa from two groups of cf patients: one with positive serum levels of bpi-anca and deteriorating lung disease, and one with negative bpi-anca levels and stable clinical conditions. epithelial cells (a549) and isolated polymorphonuclear granulocytes (pmns) were stimulated with the clinical isolates and cell death was analyzed with flow cytometry. interleukin-8 (il-8) released into the supernatant was measured by elisa. we found that the anca associated strains in most cases showed a pyocyanin negative phenotype. these strains also induced less inflammatory response than the non-anca associated strains as shown by the number of necrotic cells and il-8 release, yet elevated compared to control. we conclude that colonization with strains of p aeruginosa that induce a weak inflammatory response is associated with unfavourable outcome in cf. we speculate that inadequate control of pathogen proliferation through an insufficient inflammatory response results in a slowly increasing number of bacteria and accumulation of dying pmns in the airways, contributing to the progressive lung disease seen in many cf patients. 2 1. pulmonary critical care, northwestern university, chicago, il, usa; 2. microbiology/immunology, northwestern university, chicago, il, usa; 3. pulmonary medicine, children's memorial hospital, chicago, il, usa; 4. pediatrics, children's memorial hospital, chicago, il, usa purpose: most cystic fibrosis (cf) patients are infected with pseudomonas aeruginosa (pa) and have progressive loss of lung function. among individual patients, however, there are marked differences in rates of lung function deterioration. although the reasons for this variability are not completely understood, it is likely that microbiological factors play a role. type iii secretion in pseudomonas aeruginosa (pa) isolates from non-cf patients have been associated with poorer outcome. the aim of this study was to determine whether there is an association between type iii secretion properties and deterioration in lung function in cf. methods: we prospectively enrolled cf children and adults over 3 years. demographics, clinical characteristics, spirometry and a respiratory culture were obtained at the first visit. the age at time of first postive pa culture was also determined. subsequently, spirometry and respiratory cultures were obtained and clinical characteristics recorded every 6 months until december 2006. from each sputum culture 5 individual isolates were selected for evaluation and type iii secretion was evaluated for each isolate by western blot analysis. the children were subcohorted into those newly infected (1st positive pa culture) or those chronically infected (minimum 2 sputum pa (+) >1 year duration). results: there were 100 patients evaluated, of which 31 were adults and 69 were children. ninety percent of our population cohort was caucasian and 53% were female. overall 63% of the patients were on tobi and 72% on macrolides chronically. the mean age at time of a first pa positive culture was 11 years for the chronically infected subjects(adults and children) and 6 years for the 1st time infected subcohort. a total of 2409 sputum samples were collected for the current study. the prevalence of type iii secretion properties for the overall population was 21.11%. foe adults, 73 of 807 (9.7%) pa isolates were type iii secretion positive compared to 346 of 1602 isolates (26.8%) in chronically infected children and 48 of 90 isolates(53.3%) from newly infected children. at study entry the mean fev1 % predicted was 68 +/-24 for the whole cohort. for the adult subcohort the fev1% predicted was 60% +/-26%, for chronically infected children it was 72% +/-21% and newly infected children is was 82%+/-22%. the average annual rate of fev1 % change was -0.67% for adults, -2.42% for chronically infected children and +4.62 for newly infected children. analysis on the relationship between type iii secretion pa isolates and fev1% decline is ongoing and will be presented at nacf. conclusion: prevalence of type iii secretion in pa isolates from cf patients decreases with age. there is a slower decline in fev1% in chronically pa infected adults compared to chronically pa infected children. the relationship of type iii secretion to fev1 % change and pulmonary exacerbations will be presented at nacf. a limited number of bacterial species -in example pseudomonas aeruginosa, staphylococcus aureus, burkholderia cepacia complex, stenotrophomonas maltophilia -is typically found in the cf lung. these facultative anaerobic bacteria exhibit the capability to switch from aerobic to anaerobic metabolism, which enables them to survive in the hypoxic environment in the cf lung [1] . absence of oxygen would also favour the growth of fastidious anaerobes and, indeed, sequencing of 16s rrna resulted in the detection of strict anaerobic species in cf sputum [2] . thus, we identified and quantified strict anaerobes in cf sputum using conventional anaerobic microbiological methods, examined the recovery rate of identical species in sputum samples from the same patients, and determined the antibiotic susceptibilities. 92 sputum samples were collected from 8 cf children and 31 cf adults (mean age 25.6 ± 8.5 yrs). samples were incubated aerobically on columbia agar, supplemented with 10% sheep blood, and anaerobically on brain heart infusion agar and schaedler agar supplemented with 5% mutton blood, for up to 7 days. bacteria were identified (rapid ana ii identification system, remel, lenexa, ks) and cfu's were determined by dilution plate counting. fastidious anaerobes (141 strains) were submitted to e-test ® susceptibility testing (ab biodisk, solna, sweden) using the anaerobically active antibiotics ceftazidime, clindamycin, meropenem, metronidazole, and piperacillin/tazobactam. in 75.0% of the patients, the following strict anaerobes were detected with a mean of 6.3x10 6 cfu/ml (range 2.5x10 4 to 2.5x10 7 cfu/ml): peptostreptococcus spp., clostridium spp., actinomyces spp., prevotella spp., wolinella spp., propionibacterium spp., streptococcus spp., lactobacillus spp., gemella spp., bacteroides spp. and eubacterium spp., whereas p. aeruginosa, s. aureus and b. cepacia revealed 7.1x10 7 cfu/ml (range 5.0x10 5 to 3.5x10 8 cfu/ml). in 30 sputum samples contaminated with p. aeruginosa together with strict anaerobes 5.2x10 7 ± 7.8x10 7 cfu/ml p. aeruginosa were counted, whereas in 9 samples with p. aeruginosa without strict anaerobes only 1.7x10 7 ± 1.8x10 7 cfu/ml were found (p=0.03). for s. aureus, no significant difference was observed. identical strict anaerobic species were detected in 12 out of 19 patients with two or more repeated sputum samples (63%). e-test® sensitivity testing for strict anaerobes yielded high sensitivity for meropenem (only 5.7% resistant strains), piperacillin/tazobactam (20.6%), and clindamycin (24.8%), but not ceftazidime (49.0%) or metronidazole (50.4%). high numbers of strict anaerobes are present in the majority of cf patients. possibly, strict anaerobes promote growth of p. aeruginosa but not s. aureus. the high persistence of identical anaerobic strains reflects chronic lung infection and may be caused by their increased resistence against standard antibiotics such as ceftazidime. [ in sputum in the cf lungs bacteria such as pseudomonas aeruginosa have to metabolize anaerobically [1] . for anaerobic energy generation, p. aeruginosa can use nitrate and arginine, but also pyruvate which is produced from glucose via anaerobic glycolysis [2] and can be metabolized to lactate and vice versa. in order to investigate if p. aeruginosa may benefit from externally produced lactate, we measured the concentration of lactate in p. aeruginosa, staphylococcus aureus, burkholderia cenocepacia and polymorphonuclear neutrophils in vitro and in cf sputum. in sputum samples of 25 cf patients and in neutrophils (3 x 10 7 /ml) from healthy donors lactate concentrations were determined. in addition, p. aeruginosa (starting with 1 x 10 7 cfu/ml in tryptone soy broth), s. aureus (3 x 10 6 cfu/ml), and b. cenocepacia (8 x 10 6 cfu/ml)were grown aerobically (0 through 24 hrs) and anaerobically (1 through 4 days). l-lactate was measured spectrophotometrically (detection limit: 0.1 mmol/l), and total lactate gaschromatographically. aerobic and anaerobic gene expression of p. aeruginosa strain pao1 was determined using affymetrix® microarrays. lactate concentrations in cf sputum amounted to 3.0 ± 3.1 mmol/l (range 0.2 to 14.1 mmol/l). concentrations were similar in sputum samples colonized with p. aeruginosa, s. aureus (3.3 ± 3.7 vs. 2.8 ± 2.3 mmol/l, p=0.67) and b. cenocepacia (1.9 mmol/l). neutrophils produced 3.2 mmol/l. in all samples exclusively l-lactate was found. during in vitro experiments, p. aeruginosa did not generate any lactate at all, neither aerobically nor anaerobically. in contrast, anaerobically grown s. aureus produced up to 2.8 mmol/l lactate, and b. cenocepacia up to 10.0 mmol/l. a p. aeruginosa suspension [1x10 7 cfu/ml] spiked with 10 mmol/l l-lactate did not change its concentration, indicating that p. aeruginosa does not metabolize lactate. similar results were obtained in our gene chip experiments: after three days of anaerobic growth, the genes encoding for the lactate dehydrogenases were downregulated (pa0927 ldha -10.78 fold, pa2382 llda -2.81 fold) or unchanged (pa4771 lldd 1.2 fold). in contrast, the genes encoding for pyruvate decomposition to acetyl coa (pa3416 and pa3417, both encoding for pyruvate dehygrogenase e1 components) were upregulated by 208 and 47 fold, respectively. we could demonstrate that p. aeruginosa does not benefit from externally produced lactate. we confirmed the important role of pyruvate metabolism for anaerobic p. aeruginosa energy generation. whether lactate production of neutrophils, s. aureus or b. cenocepacia contributes to cf lung pathophysiology still remains to be investigated. references: [1] macleod, d. 1 ; barker, l. 1 ; gurgel, j. 1 ; kenney, t. 1 ; burns, j. 2 ; baker, w. 1 1. gilead sciences, inc., seattle, wa, usa; 2. university of washington, seattle, wa, usa antibiotic resistance may severely limit therapeutic options in individuals with cystic fibrosis (cf) or bronchiectasis. because of frequent antibiotic treatment courses, resistance continues to emerge, even to newer agents. treatment with multiple antibiotics in a single aerosol formulation may be a promising approach to slow development of resistance. fosfomycin is a phosphonic acid antibiotic that is bactericidal against both gram positive and gram negative organisms. fosfomycin inhibits the first committed step in the synthesis of peptidoglycan, suggesting cross resistance to other cell wall acting antibiotics will not occur. the aminoglycoside tobramycin is one of the most commonly used antimicrobials in cf, with potent activity against gram negative bacteria and the majority of staphylococcus aureus isolates. a 4:1 (wt/wt) fixed combination of fosfomycin:tobramycin (gs-9310/11) was used to determine the in vitro susceptibilities of a panel of respiratory pathogens: cf pseudomonas aeruginosa (100), s. aureus (16), haemophilus influenzae (16), stenotrophomonas maltophilia (17) and burkholderia cepacia complex (20), including minimal inhibitory concentration (mic) and time-kill experiments in the absence and presence of 2% porcine mucin. synergy was evaluated using the checkerboard method, and spontaneous resistance mutation frequencies were determined in antibiotic-containing agar (4x, 8x and 16x mic). in vivo drug efficacy was examined using a rat agar bead pneumonia model of either p. aeruginosa or s. aureus. all experiments compared gs-9310/11 to fosfomycin and tobramycin as single agents. gs-9310/11 had a lower mic 90 than tobramycin for the s. aureus strains, 75% of which were methicillin resistant (mrsa). for p. aeruginosa, gs-9310/11 had a lower mic 50 and mic 90 than fosfomycin alone, but tobramycin was more active than either. for h. influenzae and s. maltophilia, gs-9310/11, fosfomycin and tobramycin had similar mic 50 and mic 90 . b. cepacia complex were resistant to all three drugs. results in the presence of mucin were similar. time-kill studies showed a more rapid and prolonged killing of s. aureus and p. aeruginosa by gs-9310/11 compared with either agent alone at the same drug concentrations. gs-9310/11 was bactericidal and exhibited concentration-dependent killing. synergy studies showed no antagonism between fosfomycin and tobramycin, and the majority of p. aeruginosa and all of the s. aureus tested demonstrated indifference for the combination. at 4x mic concentrations the mutation frequency of gs-9310/11 was at least 2-3 logs lower than tobramycin and 2-6 logs lower than fosfomycin alone for s. aureus. for p. aeruginosa the mutation frequency of gs-9310/11 was 2-3 logs lower than fosfomycin and 1-2 logs lower than tobramycin. in the rat pneumonia model, gs-9310/11 and tobramycin alone demonstrated bactericidal killing of p. aeruginosa; both were more active than fosfomycin alone. in vivo killing of s. aureus by gs-9310/11 was also demonstrated. gs-9310/11 appears to have advantages over single agents for the treatment of both gram positive and gram negative bacterial lung infections in cf and bronchiectasis. britton, l.j. 1 antibiotic resistance is becoming a major problem in the treatment of pulmonary exacerbations in cystic fibrosis (cf). organ-isms that are resistant to multiple antibiotics infect the airways of an estimated 25-45% of adults with cf.1 as a result of the growing resistance in cf patients, many centers have been performing synergy testing of sputum cultures in addition to the conventional culture and sensitivity testing. the purpose of our investigation was to determine if antibiotic synergy studies and the use of synergistic combinations of antibiotics improve therapeutic outcomes in cystic fibrosis patients with acute pulmonary exacerbations. methods: this study is a retrospective chart review of cf patients who had antibiotic synergy testing performed while hospitalized for respiratory tract infections. eligibility criteria included cf patients hospitalized with respiratory tract infections from 1998 to 2005. laboratory data from each hospital admission was reviewed for synergy among those antibiotics commonly used against pseudomonas aeruginosa. a review of medical charts ascertained each patient's pulmonary function (determined by fev1 before and after antibiotic therapy), weight z-score, organism cultured from sputum sample, time to next hospital admission, and the antibiotic(s) actually prescribed. primary endpoints were determined to be the change in fev1 and the time to next admission. results: four hundred seventy-five hospital admissions were analyzed. a total of 48 cystic fibrosis patients, age birth to 36 years, were included in the study. patients receiving antibiotic synergy experienced a significant decrease in mean time to next admission (50 days with synergy vs. 68 days without synergy, p=0.004). no significant difference was found in the change in fev1 before or after antibiotic therapy, with an increase of 8.72% with synergy vs. 9.28% without synergy (p=0.585). patients infected with non-mucoid p. aeruginosa experienced 60 days to the next hospital admission, while patients infected with mucoid p. aeruginosa experienced 56 days to the next hospital admission (p = 0.45). no statistically significant difference was observed between the synergy and non-synergy groups in regards to nutritional status and lung function prior to antibiotic therapy. speculations: antibiotic resistance in cystic fibrosis patients increases the morbidity and mortality caused by this disorder with each exacerbation the patient experiences. synergistic antibiotic therapies should improve patient outcomes through more efficient bacterial eradication and increased time to next hospital admission; however, we were unable to substantiate this assumption based on the results from this study. synergistic antibiotics did not show an improvement in the therapeutic outcomes of days to next admission or change in fev1. background: expectorated sputum (es) technique is currently the most frequently used method for routine assessment of lower airway infection in patients with cystic fibrosis (cf). induced sputum (is) using hypertonic saline (hs) has been successfully used in cf patients unable to produce sputum spontaneously, but only limited data are available comparing the diagnostic yield of expectorated versus induced sputum in cf patients. while ultrasonic nebuliser have been used in the majority of studies, new high output jet nebulisers may offer a suitable alternative technique to induce sputum expectoration. aim: to assess the feasibility of sputum induction using a pari e-flow nebulizer and to compare the diagnostic yield of is and es in children with cf . methods: this is a preliminary report of an ongoing study in routine clinical care in sputum producing children with cf. es is being obtained before sputum induction. subsequently, sputum induction is performed using stepwise inhalation of nebulized 5 ml of 3%, 4% and 5% hypertonic saline with an e-flow nebulizer (pari, starnberg, germany). lung function is assessed by portable spirometry before the procedure and after inhalation of each saline concentration. results: so far 40 cf patients (18 females) with a mean age of 13.6 years (range 8 to 18 years) and fev1 between 38-121% predicted (mean 82%) have been included in the study. all subjects provided es samples and all produced sputum after induction. discrepancies in cultures between es and is samples were seen in 9 cases (22.5%). in 6 cases additional cf pathogens were found in is samples, whereas in 2 cases es yielded additional organisms compared to is. the number of distinct pathogens was similar in the remaining patient, but different bacteria were found with the 2 techniques. in 4 cases p. aeruginosa was detected only with one of the 2 techniques (2 es versus 2 is). the spectrum of side effects was similar to previous reports using other nebulizer systems, with throat irritation being the most common adverse event of the is technique. all patients who had symptoms during the procedure became asymptomatic at the time of discharge from the clinic 20-30 minutes after the procedure. 4 patients did not finish all 3 cycles of sputum induction procedure due to symptoms of shortness of breath and/or drop in fev1>20% from baseline (in 3 patients); all reversed after salbutamol inhalation. vomiting occurred in one patient. 3 patients refused to complete the procedure due to unpleasant taste and/or throat irritation. conclusion: these preliminary results show discrepancies between expectorated sputum and induced sputum cultures in a significant proportion of cf patients. this may be explained by the previously described regional differences in lower airway infection in cf airways rather than by a higher diagnostic yield of one of the techniques. the results also demonstrate that the e-flow system is an efficient and safe device for sputum induction in sputum producing children with cf. seidler, m.j.; salvenmoser, s.; müller, f.c. dept. pediatrics iii, university heidelberg, pediatric pulmonology, cystic fibrosis centre & infectious diseases, heidelberg, germany background: the preferred growth form of bacteria is a biofilm. s. aureus, h. influenzae, and p. aeruginosa can produce an extracellular matrix (ecm) with implications in cystic fibrosis (cf) lung disease. the biofilm can protect against host defenses and antimicrobials. a. fumigatus is a frequent colonizer of the cf respiratory tract and can cause allergic bronchopulmonary aspergillosis (abpa). while antifungals in vitro are active against a. fumigatus, in vivo antifungal therapy is often complicated or resistance is observable. the aim of this study was to investigate the ability of a. fumigatus to form a biofilm-like matrix in vitro on polystyrene (ps), human bronchial epithelia cells (16hbe) and human bronchial epithelia cells with f508del/f508del (cfbe41o-). methods: a. fumigatus atcc #9197 was incubated in rpmi at different ph and concentrations of fbs. temperature, production time and different flow conditions were varied on ps, 16hbe and cfbe41o-. dry weight measurement and antifungal drug susceptibility testing was performed. scanning electron microscopy (sem) and confocal scanning laser microscopy (cslm) images were analyzed. results: the thickest biofilm was produced on ps with rpmi (+10% fbs, ph=6.5) at 35°c for 72h slightly rocking. biofilm dry weight on ps was 2.2 mg after 4 h and 8.3 mg after 72 h. the dry weight of produced biofilm exceeded 7.4 mg on 16hbe and 7.7 mg on cfbe41o-cells after 72h of biofilm production. there was no significant difference in dry weight increase between the cell lines and ps. planktonic a. fumigatus was susceptible to itraconazole (0.125µg/ml), voriconazole (0.063µg/ml) and amphotericin b (1µg/ml). aspergillus in biofilm was resistant against all drugs (>32µg/ml). the sem pictures displayed a network of hyphal structures and matrix at 12h. characteristic flow channels were observed at 72h. cslm images displayed conidia and hyphal structures embedded in matrix formations. a-alexafluor dyed polysaccharides of the cell wall and of the ecm in the biofilm. three dimensional constructs of the cslm pictures displayed biofilm on 16hbe and proofed viability of the cells after 48h co-incubation. differences in biofilm production between 16hbe and cfbe41o-were not significant. conclusions: a. fumigatus is able to form a biofilm structure in vitro on ps, 16hbe and cfbe41o-. a biofilm-like matrix produced by a. fumigatus was evidenced by dry weight measurement, sem, cslm and antifungal drug resist-ance in comparison to planktonic cells. potential clinical implications of a. fumigatus biofilm formation in vivo require further attention and investigations. etherington, c. 1 ; peckham, d. 1 ; conway, s. 1 ; hall, m. 2 ; denton, m. 2 1. seacroft hospital, regional adult cf unit, leeds, united kingdom; 2. microbiology department, leeds teaching hospitals, leeds, united kingdom susceptibility testing results are not predictive of clinical response to antibiotic therapy in chronic pseudomonas aeruginosa infections in cystic fibrosis. we assessed the impact of reducing the number of routine susceptibility tests performed on clinical outcome in these cases. in june 2006 we introduced a protocol of limiting susceptibility tests to p. aeruginosa isolates obtained from respiratory samples taken at the commencement of antibiotic therapy, when there was evidence of clinical failure, or routinely if not tested in the previous three months. at all other times, isolates were identified and reported as normal but p. aeruginosa isolates were not subjected to susceptibility tests. between 1st june and 30th november 2006 p. aeruginosa, was isolated on at least one occasion from 193 patients attending our adult cf unit. in this six month period we reduced the number of susceptibility tests by 56% (from a projected 2,231 tests on 872 samples to an actual 972 tests on 427 samples). this resulted in projected savings of $4,777 in consumables and 170 hours (costed at $8,871) of laboratory staff time per annum, a total saving of $13,649 (£6,500) per annum. we assessed the response to intravenous antibiotic treatment between the study period in 2006 and the same period in 2005. no significant differences in median change of fev1, fvc, crp, white cell count, weight, or duration of intravenous antibiotics were observed. for cf units sending regular, routine sputum samples, a reduction in the number of susceptibility tests performed in cases of chronic p. aeruginosa can be carried out without impacting on clinical outcomes. we report our preliminary results for a total of 233 strains obtained from 112 patients (85 patients harbored more than 2 strains). these patients were classified in 3 groups of respiratory insufficiency according to their fev1: severe in 31 patients (fev1<40%), moderate in 36 patients (fev1: 40-60%) and mild in 45 patients (fev1>60%). the clonal distribution was analyzed for the different strains of sa (1 to 6) isolated from patients sputum. these strains were analyzed for their antibiotic susceptibility and typed by pulsed-field electrophoresis gel (pfge) after smai digestion of chromosomal dna. thirty seven patients (33%) were colonized with mrsa. nine patients were both colonized with mrsa and mssa. among mrsa strains, 43/61 (70%) were also resistant to more than three other antibiotic family. strains harboring minor differences in the banding pattern (>80% similarity as assessed by the dice coefficient) were considered clonal. our results show that 83% of the patients were colonized with a single persistent strain during the year of follow-up. consecutive isolates with different pfge profiles were obtained from only 14/80 patients (17%). pfge analysis revealed that mrsa isolated from 27 patients were grouped in 8 clusters. these results revealed a possible clonal relationship between mrsa isolated from different patients with cf. we did not find any difference in the distribution of sa strains in our cf patients among the 3 groups of respiratory insufficiency. the study is ongoing in our adult cf population and in necker pediatric cf centre in paris. giusti, r. 1 ; furfaro, s. 2 1. pediatrics, long island college hospital, brooklyn, ny, usa; 2. research, lumina fund, new york, ny, usa palivizumab(synagis) is a humanized monoclonal antibody to rsv. the 2006 redbook acknowledges that some patients with cystic fibrosis may be at increased risk of rsv infection but that there is insufficient data to determine the effectiveness of this therapy. the objective of this study was to assess: 1)practice patterns of cf physicians in the us and canada 2)the severity of rsv disease in cf infants during the past rsv season. 3)if there is a standard of care concerning the use of synagis for cf infants. methods a questionnaire was developed using a web-based commercial vendor. an embedded web link was distributed via an e-mail sent to all us and canadian pediatric cf center directors. respondents clicked on a link to respond to the survey and results were automatically tabulated in real-time. completed responses were received from 84 center directors (73 us and 11 canadian) for a response rate of 63% in us and 44% in canada. most responders (70%) have prescribed synagis for infants with cf in the first rsv season, however only 38% routinely prescribed synagis for all infants with cf. only 35% expressed having had difficulty in obtaining insurance approval for this medication. many physicians indicated that synagis was frequently prescribed by the general pediatrician and that infants living at long distances from a cf center may be hospitalized at local hospitals. these issues may affect the accuracy of the data and result in an underestimate of actual synagis prescription and hospitalization rates. there were 456 infants diagnosed with cf in the past year and 172 of these infants were reported as symptomatic. there were 74 cf infants (16%) reported as having a documented rsv infection and 13 (18%) of these infants had received synagis. there were 31 infants (42%) with documented rsv infection who responded to outpatient management.there were no deaths but 40 patients were hospitalized and 8 of these had received synagis. of the 3 infants that required admission to an icu 2 had received synagis. of the 12 patients who were noted as having persistent chest x-ray changes 5 had received treatment with synagis. there were 36 infants with persistent wheezing and 13 of these infants had been treated with synagis. conclusions despite the limitations of a retrospective survey, this data demonstrates that rsv can cause significant and prolonged pulmonary disease and is a significant precipitating factor resulting in hospitalization of cf infants. the data also notes that many cf infants infected during the past rsv season have a mild illness and respond to outpatient management. a surprising finding of this survey is that infants at cf care centers where synagis was prescribed for all cf infants continued to have significant rsv related hospitalizations, persistent wheezing or prolonged chest x-ray abnormalities. there are different opinions among cf physicians concerning the routine use of synagis and currently the data suggests that there is not a standard of care concerning the prophylaxis of cf infants with synagis. this results of this survey should encourage physicians to prospectively study the efficacy of synagis prophylaxis in preventing hospitalization, persistent wheezing and chest x-ray abnormalities in cf infants. milani, a. 1 ; cisbani, g. 1 ; macchi, r. 1 ; vidal-aroca, f. 2 ; bertoni, g. 1 1. biomolecular sciences and biotechnology, university of milan, milano, italy; 2. basilea pharmaceutica ltd., basel, switzerland with ever increasing frequency, we now observe several examples of bacteria being resistant to every clinically available drug. therefore this urgently calls for the development of novel and improved antibiotics that may escape the extant mechanisms of bacterial resistance. one recent and promising development of the genome-wide search for target functions for antibiotics led to the identification of essential genes of pathogens by interfering antisense rnas. the first step is the construction of shotgun antisense libraries (sals) as follows. genomic dna is extracted from the bacterial strain of interest, fragmented by shearing into short pieces of dna, blunt-ended and cloned in an expression vector under the control of a regulatable promoter. the library is then reintroduced into the cognate bacterial strain and screened by replica plating colonies both in the presence and the absence of an inducer of the vector promoter. by this method, insert sequencing of clones showing conditional growth phenotypes is expected to lead to the identification of essential genes that can be silenced via antisense rna activity. we adopted this technology in order to generate a panel of essential functions of the cystic fibrosis-related opportunistic pathogen pseudomonas aeruginosa. so far, we tuned the protocol for sal generation in p. aeruginosa and identified a number of sequences conferring different levels of growth inhibition we are now characterizing these putative antisense rnas in order to define the minimal sequence able to cause the toxic effect and, on the other hand, to understand the cellular role of their targets. pseudomonas aeruginosa (pa) is an extremely versatile microbe with a vast array of pathogenic and metabolic mechanisms that allow it to form persistent infections in select patient populations, especially patients with cystic fibrosis. an ineffective immune response is considered partially to blame for failure of pa eradication. we have discovered that some strains of pa express peptidylarginine deiminase (padi) activity. padi is an enzyme that post-translationally modifies peptidylarginine to peptidylcitrulline with ammonia as a byproduct. our lab has shown that human padi can modulate the immune system through downregulation of tlr4 and ikk-gamma signaling. characterization of the pa padi will offer insights into a completely novel method of immune modulation by pseudomonas aeruginosa. using a widely published colorimetric assay for padi activity we have found the specific activity of crude pa cell lysates is very low. however this is similar to the only other known prokaryotic padi described in porphyromonas gingivalis. a protein homology search with the porphyromonas padi has revealed the likely genetic locus of pseudomonas padi in a 4.5 kb operon that appears in the genome of the pathogenic pseudomonas isolate pa14. this operon appears to contain two candidate genes for padi activity based on conserved motif searches (padi 1 and padi 2). both have been cloned into expression vectors, partially purified by affinity tag technology and tested in the colorimetric padi assay. curiously padi 1 autocitrullinates itself while padi 2 has not shown activity. reaction conditions and substrate specificities for pa-padi are not like either the human or porphyromonas padi. furthermore analysis of up to 64 clinically diverse strains has demonstrated 15% carry the gene for padi 1. this work is only the second description of padi activity in any prokaryote. pa padi could clearly have a dramatic impact on the local inflammatory milieu if it can access the same targets as human padi. the description of this activity in pseudomonas will advance our knowledge of the human-pathogen relationship and give insight into new therapies. which functions by translocating toxins into the cytoplasm of host cells. these toxins cause disease by damaging the surrounding host tissue, promoting dissemination of the organism and paralyzing the phagocytic mechanism of macrophages. pcrv is a factor required for the translocation of the toxins. the bases of these studies were to evaluate pcrv as a protective antigen in "p. aeruginosa" pneumonia and cldc as a vaccine adjuvant. methods: mice were vaccinated 1, 2, or 3 times with the pcrv antigen combined with either aluminum hydroxide (alum) or cldc by various routes of administration. efficacy of vaccination was evaluated by challenge with "p. aeruginosa" and evaluation of survival and/or measurement of various parameters associated with lung injury. results: increase in median survival time was highly significant when cldc/pcrv was compared with cldc or pcrv alone. following 2 subcutaneous administrations cldc/pcrv showed an increase in median survival time (58 hours versus 43 hours) and overall survival benefit following 3 intraperitoneal administrations (100% versus 80%). mice with anti-pcrv antibody levels above 10µg/ml were significantly protected. conclusion: the investigators establish the efficacy of cldc/pcrv vaccines via several parenteral routes of administration compared to no treatment as well as cldc and pcrv-only controls. differences were demonstrated between performance of cldc/pcrv and alum/pcrv in measures of lung injury, median survival, and overall survival. the results correlated with antibody levels and histological examination of the lung tissues. importantly, these studies indicate that protection can be achieved against "p. aeruginosa" infection by targeting an antigen associated with the type iii secretion system. background: there has been a recent increase in the number of reported cases of acute renal failure (arf) in cystic fibrosis (cf). our group have undertaken a national survey, which measured the incidence risk of arf in cf patients at between 4.6 and 10.5 cases / 10,000 cf patients / year. we have now conducted a case control study to determine which factors which are associated with an increased risk of arf. methods: in our initial survey we confirmed 24 cases of arf, in cf patients from 20 uk cf centres, presenting between 1997 & 2004. using the uk cf database, we identified sex and age (within 6 months) matched controls. informed consent was sought from the control patients, or their parents, for access to the case notes and clinical data were extracted. analysis of risk factors was by conditional logistic regression, using stata (version 9) and by fisher's exact test. results: there were 24 cases of arf (12 male, median age 10y, range 4m-32y) and 28 controls (17 male, 9y, 10m-32y). in the group of patients with arf, 21/24 had received an aminoglycoside at the time of their episode of arf or in the preceding week, compared with only 3 of the controls for the same time period (p<0.001). the median number of days of aminoglycoside in the year prior to the index case developing acute renal failure was 24 (7-99) for cases and 0 (0-47) for controls. conditional logistic regression showed that the odds ratio for arf per each day of aminoglycoside was 1.06 (95% ci 1.01 to 1.11, p=0.02 it is well-known that pa senses the environment and changes its phenotype. for instance, it produces greater amounts of the extracellular polysaccharide alginate in the cf lung, characterized by a microaerobic environment. little is known about the changes in protein secretion induced by oxygen limitation in pao1, the proto-typical pa laboratory strain. no data are available on this regard about pa clinical strains. our work was aimed to study the differential regulation of proteins secreted by pa strains grown in microaerobic or aerobic conditions. a pa clinical isolate and pao1 were grown overnight in aerobiosis and in microaerophilic conditions. the supernatants were collected and proteomic analysis was carried over by two-dimensional capillary chromatography -tandem mass spectrometry (mauri et al., faseb j 2005) to evaluate the differential protein expression. in the pa clinical isolate, we identified 66 proteins down-regulated and 42 proteins upregulated in aerobic conditions in comparison with microaerophilic culture while in pao1 16 proteins were down-regulated and 30 were up-regulated. 6 proteins were down-regulated and 12 up-regulated both in the clinical and in the laboratory strain. these proteins can mediate different biological functions since they are enzymes, heat shock proteins, chaperones, proteins involved in adaptation, motility and in the transport of small molecules. among all these proteins, as the alkaline metalloproteinase is associated with tissue invasion not only by causing rupture of epithelial tight-junctions but also by degrading several chemokines, we decided to validate its up-regulation observed in aerobic conditions by zymography. we found that the proteolytic activity of the supernatants of pa grown in aerobic conditions was higher than in microaerophilic culture both for the laboratory and clinical strain, indicating the functional relevance of data obtained by proteomic analysis. the identification of proteins differentially regulated in aerobiosis and oxygen limitated conditions in pa laboratory and clinical strains might be helpful for the knowledge of the mechanisms of colonization and lung damage due to pa in cf patients. for instance, the validation of the upregulation of the alkaline metalloproteinase in aerobic condition may shed light on these mechanisms of cf lung disease. further studies are in progress to evaluate the function of other bacterial exoproducts regulated in this model and to extend the analysis to other clinical strains. supported by the italian cystic fibrosis research foundation (ffc-grant#17/2006), comitato di vicenza dell'associazione veneta per la lotta contro la fibrosi cistica and azienda ospedaliera di verona, italy. cystic fibrosis (cf) sufferers are subject to repeated lung infections most commonly with the bacterium pseudomonas aeruginosa. in spite of antibiotic treatment p. aeruginosa tends to become established giving rise to persistent chronic infection. in the lung environment, the bacterium grows as a highly structured biofilm consisting of a complex community of cells embedded within a self-secreted polysaccharide matrix. investigations of p. aeruginosa biofilm growth using model strains have elucidated mechanisms which appear to govern the biofilm life-cycle. our research aims to test whether observation of these mechanisms in planktonic culture can be related to the efficiency of biofilm formation. biofilm initiation has been linked to cell motility and in particular to the presence of flagella and pili, which are thought to be important for cell attachment and the formation of microcolonies. on testing a large, genetically diverse group of clinical p. aeruginosa isolates retrieved from the lungs of cf patients we found no definitive correlation between the degree of motility of an isolate in planktonic culture and its ability to form a biofilm in vitro. the development of biofilm architecture has been demonstrated in model systems to be coordinated by the production and secretion of n-acylhomoserine lactone (ahl) quorum sensing molecules. however among the clinical isolates tested we observed no obvious correlation between the amount of ahls produced in planktonic culture and the extent of biofilm formation. overall we have found observation of phenotypic characteristics in planktonic culture to be poor predictors of efficient biofilm formation. a proteomics approach was adopted to provide further insight into the physiology of biofilm growth of p. aeruginosa isolates by comparison with planktonic growth of the same isolates. two genetically unrelated clinical isolates, demonstrated as being capable of efficient in vitro biofilm formation, were selected from our culture collection on the basis of their diverse phenotypic characteristics when cultured planktonically. one displays both twitching and swimming motilities, is mucoid and expresses ahls, while the other is non-motile, non-mucoid and no ahls have been detected in planktonic culture. we have developed a simple flow-through bioreactor to provide sufficient quantities of biofilm for proteomic analysis. gel-based and gel-free techniques were employed to study protein expression patterns for both biofilm and planktonic cultures of each isolate by mass spectrometry. proteins specific for each growth phase could be detected and may prove suitable biomarkers for monitoring the physiological status of biofilm forming strains when a larger bank of clinical isolates are examined. liposomal amikacin (arikace tm ) is a liposome-encapsulated form of amikacin that is formulated to treat chronic p. aeruginosa infections in cystic fibrosis patients. these liposomes carry a zwitterionic surface charge and are composed of lipids found naturally within the lung. a key aspect of the activity of the formulation is the ability to penetrate to the sites of pseudomonas biofilm-like growth in the lung. experiments were designed to investigate the penetration of liposomes into p. aeruginosa biofilms and in vitro activity. methods and results: model liposomes of the same size and lipid composition as liposomal amikacin (arikace tm ) were prepared with membrane-associated or encapsulated fluorescent labels, a hydrophobic carbocyanine dye and calcein, respectively. a mucoid strain of pseudomonas aeruginosa (pa3064) was used to establish biofilms in rectangular optical grade glass flow cells. biofilms were observed after four days of growth by confocal laser scanning microscopy using a focal plane set to view within the biofilm cluster or outside as a control. time dependent accumulation of fluorescent liposomes within the biofilms was measured by the spatial distribution of fluorescence intensity in regions within or outside of the biofilm. images indicated significant penetration of liposomes into the interior of biofilms under these conditions. the rate of penetration was considerably slower than typical rates for small molecules, consistent with the size of the liposomes. liposome concentrations were higher near the periphery than the interior. however, even the interior concentration was at least as high as the concentration of liposomes in the fluid outside of the biofilm, suggesting some binding or trapping of the liposomes within the biofilm. penetration of liposomes was observed under flow or static conditions. in a "washout" experiment, where medium is passed through the biofilms previously treated with liposomes, a significant portion of the liposomes remained associated with the biofilms for an extended period of time. the penetration of liposomes was reflected in the observation of killing of bacteria in colonies in the interior of agar beads. exposure of these cultures to liposomal amikacin resulted in a large reduction of viable bacteria throughout the beads as monitored by a fluorescent dna content assay. similar colony forming unit reductions in animal models (to be shown in other poster presentations) suggest that these principles also operate in vivo. liposomes similar to liposomal amikacin (arikace tm ) readily penetrate into biofilms of pseudomonas aeruginosa and may even have enhanced binding to biofilms. this binding along with localized release can explain the substantial efficacy observed in animal models. coates, a.l. 1 adherence to recommended therapy in cf has always been a challenge, in part, due to the time demands of the daily therapy. while twice daily inhaled tobramycin for those infected with pseudomonas aeruginosa (pa) has become an accepted standard of care, as much as 40 minutes a day may be consumed inhaling 300 mg in 5 ml of tobramycin (tobi ® ) from the pari lc plus ® breath enhanced jet nebulizer. the purpose of this study was to determine if equivalent levels of pulmonary deposition could be achieved in a much shorter time period using 1.5 ml of a more concentrated (100 mg/ml) tobramycin solution delivered by a perforated vibrating membrane nebulizer (eflow ® membrane configuration 35l) both, developed by pari pharma; germany. methods with a goal to study 7 children and 8 adults, to date, the subjects are 4 children 10 years and older and 2 adult males, all with an fev1 > 50% predicted, with stable cf. all were receiving inhaled tobramycin for positive sputum cultures of pa. following pretreatment with albuterol, they inhaled both preparations on two occasions with 99m tc-dtpa added to the tobramycin in the nuclear medicine facility. in vitro preliminary work demonstrated that the radiolabel tracked with both formulations of tobramycin. deposition was measured by a gamma camera taking both tissue attenuation and mucociliary clearance during nebulization into account (pediatric pulmonol suppl 29: a343; 2006) . in order to have a continuous rate of deposition, the pari lc plus ® was run for a timed 10 minutes and then both the total deposition and time of nebulization "scaled up" from in vitro testing when the nebulizer was run to dryness. this was done by multiplying the deposition by the total output when run to dryness divided by the total output in 10 minutes. (blood samples were taken for quantification of tobramycin in the serum but not yet analysed). the rate of output per minute was calculated from the 10 minute run and the total time was total output from in vitro testing divided by the rate of output. the eflow ® pro-vides a continuous output and stops automatically at dryness. quality assurance was the agreement between total radioactivity pre nebulization (in the nebulizer) and post which included the subject, the nebulizer, the connectors and the expiratory filter. the pari lc plus ® delivered 38.1±6.1 mg in 15.8±1.6 minutes compared to 42.7±10.2 mg in 4.2±1.2 minutes for the eflow ® . only the time of delivery was significantly different with p<0.0001(paired t-test). tolerability of the treatment was comparable for both inhalation regimes, but the shorter treatment was preferred by all patients. these results demonstrate the possibility of delivering equivalent levels of tobramycin in much shorter periods of time into the lungs of cf patients when using eflow ® , a very efficient electronic nebulizer. this time saving may improve adherence to recommended therapy. (pediatrics 2006; 118:1260) . in order to properly interpret op cultures from nbs infants, especially those with non-classic cf mutations, we need to know the op flora of non-cf infants. we obtained op cultures from 100 healthy infants under 1 yr of age. op specimens were plated on standard cf culture media. exclusion criteria included a first degree relative with cf, respiratory illness at the time of culture, or positive newborn screen for cf. data on cigarette smoke exposure, animal exposure, and exposure to hot tubs/swimming pools was collected. 57 samples have been collected to date. in healthy, non-cf infants, the most common finding is non-specific mixed gram negative and positive growth. however, 3 infants have grown pa (ages 3 months, 10 months, and 10 months). many infants have grown multiple organisms. the following bacteria have been found in (n) number of infants: s. aureus (10), e. coli (8), e. cloacae (6) , h. flu (4), klebsiella (4), pseudomonas aeruginosa (3), h. parainflu (3), unidentified non-lactose fermenting (4), other (9). data on 100 infants including correlation to environmental factors will be presented. conclusion: non-cf infants commonly have s. aureus and many gram negative organisms including pa in their oropharynx. these results may have some bearing on interpetting colonization and clearance of pa in infants identified through nbs and in epic study participants. #300) ). identification of smg by sputum cultivation represents a significant challenge because the organisms are phenotypically diverse, grow poorly on routine culture media, and are very difficult to discriminate from other members of the oropharyngeal flora. we have developed a solid media for the selective isolation of smg from sputum. the value of the media is highlighted by the identification of smg as the quantitatively dominant organism in sputum samples of three cf patients admitted to hospital for an acute pulmonary exacerbation. in all three, the smg species failed to be identified on routine or selective media currently described for the culture of cf-specific sputum pathogens. antibiotic treatment directed against the smg correlated with clinical resolution of acute symptoms as well the reduction of smg on daily serial sputum cultures during hospitalization. this novel selective media makes use of antibiotics (colistin, sulfadiazine and oxolinic acid) inhibitory to the growth of principal cf pathogens and much of the usual oropharyngeal flora. smg agar utilizes a colorimetric indicator to uniquely identify smg colonies. the sensitivity and specificity of the selective media has been evaluated by molecular methods using terminal restriction fragment length polymorphism analysis. smg organisms do not respond well to anti-pseudomonal therapy, therefore proper detection and culture-directed antibiotic therapy is paramount. we believe that smg represent significant respiratory pathogens in cf, and because of the inability to effectively culture and identify smg they have largely gone unrecognized. pseudomonas aeruginosa releases substantial amounts of the blue antibiotic pigment pyocyanin. in presence of a reductant (such as nadph), pyocyanin redox-cycles and generates superoxide and h 2 o 2 . in infected cf airways, pyocyanin concentrations can reach high micromolar levels and contribute to oxidative stress of the airways. the structural basis of the pyocyanin molecule that underlies the redox-cycling with reductants of the airways is not clear. we therefore investigated i) the ability of physiologically or pharmacologically relevant reductants of the airways to support the redox-cycle activity of pyocyanin, and ii) the molecular features of pyocyanin that support redox cycling. dose-and time-dependent h 2 o 2 production by pyocyanin was measured by amplex red oxidation in presence of horseradish peroxidase. rates of h 2 o 2 production by 100 µm pyocyanin in presence of 150 µm reductant were: nadph (392 pmole/min) > l-ascorbate (108 pmole/min) > reduced glutathione (41 pmole/min) > α-tocopherol (12 pmole/min). in contrast, lipoic acid, genistein, or resveratrol did not significantly support pyocyanin-mediated h 2 o 2 production. in absence of a reductant, pyocyanin showed no measurable formation of h 2 o 2 . to identify the structural characteristics of the pyocyanin molecule that allow for its redox-cycling activity we synthesized a number of new pyocyanins containing electron-donating or electron-withdrawing substituents. functional assays were performed in presence of l-ascorbate as reductant. molecular substituents that donated electrons to the positively charged core of pyocyanin, either by hyperconjugative or resonance effects, reduced the h 2 o 2 output of the corresponding pyocyanin. in contrast, a closely related analog (2-hydroxyphenazine-5,10dioxide) showed significantly increased activity (2.2x compared to pyocyanin) suggesting that the electron-withdrawing effect of the n-oxide functionality led to an increase in the redox-cycle activity. these data indicate that the functional characteristics of pyocyanin as a redox-cycling compound are governed by its positively charged core. in the airways, pyocyanin is predicted to utilize several reductants that are present in the airway surface liquid or intracellularly, thus contributing to cf airway disease. because pyocyanin utilizes a variety of reductants, it appears prudent to test whether inhaled small-molecular cf therapeutics support pyocyanin function. supported by nih (hl-071829, p01 at002620), cfri, philip morris usa inc and philip morris international, and cff (fischer07g0). taccetti, g.; braggion, c.; ravenni, n.; zavataro, l.; neri, a.; festini, f.; campana, s. meyer hospital, university of florence, cf center of tuscany, florence, italy for practical purposes, after early eradication treatment, at least three consecutive negative respiratory cultures over a 6-month period would indicate that the organism has been eradicated (cf trust guidelines). this recommendation is based on opinion/clinical experience of respected authorities in the absence of directly applicable studies of good quality. aims: using molecular biological techniques, we evaluated whether this 6-month interval is really trustworthy for distinguishing between regrowth of the same strain, suppressed but not eradicated by treatment, and new pseudomonas aeruginosa (pa) colonization. patients and methods: cystic fibrosis (cf) patients were treated with oral ciprofloxacin and nebulized colistin at detection of pa. all pa colonization episodes were recorded in an appropriate database. molecular study of each bacterial isolate from each colonization episode was performed with the rapd-pcr. results: between 1998 between -2006 of patients in follow-up in our center had repeated pa colonization. the patients' mean age at first pa colonization was 78 ± 4.9 months. a total of 78 episodes was observed (mean of 2.75 episodes per patient, median of 2, range 2-7). molecular typing on 78 strains indicated that 66 (84.6%) were a different genotype from later colonization episodes while 12 (15.4%) isolates had the same genotype as those of the preceding episode. the same genotype as preceding colonization was observed in 7 (46.6%) of 15 isolates from patients in which a successive colonization was verified in less than 6 months, and was verified in 8 (12.6%) of 63 strains in patients having a successive colonization in over 6 months (or=6.01). colonization was due to a genotypically diverse strain in 54% of cases where colonization occurred within 6 months of eradication. during the observation period 7 (25%) of 28 patients acquired chronic pa infection. conclusion: re-colonization by pa following eradication therapy is mostly (84%) caused by strains with a different genotype, suggesting acquisition from an external source. a short pa-free period is mainly due to transient suppression of pa growth, and true eradication followed by acquisition of a new pa genotype occurred in most cases only after a pa-free period longer than 6 months. those patients with a pa-free interval of less than 6 months had a six-times higher chance that the pa was not eradicated compared to those with a germ-free interval of over 6 months. this evidence demonstrates that the definition of successful eradication should be reconsidered, taking into account additional parameters such as molecular analysis. cystic fibrosis (cf) patients appear to have an increased risk of urolithiasis. while a number of possible explanations for this have been proposed and investigated, no definitive mechanism has yet been demonstrated. as cf patients frequently get respiratory tract infections they regularly receive ciprofloxacin treatment, often given in doses well in excess of conventional prescription regimes. there are occasional reports of ciprofloxacin crystals urine and stones in the urinary tract. here we investigate the hypothesis that ciprofloxacin excreted in urine might act as a promoter of crystallisation of calcium or magnesium salts and thereby increase the risk of kidney stone disease. the effect of ciprofloxacin was tested in artificial urine (au). in vitro crystallisation was tested using a 96 well plate turbidity method, to identify a metastable limit of oxalate concentration (ml) and a growth and nucleation parameter, the turbidity rate index (tri). the nucleation ph of urine was examined by tritrating oxalate free au through a ph range of 5.3 to 9.0 and monitoring the solution/suspension turbidity. in au at ph 6.0, ciprofloxacin, at 300, 600, 1000 or 1800mg/l, had no detectable effect on initiation of calcium oxalate crystallisation (ml) or its progress (tri) (n=6 for each concentration). when au with 6mm ca, 3mm mg and 25mm po4 was titrated there were two distinct nucleation points; a slow event starting at about ph6.7 and a much faster event at about ph 7.4. omitting ca or mg confirmed that the first event was due to calcium phosphate precipitation and the second to struvite. including ciprofloxacin at 400, 500 or 600mg/l did not alter these nucleation ph values, but the magnitude of the turbidity rise showed that the ciprofloxacin co-precipitated with the struvite. ciprofloxacin at 1000mg/l and without ca or mg began to precipitate at ph 6.2 and could be held in solution until ph 7.2 when ca and mg were included. even at high concentrations, ciprofloxacin does not influence calcium oxalate crystallisation. nor does it promote calcium phosphate or struvite precipitation; on the contrary, while the calcium and magnesium remain in solution, they help to prevent precipitation of the ciprofloxacin itself. urinary ciprofloxacin does not appear to act as a stone or crystal promoter. pseudomonas aeruginosa is a significant cause of mortality in cystic fibrosis (cf) sufferers. cf patients were thought to acquire p. aeruginosa from the environment; however genotyping over recent years has revealed clonal strains in sputa from cf patients in the uk, australia, and canada that are transmitted person to person or from a common source. one clonal strain, australian epidemic strain-1 (aes-1), (formerly melbourne epidemic strain, m16 or pi) currently infects up to 40% of patients in five cf clinics on the eastern seaboard of australia. most cf clonal strains have been associated with increased virulence not fully explained by greater antibiotic resistance. both genotypic and phenotypic differences have been postulated as important in enabling transmission of clonal strains. in order to compare the expression profile of aes-1 to the type strain p. aeruginosa pao1, the cf research group at the university of sydney compared the genome expression data of four clonal aes-1 isolates and pao1, when grown as planktonic and as 72-hr biofilm cultures. in aes-1, a set of 22 significantly differentially expressed genes (all downregulated) were identified, including the quorum sensing genes lasa, lasb and rhll. in contrast, both upregulated and downregulated genes were differentially expressed in pao1 biofilm compared to pao1 planktonic culture. expression data was validated using quantitative real-time pcr. to compare biofilm growth at the phenotypic level, the four clonal strains and pao1 were grown as 72-hr biofilms in a double-blind study, and the size of ten randomly selected biofilms per isolate, stained with syto® 9 green fluorescent stain was measured. at 72 hr, the biofilms formed by aes-1 isolates were significantly larger (ca. 18-fold) than pao1 (average size: 33389±14211µm 2 vs 1791±498µm 2 )(p<0.01). the average thickness of three biofilms per isolate, measured by confocal microscopy, showed aes-1 biofilms to be approximately 1.5-fold thicker than those formed by pao1 (12.6±0.2µm vs 8.5±1.4µm). the general gene downregulation observed in aes-1 biofilms suggests an adaptation to the cf host, while a larger biofilm would provide for more effective bacterial dispersal. thus the transmissibility of aes-1 may be linked to enhanced biofilm formation upon colonisation. background -objective: whilst influence induced by bacterial colonization in cystic fibrosis (cf) is established, risk induced by fungal colonization is less defined. prevalence of other species than aspergillus sp. or candida sp., and factors associated with fungal presence are also poorly documented. our preliminary study aimed to determine which fungal species were present in sputum collected from adult cf patients, and which factors were associated with fungal presence. methods: in a monocenter, transversal prospective study, 21 cf adult patients were included to determine fungal presence in sputum using semi selective growing media. clinical parameters (shwachman score, respiratory function, nutritional status, gastro-oesophageal reflux, pancreatic insufficiency and diabetes); therapeutics used (including oral or intravenous antibiotics, systemic or inhaled corticosteroids or bronchodilatators, antifungal treatments); microbiological data of bacterial colonization and environmental parameters (potted plants or domestic animals presence) were determined for each patient. correlation between fungal, clinical, environmental, therapeutic or microbiological data was evaluated by mann-whitney non parametric u test. results: 16 patients (76%) presented with fungal presence in sputum. 61% presented with yeasts species, 52% with moulds. aspergillus fumigatus and candida albicans were the predominant species in moulds and yeasts respectively, but less common mould species such as exophiala dermatidis or paecilomyces variotii were also recovered. factors associated with fungal presence were pancreatic insufficiency (p=0,04); malnutrition (p=0,039), bacterial colonization and inhaled corticosteroids. candida albicans was correlated with more severe shwachman score (p=0,019), bacterial colonization (p=0,017), notably with pseudomonas aeruginosa (p=0, 03) , and intravenous antibiotics use (p=0,015). moulds species were significantly associated with inhaled corticosteroids (p=0,049). antifungal use was associated with frequent resistance to azoles treatments (4 resistant isolates out of 5 patients treated). conclusion: fungal presence in cf appears frequent. some species could have been previously overlooked due to diagnosis difficulties. the effect of corticosteroids on moulds species, already found in other pathologies, appears important in cf. influence of fungal presence on cf course needs prospective studies, in order to establish if patients could benefit from antifungal treatments or preventive measures. dren on a ventilator without a previous diagnosis of cystic fibrosis is unknown. the aim of our study was to investigate the prevalence of these microorganisms in routine sputum cultures in young children on a ventilator in a pediatric intensive care unit (picu). methods: from all ventilated children aged 0-3 years admitted from 1998-2004, sputum culture results obtained from tracheal aspirates within the first week of admission were retrospectively analysed. three patient subgroups were identified: respiratory failure due to pulmonary disease (group 1), ventilation after elective surgical procedures (group 2) and other ventilated children (group 3). children with a previous intensive care admission or cf were excluded. the cf database was checked (2007) to identify any children with a new diagnosis of cf that were included in the study. results:12.1 % of all ventilated children had a positive sputum culture with one of the "cf-bacteria" s. aureus, h. influenzae and p. aeruginosa these were found mainly in group 1 (see table) . 27/54 of these children were admitted for treatment of respiratory syncytial virus (rsv) bronchiolitis. a sweat test was performed in 7 children, all admitted with pulmonary disease and because of co-existing clinical signs or symptoms. one sweat test was positive and subsequently cf was diagnosed in this child. no other children out of the study group have since then been diagnosed with cf. as the population in the northern part of the netherlands is very stable, it is unlikely a diagnosis of cf has been missed. conclusion: s. aureus and h. influenzae are cultured frequently in ventilated children without cf, especially when ventilated because of pulmonary disease. the opportunistic pathogen p. aeruginosa causes both acute and chronic human infections. the balance between systemic infection and mortality or chronic persistence and morbidity depends on complex relationships in which the immunological status and genetic potential of the host but also the bacterial biodiversity are determinant factors. p. aeruginosa extensive genetic adaptation and microevolution have been repeatedly observed in chronic infections of cf patients in contrast to what it is documented in acute infections. whether these p. aeruginosa clonal variants differ in their pathogenic potential is not yet known. a total of 19 clinical p. aeruginosa isolates from six cf patients which carried unique clonal lineage from the onset of colonization over 4-18 years were selected (bragonzi et al, 2006; montanari et al, 2007) . five p. aeruginosa environmental strains, which represent the source of acquisition for cf patients, and two laboratory strains pao1 and pa14 were also used as references. multiple genotypic analysis of sequential p. aeruginosa isolates which included pfge, atchip and multilocus snps showed intraclonal diversity with genome rearrangements, variations in pathogenic islands and acquisition of prothoadaptive mutations in the muc genes. the largest divergence was observed between the completely sequenced reference strains pao1 and pa14, the latter represented in our panel by three cf isolates. p. aeruginosa virulence has been assessed by monitoring its capacity to induce bacteremia and to establish chronic infection in a murine model (bragonzi et al, 2005) . overall, p. aeruginosa environmental strains increased five times the risk of bacteremia when compared to clinical strains (test of proportion: p<0.001). the high risk of mortality was also evidenced for p. aeruginosa cf strains isolated at the onset of the infection when compared with those isolated after years of colonization (p=0.002) indicating that environmental strains or newly acquired strains were similar in their virulence. p. aeruginosa clinical strains isolated after years of colonization increased chronic persistence and reduced the risk of bacteremia (p=0.002) when compared to strains isolated at the onset of the infection. the strain pa14 was found to be lethal in contrast to p. aeruginosa clonal strains of clinical origin that established chronic persistent infection in the murine lung. furthermore, our data showed that adaptive traits commonly associated with the chronic p. aeruginosa infections in cf patients, such as transition to the mucoid phenotype, did not confer a selective advantage to bacterial cells in colonizing the murine lung (p=0.294). these results suggest that p. aeruginosa pathogenicity is independent of the strain's genotype but rather genetic adaptation to the cf airways plays an important role in the development of persistence and in the resistance to host defences. supported by telethon and italian cf research foundation. beringer, p. the combination of antipseudomonal beta-lactam and aminoglycoside antibiotics is frequently prescribed during acute pulmonary exacerbations. since the introduction of the beta-lactam compounds there has been numerous reports citing altered pharmacokinetics of several compounds (but not all) within the beta-lactam class. recently it has been suggested that substrate specificity for the renal transporter pgp may explain the variability in renal drug clearance observed in patients with cf. pgp is structurally related to cftr and evidence suggests it serves a complementary role in modulating alternative chloride channel function. in a cftr knockout model mdr1 expression was reported to be increased nearly four-fold. in an effort to elucidate the potential contribution of pgp to the renal clearance of drugs in patients with cf, we conducted a controlled clinical trial comparing the pharmacokinetics of fexofenadine (fx) in patients with cf and age matched healthy volunteers (hv). fexofenadine is not significantly metabolized and is a relatively specific substrate for the membrane transporters pgp and oatp. probenecid (pb) is a selective inhibitor of oatp and was used pharmacologically to block the activity of this transporter in-vivo. our hypothesis is that if pgp is upregulated within the renal tubules of patients with cf we would expect to see an enhanced renal clearance of fexofenadine in these patients when compared with control subjects. methods: 16 (n=8 cf, 8 hv) subjects underwent this prospective controlled study which fx was received alone or in combination with probenecid (pb). iothalamate was given each day to measure glomerular filtration rate (gfr). blood and urine samples were obtained at specified times over 12 hours each study day for determination of fexofenadine and iothalamate concentrations. fexofenadine and iothalamate concentrations were assayed using liquid chromatography-tandem mass spectrometry and hplc-uv respectively. pharmacokinetic analysis was performed using a 1-compartment cumulative urinary excretion model with the adapt ii software. differences between groups were determined using a paired t-test or mann whitney-u. results: 16 patients were included, cf patients were slightly older and had lower body mass index when compared to hv, (mean±sd, 27±5.6 vs. 33.4±7 years p=0.0499, 20.03±1.6 vs. 25.2±2.2 p=0.0019 respectively) but did not differ in gfr (120±21.7 vs. 108±25 ml/min-1.73m 2 p=0.38 respectively). no significant differences were found between cf and hv in clr of fx alone and fx in combination with pb clr (93.3±20 vs. 83.3±23.3 ml/min-1.73m 2 p=0.31 and 38.3±10 vs 36.7±23.3 ml/min-1.73m 2 p=0.79 respectively). conclusion: the results of this study indicate the enhanced clearance of certain antibiotics previously reported in patients with cf do not appear to be due to upregulation of renal tubular pgp or increased gfr. maceachran, d.p. department of microbiology and immunology, dartmouth medical school, hanover, nh, usa p. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (cf) patients. shortly after birth cf patients are colonized by p. aeruginosa, which leads to a lifelong chronic infection. the mechanisms behind p. aeruginosa colonization of the cf lung are still poorly understood, however several secreted toxins have been associated with this phenomenon. cf is characterized by a loss of mucocilliary clearance, a key component of innate immunity in the lung, and an increase in sputum viscosity as a result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (cftr). we have previously shown that the novel p. aeruginosa secreted protein cif is capable of reducing apical membrane expression of cftr. we have begun to characterize cif expression and have identified a key regulator of cif transcription. mutations affecting the divergently transcribed putative tetr family repressor encoded by the pa2931 gene result in a significant increase in cif expression. furthermore, we have demonstrated that the pa2931 gene product is capable of binding the promoter region located immediately upstream of the cif-containing operon. this work has been supported by the nih t32-dk007301-29. ryall, b. 1 ; davies, j.c. 2, 4 ; wilson, r. 3 ; shoemark, a. 3 ; williams, h.d. 1 1. division of biology, imperial college london, london, united kingdom; 2. department of gene therapy, imperial college london, london, united kingdom; 3. host defence unit, royal brompton hospital, london, united kingdom; 4. paediatric respiratory medicine, royal brompton hospital, london, united kingdom pseudomonas aeruginosa is the most important respiratory pathogen in patients with cystic fibrosis (cf) and non-cf bronchiectasis. the factors which enable p. aeruginosa to establish chronic, debilitating respiratory infections are unclear. however p. aeruginosa is one of a limited number of bacteria that is able to synthesise hydrogen cyanide, a potent inhibitor of cellular respiration. this study examined whether hydrogen cyanide is produced by p. aeruginosa in cf and non-cf bronchiectasis airway infection. cyanide concentration was measured in fresh sputum from cf and non-cf bronchiectasis patients with and without p. aeruginosa lung infection using a cyanide ion sensing electrode. cyanide was detected in sputum from 15/25 cf and non-cf bronchiectasis patients with current p. aeruginosa infection, whereas it was not detected in any of the 10 patients without this organism (p<0.01). maximum lev-els were 130µm (mean ±se: 72 ± 6.6 µm), which compares with levels of above 40µm which would be considered toxic in blood. concurrent lung function data were available on all 21 p. aeruginosainfected cf patients; the group with measurable sputum cyanide (n=11) was not different from those without (n=10) on the basis of age, gender or p. aeruginosa phenotype (mucoid/ non-mucoid). however, those with detectable cyanide had significantly poorer lung function than those without (fev1% predicted: 26.8±3.8% versus 46.0±6.7%, p<0.01; fvc% predicted: 44.4±4.9% versus 60.1±7.7, p<0.05). we have shown that cyanide is detectable at clinically significant levels in sputum from cf and non-cf bronchiectasis patients infected with p. aeruginosa and is associated with significantly impaired lung function. we have recently reported a dose regimen for once-daily tobramycin dosing in patients with cystic fibrosis which was based on a retrospective study (lam w et al., j antimicrob chemother 2007) . we present here an interim analysis of an ongoing prospective study to evaluate the proposed dosing in paediatric patients with cystic fibrosis. as part of this protocol pharmacokinetic parameters were determined in cystic fibrosis (cf) patients receiving intravenous antibiotic therapy for a pulmonary exacerbation. serum creatinine, audiometric testing, respiratory cultures and fev 1 were assessed at baseline and after a 2 week treatment period. serum tobramycin concentrations were interpreted by clinical pharmacists performing therapeutic drug monitoring (tdm) to optimize dosing. serum tobramycin levels were drawn after the first dose, repeated with each dose adjustment and weekly thereafter. the proposed sample size of this study is 60 patients; an interim analysis was performed to identify trends for pharmacokinetic and safety outcomes from 18 patients admitted from november 2006 through april 2007. seven (38.9%) patients achieved the target c max of 25-35mg/l, 10 (55.6%) patients remained under the target range and 1 patient was above the target range with the 1st dose. with 2nd, 3rd, and 4th tdm performed for either routine weekly levels or dose adjustment levels, 6, 16, and 17 patients met the target c max respectively. the percentage of patients who achieved target c max increased to 88.9 % with the 3rd set of tdm levels and 94.44 % with the 4th set of tdm levels (p-value<0.0001). there were no significant changes in k e , t 1/2 and v d /kg for each patient from 1st to 2nd set of tdm levels over time. compared to the previous retrospective study the mean v d /kg was significantly higher (0.327l/kg versus 0.267l/kg, p=0.0021) and the mean t 1/2 was significantly shorter ( 1.73 hrs versus 2.14 hrs; p<0.0015). none of the patients' serum creatinine increased ≥ 50 % from baseline. baseline audiometric tests were within normal limits in 12 (70.6 %) patients. p. aeruginosa was isolated from respiratory specimens of 6 patients on initial culture. of the 5 patients with follow-up cultures, 3 patients grew p. aeruginosa sensitive to tobramycin, demonstrating no decrease in sensitivity. our preliminary results suggest the pharmacokinetic data differ from the expected profile suggested by the retrospective study. all study patients treated according to the guidelines were clinically well after their course of therapy and did not experience clinically significant toxicity from once-daily tobramycin dosing. the once-daily tobramycin dosing will continue to be evaluated until the proposed sample size is achieved for final analysis. anderson, g.g.; o'toole, g.a. dartmouth medical school, hanover, nh, usa pseudomonas aeruginosa chronically colonizes the lungs of individuals with cystic fibrosis (cf). evidence suggests that this infection progresses through distinct phases, wherein initial colonizing strains undergo a switch to mucoidy that corresponds to formation of bacterial microcolonies in the airways and life-long persistence. currently, the environmental signals and mechanisms regulating conversion from the acute, highly virulent form to a less virulent biofilm state remain obscure. to investigate these issues, we developed a tissue culture model system for growth of p. aeruginosa biofilms on cf-derived human airway epithelial cells in vitro. biofilms grown on these cells appear similar to those found in cf airways as well as to abiotic biofilms. eventually, epithelial cells were killed by the p. aeruginosa biofilms. however, we discovered that addition of the antibiotic tobramycin preserved the monolayer integrity for at least 24 hours. bacteria were not completely eliminated by this treatment, suggesting that tobramycin influenced the virulence of the biofilm bacteria. microarray analysis of cf cell grown biofilms treated with tobramycin revealed marked alterations in transcriptional profiles compared to untreated controls. two tobramycin-induced genes were identified from this screen which, when deleted, exerted altered virulence phenotypes on airway epithelial cells in culture. quantification of cytotoxicity of these mutants revealed either increased or decreased ability to kill the epithelial cells. complementation with full-length copies of these genes restored wild-type function. these studies suggest a complex relationship between acute and chronic forms of p. aeruginosa, and that antibiotic treatment may influence transitions between these modes. it is this interplay that may determine the virulence status of the microorganism upon initial infection as well as during cf exacerbations. neville, m.; sardaryan, g.; ejaz, s.; scotto, a.w.; gupta, r. transave, inc., monmouth junction, nj, usa purpose: during the course of treatment, patients with cystic fibrosis and chronic pseudomonas aeruginosa infections in their lungs may be treated with nebulized tobramycin (tobi®). reduction in treatment times or dosing frequency may improve compliance and outcome. nebulized arikace™ may offer advantages to tobramycin by producing sustained lung levels of drug, reducing dosing frequency and increasing antimicrobial efficacy. herein are presented biodistribution data and efficacy data in a chronic rat model of p aeruginosa comparing arikace™, soluble amikacin and tobramycin. method: female rats (charles river) received (~6 mg / kg) of arikace™, soluble amikacin and tobramycin by inhalation. test articles were aerosolized in a pari lc star nebulizer at 30 psi using devilbiss compressors (sunrise medical) for 80 min. 3-4 rats were euthanized at time 0 and 6 hr post inhalation by co2 asphyxiation. blood was collected by a cardiac puncture. serum was collected from coagulated blood by centrifugation and stored at 4oc until analysis for drug content. in addition lungs, kidneys, intestinal contents, urine and brains were harvested. tissues and biological samples were homogenized in saline and antibiotic concentrations determined by tdx analyzer (abbott). chronic lung infections were produced in rats by intratracheal instillation of agar beads containing p. aeruginosa. three days post instillation, rats (n=12/group) were treated with aerosolized saline or once daily with arikace™, or twice daily with tobramycin (total daily dose = 6 mg /kg) for 14 days. rats were euthanized 3 days post final dose and the pseudomonas cfu counts per lung were determined. results: the biodistribution and pharmacokinetics of aerosolized arikace™ was significantly different from that of amikacin and tobramycin. the primary difference was the amount of antibiotic retained in the lungs. rats that inhaled soluble amikacin cleared approximately 84% of the amikacin from the lungs after 6 hr with concomitant 86% increase in drug levels in the urine. rats that inhaled arikace™ cleared only 41% of the deposited amikacin from the lungs after 6hrs. this initial clearance of drug most likely reflected the amikacin released during nebulization. extended clearance of arikace™ from the lungs was measured by recovery of amikacin in the urine at later time points. in a separate longer-term study comparing arikace™ and tobramycin, auc (0-168 hr)for arikace™ in the lung was 56233 ug*hr/g while only 1150 ug*hr/ g for tobramycin which demonstrates a 4-fold increase in retention of antibiotic in the lung with arikace™. eradication of p. aeruginosa from the lungs of rats with arikace™ was equal to tobramycin (given equivalent daily doses). however, arikace™ reduced the number of cfu in the lungs with a single daily exposure to the same degree as inhalation of tobramycin twice a day. conclusions: inhalation of arikace™ achieved higher concentrations and increased retention time of the antibiotic in the lungs of rats when compared to inhaled amikacin or tobramycin. it is expected that these properties of arikace™ will reduce dosing frequency and improve antimicrobial efficacy in patients. nielsen, x.c. 1 ; johansen, u.r. 1 ; nørregaard, l. 1 ; vandamme, p. 2 ; høiby, n. 1 1. clinical microbiology, rigshospitalet, copenhagen, denmark; 2. laboratory for microbiology, university of ghent, ghent, belgium background: the burhkolderia cepacia complex (bcc) is a diverse group of bacteria with at least 9 genomovars (gv) or species. accurate species identification is necessary for better understanding of the epidemiology and pathogenesis of bcc. species identification based on phenotypic characters is difficult when it is based on few if any differences. crossed immunoelectrophoresis (cie) has been performed routinely to identify all burkholderia and other difficult isolates in our department (høiby et al.: "taxonomic application of crossed immunoelectrophoresis." int. j. syst. bact. 37 (1987): 229-240.) . in this study, we compared the results obtained by cie with those obtained by the standard molecular biological method using reca gene pcr and rflp to identify bcc at the species level (golden standard). methods: between 1996 and 2005, burkholdreia isolates were recovered from 34 patients in the two danish cystic fibrosis (cf) centres. 1. cie method: immunological cross-reactivity between antigens from the reference strains and the strains isolated from our cf patients were compared by employing cie and rabbit standard-antibodies (purified igg) raised against water-soluble antigens from reference strains of the bcc. species identification was made based on matching coefficient (mc). 2. reca gene-based identification: bcc specific primers were used to amplify reca gene. species-specific pcr is then performed using b. multivorans specific primers to identify the b. multivorans gv ii. rflp patterns were generated for those non-multivorans bcc by digesting the bcc-specific pcr products with haeiii. the patterns were then compared with the patterns generated from reference strains in the database. pulsed field gel electrophoresis (pfge) genotyping was performed for all 34 strains to detect patient-topatient transmission. results: 32/34 isolates were bcc-specific pcr positive. species-specific pcr identified 29 isolates as b. multivorans (gv ii). the other three isolates were identified as b. cenocepacia (gv iii) based on rflp patterns. two isolates with negative bcc-specific pcr reactions were identified as b. gladioli by cie (confirmed by reference laboratory in belgium). results from cie showed that strains from bcc were immunologically closely related with only a few non-cross-reactive antigens. compared with the standard reca gene-based method, cie method resulted in correct identifications in 27 isolates (79.4 %), misidentifications in 4 isolates (11.8 %) and uncertain identifications in 3 isolates (8.8%). genotyping by pfge showed unique patterns for all isolates except for those from two sisters. this suggested that patient-to-patient transmission of bcc among the danish cf patients has remained very rare. conclusions: reca gene-based pcr and rflp is a quick and reliable method for identification of bcc at the species level. cie proved useful in initial classification of bcc strains, but additional information is obtained by the reca gene-based species identification and pfge method. background: in childhood, the cystic fibrosis airway is characterized by persistent inflammation with high quantities of neutrophils. in this setting, recurrent exposure to low numbers of pseudomonas aeruginosa occurs routinely. chronic infection by p. aeruginosa is associated with a significant increase in morbidity and mortality in cf, and is believed to occur by formation of a biofilm in the cf airway. previously, we have shown a significant neutrophil dependent enhancement of p. aeruginosa biofilm density that is greatest for low initial concentrations of p. aeruginosa. biofilm enhancement is facilitated via neutrophil-derived dna and f-actin, which form a framework that p. aeruginosa can exploit for growth. dna and factin polymerize via positively charged molecules such as histones. p. aeruginosa then associates with these polymers, which results in increased early biofilm density, displaying an increase of over 400 fold at 24 hours compared to p. aeruginosa in the absence of neutrophils. hypothesis: compounds that disperse dna and/or f-actin can disrupt neutrophil-enhanced biofilms. methods: poly(aspartic acid), a negatively charged amino acid chain with the capacity to disrupt f-actin, was examined singly and in combination with dnase and/or antibiotics (ciprofloxacin and tobramycin). the nunc-tsp system was used to allow for high throughput assessment of the density of biofilm formation in the presence of neutrophils and the effect of various agents in disrupting the biofilm. the biofilm assay is independent of cellular settling, as the reactor is vertically suspended in the culture. p. aeruginosa strains tested were pao1, and two isogenic cf isolates recovered from an initial infection (early), and following established infection ~4 years later (late). biofilms were formed by incubation of p. aeruginosa with human neutrophils for 24 to 48 hours in rpmi with 2% heat inactivated platelet poor plasma. results: antibiotics were incapable of disrupting biofilms. poly(asp) significantly disrupted neutrophil-induced biofilms. in the presence of neutrophil products this action is sensitive to proteolytic degradation, but can be protected by the presence of protease inhibitors. dnase also disrupts a biofilm formed in the presence of human neutrophils. the combination of poly(asp) + dnase resulted in an increase in biofilm disruption for a 24 hour biofilm. biofilms allowed to form for 48 hours were more resistant to disruption by dnase and poly(asp) as single agents, but the combination of both demonstrated a synergistic effect. similar disruption of biofilms were observed when pao1 was compared to early and late cf strains. conclusions: pseudomonas aeruginosa biofilms formed in the presence of neutrophils in vitro can be disrupted by agents that disperse the f-actin and dna framework. support: cff, max and yetta karasik foundation cystic fibrosis (cf) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. the black yeast, exophiala dermatitidis (synonym wangiella dermatitidis) has been found with increasing frequency in sputum specimens of cf patients, with reported isolation rates ranging from between 1.1 and 15.7%. at present, no species-specific diagnostic pcr exists to aid with the clinical laboratory detection and identification of this organism. a novel species-specific pcr-based assay was developed for the detection of e. dermatitidis, based on employment of rdna operons and interspacer (its) regions between these rdna operons. two novel primers, (designated exdf & exdr) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of exophiala, as well as with other commonly described yeasts and filamentous fungi within cf sputum, including candida, aspergillus and scedosporium. pcr employing exdf (forward primer [16-,mer], 5'-ccg cct att cag gtc c-3') and exdr (reverse primer [16-mer], 5'-tct ctc cca ctc ccg c-3'), was employed and optimized on extracted genomic dna from a well characterized culture of e. dermatitidis, as well as with high quality genomic dna template from a further 16 unrelated fungi, including candida albicans, c. dubliniensis, c. parapsilosis, c. glabrata, scedosporium apiospermum, penicillium sp., aspergillus fumigatus, aspergillus versicolor, pichia guilliermondii, rhodotorula sp., trichosporon sp., aureobasidium pullulans, fusarium sp., mucor hiemalis, bionectria ochroleuca, gibberella pulicaris. results demonstrated that only dna from e. dermatitidis gave an amplification product of the expected size, whilst none of the other fungi were amplifiable. subsequent direct employment of this primer pair detected this yeast in the sputum of 2/50 (4%) adult cf patients, employing a nested pcr approach with panfungal outer flanking primers of the its1-its2 region. these two patients were the only patients who were previously shown to have a cultural history of e. dermititidis from their sputum. e. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan-and multiresistant bacterial pathogens from sputum, namely pseudomonas aeruginosa and burkholderia cepacia complex organisms, hence this species-specific pcr assay may help detect this organism from cf sputum more specifically and rapidly. overall, employment of this novel assay may help in the understanding of the occurrence, aetiology and epidemiology of exophiala dermatitidis, as an emerging fungal agent in patients with cf. cystic fibrosis patients are particularly susceptible to infection by strains of pseudomonas aeruginosa and burkholderia cepacia complex. since 2001, the pseudomonas genome database has been a resource for peer-reviewed, continually-updated annotations for the pseudomonas aeruginosa pao1 reference strain's genome and, more recently, comparative analyses to several closely-related pseudomonas species. in order to facilitate better cross-strain and cross-species genome comparisons, we have developed or are incorporating methods to improve the identification of orthologs (genes diverged due to speciation) and identify genes undergoing unusual selection. our analysis of several completely sequenced pseudomonas genomes has revealed that approximately 5% of ortholog predictions by the classic "reciprocal best blast hit method" are likely incorrect. we have also performed an analysis of unusually large intergenic regions in p. aeruginosa pao1 that appear to be essential (according to saturation transposon mutagenesis) or involved in virulence (according to signature tagged mutagenesis). we identified at least 20 new putative protein-coding regions and non-coding rnas that were not previously annotated and may play critical roles in microbial pathogenesis or viability. we are also focusing our attention on facilitating better cross-strain comparisons between recently sequenced burkholderia cepacia complex genomes through development of a new online burkholderia genome database. in addition to improved ortholog prediction, this new database will provide access to a very flexible boolean search feature that allows researchers to search and compare annotations within or between the genomes of burkholderia strains, returning annotations from multiple genomes suitable for simultaneous viewing and downstream analyses. we have also included new, very accurate protein subcellular localization predictions for the deduced proteome from each of these genomes -predictions that can aid the identification of new cell surface or secreted therapeutic targets or vaccine candidates. further comparative analyses with other newly-sequenced, related strains should provide insight into strain-specific features that may be exploited to better understand virulence and antimicrobial resistance exhibited by cf-relevant pathogens. funding provided by cystic fibrosis foundation, therapeutics (usa). popova, a.p.; verma, r.; zanni, r.l.; sembrano, e. pediatrics, monmouth medical center, long brnach, nj, usa background: cystic fibrosis (cf) is a chronic, progressive, genetic disease leading to poor lung function, chronic bronchial infection with certain bacteria and recurrent pulmonary exacerbations. despite evidence of inflammation within the airway, it is unclear which circulating inflammatory biomarker best reflects the airway inflammation and lung function in cf. objective: to determine the utility of c-reactive protein (crp) value in evaluating patients with cystic fibrosis pulmonary exacerbation. design: a retrospective chart review was carried out on 87 patients followed at the cystic fibrosis center at the children's hospital. information about serum crp values, pulmonary function test results(pft), presence of pulmonary exacerbation as documented in the chart, sputum culture results, current medications, as well as demographic data, genetic compositions and weight percentiles between january 2005 and march 2007 was collected from the charts and was analyzed. results: of 87 patients whose charts were reviewed, 5 were excluded because they underwent lung transplant, 3 were excluded because they had other mild infection, not diagnosed as pulmonary exacerbation. for 31 patients crp was not measured and they were also excluded from the analysis. total of 48 patients were included in the study. for 9 patients information about crp was obtained on more than one occasion. the patients were divided into groups based on the presence of pulmonary exacerbation and elevation of crp values -in the group with pulmonary exacerbation, 10 patients had elevated crp and 23 had normal crp and in the nonexacerbation group 1 had elevated crp and 26 had normal. fisher's exact test (two -tail) was applied (p=0.043) and confirmed statistically significant elevation of crp in patients with pulmonary exacerbation of cystic fibrosis. the means and standard deviations of the crp values for the patients without pulmonary exacerbation and the patients with pulmonary exacerbation were calculated and analyzed using independent group t-test (two tail). mean crp value for the exacerbation group was 16.34 with standard deviation of 33.72 and mean crp value for the non-exacerbation group of 1.83 with a standard deviation of 2.43. statistically significant difference between the two means was noted (p=0.019). conclusion: serum crp values in patients with cf pulmonary exacerbation were significantly higher than in patients without pulmonary exacerbation. serum crp may be a useful marker of pulmonary exacerbation in children. in the absence of pulmonary exacerbation, cf is not associated with elevated crp values in children. the findings of this study could aid in identifying patients with pulmonary exacerbation earlier and initiate appropriate therapy sooner. further studies are needed to determine whether crp values are associated with the severity of the underlying disease, to what extent the pft are affected, the age of the patient, the type of bacterial pathogen, and genetic factors. background pulmonary infections with nontuberculous mycobacteria (ntm) are commonly associated with structural lung diseases or airways clearance disorders such as cystic fibrosis and primary ciliary dyskinesia. we follow approximately 100 individuals with ntm pulmonary disease who share common clinical features but have no identifiable systemic immune defect or clearly defined predisposing conditions. the pulmonary disease and clinical presentation suggest these individuals may have disorders of ion transport or ciliary function. while some cftr mutations and ciliary abnormalities are difficult to detect through standard genetic and ultrastructural analyses, in vivo physiologic tests of ion transport and measures of ciliary function may allow us to identify ion transport abnormalities in subjects with no identified cftr abnormalities, determine the functional significance of novel cftr mutations, and distinguish disorders of ciliary function from other airways clearance disorders. methods we evaluated the medical and family histories, chest and sinus ct's, sputum cultures, cftr genotypes, and sweat chloride levels of 15 individuals with nontuberculous mycobacterial pulmonary disease and 3 individuals with chronic airways disease and bronchiectasis. we systematically tested the pulmonary function (pft), nasal nitric oxide production (nno), and nasal potential difference (npd) of each subject. nasal mucosal scrape biopsies were performed and sent to unc-chapel hill for assessment of ciliary ultrastructure as part of the genetic diseases of mucociliary clearance consortium. pulmonary function, nno, and npd were measured on 8 healthy controls. results all subjects had sino-pulmonary symptoms consistent with those associated with cf and pcd. 5 subjects had low nno (27.2 ± 20.7 nl/min) consistent with levels reported in subjects with pcd (<100 nl/min, noone 2004). one subject with variant cf (∆f508/r347h) displayed low basal pd (-46.1 mv), elevated na+ absorption (34.2 mv), and no cftrmediated cl-conductance (-0.6 mv). 5 subjects with cftr mutations, 1 subject with isolated elevated sweat chloride (72 mmol/l), and 1 subject with isolated borderline sweat chloride (44 mmol/l) displayed normal basal potential difference (-12.4 ± 5.1 mv) and na+ absorption (8.0 ± 4.4 mv) but decreased cftr-mediated cl-conductance (-9.3 ± 5.5 mv) relative to controls (-15.0 ± 9.1 mv). conclusions ntm patients heterozygous for cftr-mutations or with elevated sweat chloride (and no identified mutations with coding region sequencing of cftr) may have detectable subtle differences in cftr clconductance, while measurement of nno may identify disease associated with ciliary abnormalities. these findings suggest subtle abnormalities in airways clearance may predispose to airway infection with environmental organisms such as ntm. support: intramural funds, national institute of allergy and infectious diseases muhlebach, m. 1 ; goodrich, j. 2 ; sutton-shields, t.n. 2 ; wedd, j.p. 3 ; henegar, c. 1 ; miller, m.b. 4 ; gilligan, p.h. 4 1. dept. of pediatrics, unc chapel hill, chapel hill, nc, usa; 2. university of north carolina hospitals, chapel hill, nc, usa; 3. school of public health, university of north carolina hospitals, chapel hill, nc, usa; 4. dept. pathology and laboratory medicine, university of north carolina hospitals, chapel hill, nc, usa introduction: proportion of methicillin resistant (mrsa) vs. susceptible sa infections increased from 36% in 1992 to 64% in 2003 in us hospitals. similarly, the prevalence of mrsa in the community has risen in recent years. these community acquired (ca) mrsa isolates are genotypically different, have different antibiotic susceptibility, and often carry the pvl virulence gene. this virulence gene is associated with more invasive, mostly skin infections in healthy individuals. reports on impact of mrsa in cf show conflicting data. 1 , 2 . the aim of this study was to determine the prevalence of ca-mrsa strains at our pediatric and adult cf center and to assess the impact of these infections on clinical outcome. methods: all mrsa isolates recovered from routine clinical cultures, were prospectively collected and analyzed for 18 months (10/05 -4/07). using molecular assays to determine the pvl status and to identify the sccmec (staphylococcal cassette chromosome) type, the organisms were classified as ca or ha. classification as ca mrsa was based on presence of pvl and sccmec iv type. clinical outcomes included lung function measurements in patients reliably performing spirometry and nutritional parameters (bmi%). patients who had undergone lung transplant were excluded as their lung functions vary depending on transplant outcome. results: of the 104 patients identified in this 19 month period, 83% had ha and 17% ca mrsa. neither current age (mean 18.1 yrs. in ha vs.14.6 years in ca) nor age at acquisition of mrsa (14.6 vs. 13 years) was significantly different. sixty-eight % of all mrsa infected patients had chronic p. aeruginosa infection, (70% in the ha and 51% in ca, relative risk 2.3 of having ha in presence of chronic p. aeruginosa infection). crosssectional comparison of patients infected with ca vs. ha acquired strains showed no difference in nutritional status (bmi 37 vs. 38%). lung function (fev1 and fef25-75) was not different in ca vs. ha patients, neither when including all mrsa infected patients or those who had no concomitant infection with other organisms (n=22). we will present further comparisons to age and gender matched children with ossa infection in the final report. conclusion: comparison to ossa in matched patients will assess clinical outcomes of infections with mrsa. initial data collected at our center did not show a significant difference in outcomes between patients infected with ca vs. ha mrsa. a multicenter study would most efficiently assess the impact on clinical outcomes and determine regional differences of mrsa patterns (ca vs. ha) in different cf centers. ref : the stringent response is a global regulatory mechanism used by bacteria to adapt to nutrient limitation and other environmental stresses, and is mediated by the signaling molecules (p)ppgpp (phosphorylated guanosine nucleotides). increases in (p)ppgpp repress metabolic processes and growth, and regulate genes involved in long-term stress and starvation survival in several species ((i)e. coli, m. tuberculosis(i)), bacterial multicellular behavior ((i)myxococcus xanthus(i)), and pathogenesis ((i)p. aeruginosa(i)). while many studies have investigated bacterial functions (such as attachment and motility) involved in biofilm development, less is known about the physiological adaptation occurring in biofilm growth. when cells grow to high densities entrapped in a polymer matrix, they must adapt to heterogeneous micro-environments where gradients in nutrients, oxygen and metabolic waste exist. nutrient may thus become depleted as they are consumed by overlying cells, and some biofilm subpopulations are likely starved. we hypothesized that the stringent response mediates adaptation to nutrient starvation in (i)p. aeruginosa(i) and is important for biofilm formation. we tested a (i)rela(i) deletion mutant unable to synthesize (p)ppgpp in response to amino acid starvation, and its isogenic wildtype parent pao1. we measured the starvation survival of planktonic bacteria in phosphate buffered saline and observed that the (i)rela(i) has a 10 to 100fold greater loss in viability compared to wild type after 48h. we also tested biofilm formation in a static biofilm assay using polystyrene peg lids and estimated biomass accumulation by crystal violet staining. in this system, the (i)rela(i) accumulates 5 times less biomass than wild type. in a flow-cell reactor system, the wildtype strain forms complex mushroom-like biofilm structures, but the (i)rela(i) mutant forms flat, thin biofilms. we determined cell attachment to polystyrene by crystal violet staining, and to glass surfaces by direct visualization under microscopy. the attachment to both surfaces is similar in the (i)rela(i) and wildtype strains, and the strains have identical planktonic growth rates. our findings suggest that the stringent response is involved in starvation survival, a physiological condition relevant to growth in biofilm. furthermore, (i)rela(i) inactivation impairs biofilm formation, suggesting that the stringent response may be an important stress adaptation in biofilm. belfast, united kingdom; 2. northern ireland public health laboratory, belfast city hospital, belfast, united kingdom; 3. northern ireland regional adult cf unit, belfast city hospital, belfast, united kingdom; 4. department of respiratory medicine, queen's university, belfast, united kingdom pseudomonas aeruginosa (pa) is a clinically significant bacterial pathogen responsible for increased morbidity and mortality in patients with cystic fibrosis. small non-coding micro(mi)rna species (generally 80~100nts or less) in prokaryotes are involved in numerous cellular processes as is the case with eukaryotes. as in eukaryotes, these mirnas act by base pairing with target mrnas imposing translational and stability changes of mrnas and thus culminating in the control and regulation of target mrnas, crucial for bacterial stress responses and virulence to changing environments surrounding the infected zone or host cell (e.g. human airway epithelial cells). we recruited a commercial kit (biodynamics dynaexpress mirna cloning kit av, tokyo, japan) and isolated mirnas from a variety of pseudomonas pathogens from plant, soil / agricultural environment (pseudomonas syringae -ps), type strain (pseudomonas aeruginosa -pa) and a collection of clinical isolates of pseudomonas aeruginosa (cfpa) from adult cf patients, who were chronically infected with p. aeruginosa. the results on the mirna clones obtained exhibited a wide variation in the occurrence of mirnas in this bacterium. the putative mirnas obtained, cfpa in particular, yielded small non-coding rnas of sizes from as little as 16nts to well over 45nts. an initial search using the bioinformatic tool srnapredict (www.waldorlab/tufts.edu) has not as yet, fully revealed the genomic annotations for these novel srna sequences in the inter-generic regions of the pathogenic pseudomonad group of bacteria. we present our data on these unknown cellular mirnas cloned from cfpa isolates from cf patients and discuss their significance as novel regulators to bacterial stress responses and in the context of the bacterium's involvement as the predominant pulmonary pathogen, associated with cystic fibrosis. airway function is diminished in infants with cf diagnosed clinically but whether this is true for those identified by newborn screening remains unclear. we investigated whether lung function is diminished in infants with cf diagnosed by newborn screening and if this occurs in association with pulmonary infection. methods:lung function was measured in asymptomatic infants with cf following sedation with chloral hydrate (60-100mg/kg) using the raised volume rapid thoracic compression technique at an inflation pressure of 30cmh 2 0. we measured forced vital capacity (fvc), forced expired volume after 0.5 seconds (fev 0.5 ) and forced expired flow at 75% expired fvc (fef 75 ) which were then expressed as z scores. 1 broncho-alveolar lavage (bal) was performed under general anesthesia within forty-eight hours following infant lung function testing. three separate aliquots of 1ml/kg sterile saline were instilled into the right middle lobe and a further aliquot in the left lingula and the bal fluid analyzed for bacterial, fungal and viral pathogens using routine culture techniques. results: bal was successful in all 24 infants studied aged 0.2 to 2.1 yrs (median 1.0 yrs) and lung function in 21 infants. median (range) z scores for fvc, fev 0.5 and fef 75 were -1.3 (-2.7 to 1.6), -2.0 (-5.0 to 0.6) and -1.7 (-3.5 to 0.47) respectively. these decrements in lung function were all significant (p<0.01). bal cultures were positive in 12 (50%) infants of whom 4 (17%) had evidence of infection with s. aureus and 1 (4%) with p. aeruginosa. in 12 infants there was no growth in bal or growth only of mixed oral flora. viruses were not identified in any infants. there was no association between any of the lung function parameters and pulmonary infection detected by bal. lung function was successful in a subgroup of 7 of 9 infants who were less than 0.5 yrs (median age 0.3 yrs) when tested. diminished airway function was identified in this younger subgroup even though only one infant had evidence of pulmonary infection with respiratory organisms in bal. in this younger subgroup median fev 0.5 z score was -1.6 which was significantly below that predicted (95% ci: -2.3,-0.2 and p=0.03). conclusions: airway function is diminished in infants with cf diagnosed by newborn screening and occurs irrespective of infection identified by bal. lung function is abnormal in very young infants before pulmonary symptoms develop and when no evidence of pulmonary infection can be detected in bal. 1 inhaled high-dose tobramycin appears to transiently clear p. aeruginosa (pa) from airway secretions in young children with cf, though inflammation may not be markedly reduced (am j resp crit care med 2003; 167:841) . because inhaled antibiotics may not reach some areas of infection, we hypothesized that intravenous (i.v.) antibiotics may be more effective than inhaled high-dose tobramycin for reducing lower airways inflammation in cf children with early pa infection. study design: we initiated a single-center, randomized, prospective study comparing the effects of two antibiotic treatment regimens on bronchoalveolar lavage fluid (balf) inflammatory markers. clinically stable cf children with a recent isolate of pa from surveillance cultures are randomized to receive 4 weeks of tobi® (group 1) or 2 weeks of i.v. ceftazidime and tobramycin (group 2). balf is obtained just before treatment, and repeated 4-6 weeks after completion of treatment. the primary study outcome is change in balf % neutrophils (pmn). multiple secondary outcomes are assessed including quantitative bacterial cultures and cytokines in balf. this report summarizes interim analysis at the halfway point of the study. results: to date, 18 subjects have enrolled. five subjects (3 in group 1, 2 in group 2) dropped out after the initial bronchoscopy due to a decision by the primary cf physician to use other treatments based on balf culture results (2) , or because of the development of a new respiratory illness before the second bronchoscopy (3). a total of 5 adverse events (1 significant, unrelated to protocol) were reported in 3 subjects, none resulting in a change of protocol request by the irb or data safety monitor. of the 13 subjects completing the protocol to date, 5 were in group 1, and 8 were in group 2. the groups were well matched in terms of age, pa in initial balf, and initial balf %pmn. a majority of subjects in each group were children with their first isolation of pa on surveillance cultures. there was a tendency for group 2, but not group 1, to have mild reductions in balf %pmn (-9 ± 6%) after treatment. interim efficacy analysis did not suggest a significant difference between the treatment groups at this stage. similar changes were seen in group 2 for pmn/ml (-589 ± 351 x 10 3 /ml) and several cytokines including gm-csf (-54 ± 27 pg/ml) and mcp-1 (-368 ± 231 pg/ml). two group 2 subjects were unable to have stable i.v. access established and were treated with oral ciprofloxacin and inhaled tobi® for 2 weeks as alternative systemic antibiotics, per protocol. dropping these subjects from group 2 data did not change data trends. conclusions: the protocol appears to have adequate safety. parents of children with first-time pa infection may be more likely to consent to treatment randomization, than those with repeated pa infection. interim data analysis suggests that continuation of the study toward the original sample estimate is appropriate. acknowledgements: supported by cff(noah04a0) and nih (gcrc r00046). we thank tracy callahan rn, lupe haynes rrt, justin hubbard, paula murphy, cassidy henegar, nancy lee rn, benjamin butler rn, and sheree berckmans rn for logistical and technical support. methods: our local cf database was searched for patients born 1985-2000. patients were included in the analysis if they were followed in our cf center before the age of 5. patients were separated into two birth cohorts, born 1985-1992 (cohort 1) and 1993-2000 (cohort 2). yearly peak fev1% predicted from ages 6-12 were obtained using knudson reference equations. yearly peak bmi% and weight-for-age% at each age were also obtained. type 3 tests of fixed effects and repeated measures analyses were used to assess mean peak bmi%, mean peak weight-for-age%, mean peak fev1% predicted at first pft, mean peak fev1% predicted from ages 6-12, and mean rate of change (slope analysis) in peak fev1% predicted from ages 6-12. chi-square test for association was used to analyze differences in demographic parameters. wilcoxon rank-sum test was used to assess differences in age at diagnosis. wilcoxon non-parametric test was used to analyze differences in mean clinic visits per year. chi-square testing with a two sided alpha of p <0.05 was used to determine significance. results: 144 cf patients were included in the analysis. there were no significant differences between birth cohorts 1 and 2 for age of diagnosis (p=0.94), sex (p=0.3792), race (p=0.35), genotype (p=0.16), pancreatic insufficiency (p=0.61), or the mean number of clinic visits per year from ages of 0-5 (p=0.33). there was a significant difference in the mean number of clinic visits per year from ages of 6-12 (p=.0009; see table 1) . birth cohort had a significant effect on the rate of decline in lung function from ages 6-12. birth cohort and age had significant effects on the mean weight-for-age%, mean fev1% predicted at first pft, and mean fev1% predicted from ages 6-12. sex had a trend for significant effects on lung function parameters. adjusted for age and sex, there were significant differences between birth cohorts 1 and 2 in mean bmi% and mean weight-for-age% at ages 6 and 12, mean fev1% predicted at first pft, mean fev1% predicted from ages 6-12, and in the mean rate of decline in fev1% predicted (see table 1 ). conclusions: improvements in lung function and nutritional outcomes in our cf center are associated with an increase in the mean number of clinical visits per year for cf patients ages 6-12. the frequency of clinical follow-up is a marker of cf care that may have positive impacts on cf outcomes. background: current management of cf airway disease includes the use of key pharmacological therapies designed to combat the chronic cycle of obstruction, infection, and inflammation. randomized controlled trials have demonstrated that maintenance therapy with azithromycin (azm), dornase alfa (dnase), and tobramycin for inhalation (tob) each improves pulmonary function and reduces the frequency of exacerbations in patients with p. aeruginosa. the purpose of this study was to determine if cf patients in an uncontrolled environment (clinical setting), experience a reduced rate of pulmonary function decline when receiving azm + dnase + tob compared to those not on all three therapies. methods: adult cf patients were selected based on positive sputum cultures for p. aeruginosa, pulmonary function test results (fev 1 % predicted) for the past year, and receiving one of the key pharmacological therapies. in addition data was collected on cfrd, depression, medication adherence, airway clearance, and nutritional status. patients were stratified according to baseline pulmonary function: mild (fev 1 ), moderate (fev 1 of 40-70%), and severe (fev 1 <40%). patients were then sub-grouped as to whether they were receiving all 3 or ≤ 2 of the key therapies. the annual rate of decline in % predicted fev 1 was determined using linear regression on non-exacerbation data and was categorized as stable (<2.3% decline), intermediate (2.3-4.1%) , or rapidly declining (> 4.1%). time to intermediate or rapid decline in pulmonary function was determined using cox proportional hazard modeling. results: 107 patients (54 males/53 females, median age 28 years) were included in this study; 28 with mild, 42 with moderate, and 35 with severe pulmonary disease. overall, the mild and moderate groups experienced median declines of 2.12% and 1.50%, while the severe group experienced a 2.41% improvement in fev 1 % predicted respectively. the use of regimens containing azm therapy significantly delayed time to intermediate or rapid decline in lung function in the mild group (median 210 days vs. 49.5 days, hr=0.19, p=0.013). similarly, regimens containing dnase + azm + tob significantly delayed time to intermediate or rapid decline in lung function in the mild group (median 231 days vs. 105 days, hr=0.27, p=0.048). no significant differences were seen in the moderate or severe group for patients receiving all 3 or individual therapies. con-clusion: administration of pharmacological regimens containing dnase + azm + tob, or azm + tob or azm + dnase significantly delay progression of pulmonary disease in cf patients with mild lung disease. sawhney, v. 1 ; arthur, b. 2 ; seltzer, r. 3 ; kraynack, n.c. 4 1. internal medicine, akron general medical center, akron, oh, usa; 2. pediatrics, akron children's hospital, akron, oh, usa; 3. biostatistics, neoucom, rootstown, oh, usa; 4. pulmonology, akron children's hospital, akron, oh, usa early and efficient diagnosis of pulmonary exacerbations (pex) in patients with cystic fibrosis (cf) is crucial in the management of cf. in oct 2004, we implemented a pulmonary exacerbation scoring (pes) system to uniformly and consistently identify pex in cf patients as part of a quality improvement project at the cf center at akron children's hospital. we have previously reported improved pulmonary function outcomes in pediatric patients at our center after implementation of the pes. we now describe the impact of the pes on clinical practice in the management of pex and pulmonary function in our adult cf patients. the pes was developed by our multidisciplinary cf team after an extensive literature review and was implemented in oct 2004. the pes has been previously described and consists of 13 clinical questions divided into three domains: systemic and pulmonary symptoms and signs, and objective measurements. these elements are scored individually and a cumulative pes of ≥ 5 (range 0-17) is considered a pex and treatment with antibiotics is recommended. the course and choice of antibiotic regimen is left to the physician's discretion. we examined median percent predicted fev 1 decline from quarterly percent predicted fev 1 measurements obtained from port cf. we calculated this for all the patients seen in our adult cf clinic (age>19) from oct 2004 till sep 2006 during which the pes was in use. we compared this rate of decline to median percent predicted fev 1 decline for the same cohort of patients over the preceding two years (oct 2002 till sep 2004 , during which the pes was not in use. the percent utilization of the pes was calculated individually for all adult cf care providers. the influence of individual components of pes in decision to treat was also studied. we found no significant difference between the rates of decline in percent predicted fev 1 during the pre-pes and post-pes periods (mean = -.25, sd = 1.58 vs mean = -.59, sd = 1.66, p=0.29) as measured by paired samples t-test. logistic regression analysis was done to evaluate the relative importance of each pes component in predicting decision to treat a pex. we found that changes in fev 1 (or=1.87), cough (or=2.64), sputum (or=3.28), and chest examination (or=1.51) are most likely to affect the decision to treat where as dyspnea (or=0.62) has the little effect on decision to treat. we found a high degree of correlation between adult provider use of the pes and an independent chart reviewer (correlation coefficient 0.66-0.87; p<0.001). in contrast to cf patients ages 6-18 years, we found no significant effect of use of the pes on pulmonary function in our adult population, despite the high frequency of use by our adult providers. it is unclear why this is the case given the robust effect of the pes on our pediatric population. this study suggests that a simplified score that includes fev 1 , cough, sputum, and chest exam may be useful in identifying a pex in adult cf patients. special thanks to tasha capozzi for her help with chart review. this project was supported by the akron children's hospital foundation. objective chronic pulmonary infection with pseudomonas aeruginosa is the main cause for morbidity and mortality in cystic fibrosis (cf) patients. tobramycin and colistin, widely used inhaled antibiotics in cf patients, were found to affect elastase activity in vitro. we showed recently that cxcr1 on neutrophils mediates bacterial killing, but is cleaved in cf airways by elastase. therefore, we examined the effects of inhalation with tobramycin and colistin on cxcr1 expression and antibacterial capacities by airway neutrophils in cf patients in vivo. methods cxcr1 expression was quantified by flow cytometry on neutrophils in peripheral blood and induced sputum of 25 cf patients without inhalation therapy, 25 cf patients with tobramycin and 25 cf patients with colistin inhalation therapy. the longitudinal effect of a 2 weeks tobramycin or colistin inhalation period on cxcr1 expression, free elastase levels and bacterial killing by airway neutrophils was assessed. results inhalation with tobramycin increased, while inhalation with colistin decreased cxcr1 expression on neutrophils in induced sputum of cf patients. neutrophils isolated from sputum of cf patients after tobramycin therapy had higher, whereas patients after colistin therapy had lower bacterial killing capacity and α-defensin release as compared to neutrophils before antibiotic inhalation therapy. conclusions tobramycin and colistin differentially modulate the pulmonary host defense in cf patients in vivo via cxcr1 on neutrophils. these findings may have clinical implications when considering antibiotic treatment in cf patients. sharp, j. 1 ; sheehan, d. 1 ; ren, c.l. 2 1. pediatrics, women & children's hospital, buffalo, ny, usa; 2. pediatrics, university of rochester, rochester, ny, usa background: airway infection and inflammation occur early in infants with cf. the raised volume/rapid thoracoabdominal (rv/rtc) technique is more sensitive than older infant pulmonary function test (ipft) methods in detecting airflow obstruction in these patients. however, there are limited data regarding lung function measured by rv/rtc in infants with cf nbs. we hypothesized that abnormalities of lung function measured by rv/rtc are present in cf nbs infants. to test this hypothesis, we reviewed the ipft data from cf nbs infants followed at our cf centers. methods: this study was a retrospective chart review. we identified infants with cf nbs followed at our cf centers who had had at least 1 ipft performed in the first year of life. ipfts were performed using the collins ipl. forced expiratory flows were measured using rv/rtc as described by feher et al (j appl physiol 80:2019 , 1996 and fractional lung volumes were measured by whole body plethysmography (bp) as described by castile, et al (ped pulm 30:215, 2000) . data from the cf infants were compared to normal data reported by jones, et al (ajrccm 161:353, 2000) for rv/rtc and castile, et al for bp. results: we reviewed data from 22 infants. their mean age was 9.7 m (range=4-24). pancreatic insufficiency was present in 20 (91%) infants, and 3 (12.5%) had been treated for pseudomonas infection. their pft findings are summarized in the table. fvc and fev0.5 were not significantly different from the normal predicted values. however, the mean fef75 and fef25-75 z scores were significantly below the mean of normal infants (p=0.02 and 0.03 respectively). gas trapping was present, as evidenced by significantly increased frc, rv, and rv/tlc (p<0.001 for all 3 measures). six infants (27%) had an fef75 z score of < -2.0, and 20 (91%) had an frc > 100% predicted. conclusions: infants with cf nbs have abnormal lung function early in life. our results suggest that pfts in cf nbs infants can be useful in detecting early changes in lung function. we speculate that early therapeutic interventions may improve lung function in this group of patients. table. pft findings in infants with cf nbs. forced expiratory flows are reported as z scores, while fractional lung volumes are reported as % predicted. objective: clinicians use pfts to assess effectiveness of therapy in hospitalized cf patients. however, the variability of multiple pft efforts has not been described in this group of patients. previous study of within day variation of pfts has been reported in healthy adults and children (enright, 2004, and nickerson, 1980) and clinically stable outpatients with cf (nickerson, 1980, and cooper, 1990) . nickerson and cooper found coefficient of variations (cv) for fvc of 1.9-6%, fev 1 of 2.4-5.3%, and fef 25-75 of 7.4-9.3% for adults and children with cf. no one has studied the within day variation of pfts in cf patients during pulmonary exacerbations. our objective was to determine the within day variation of pfts during hospitalizations for cf pulmonary exacerbations. we hypothesized that within day variation would improve during treatment. methods: we retrospectively reviewed pft data for all patients admitted to childrens' hospital and regional medical center for a cf pulmonary exacerbation in 2005. patients performed pfts under the direction of experienced pulmonary function technicians. the test was complete when the patient performed 3 efforts that met ats/ers criteria for acceptability. if the patient was unable to produce 3 efforts because of coughing, 2 efforts were accepted if the fvc and fev 1 were within 5% or 150 ml of each other. for analysis, we included only those pfts: (1) with at least 2-3 flow-volume curves that fulfilled ats/ers criteria, and (2) were performed within 3 days of hospital admission or discharge. for patients with multiple admissions, we used pft data for their first admission only. for patients with admission and discharge pfts available, we compared the coefficient of variation (cv) for pfts (fvc, fev 1 , ). there were at least 8 days between admission and discharge pfts. the wilcoxon rank-order test was used to compare the cv for admission and discharge pfts. data: sixty-one patients were admitted 114 times in 2005. excluding repeat hospital admissions, 17 patients had individual pft trials available at the time of admission and 13 patients at the time of discharge. seven admission and 2 discharge pfts included only 2 trials. three patients at admission, and 2 patients at discharge, were unable to produce pft curves that met ats/ers criteria, despite previously having done so when well. nine patients had paired admission and discharge pfts from the same hospitalization. the cv for fvc at discharge was significantly better than at admission, p = 0.02 (see table) . conclusions: within day variation of pfts during hospitalizations for cf pulmonary exacerbations is comparable to published outpatient data. cv for fvc improves significantly during exacerbation. changes in fvc > 4%, fev 1 > 3%, and fef 25-75 >10% can be considered a significant spirometric response to therapy in hospitalized cf patients. background: lung clearance index (lci), a measure of ventilation heterogeneity, can be calculated from multiple breath washouts of inert gas. in cross sectional studies it is a more sensitive measure of early airway dysfunction in cf than spirometry alone. in normal subjects, the technique is reproducible, with a narrow normal range of 5.9-7.5. we hypothesized that it may be helpful in detecting change longitudinally, and may have value in assessing novel therapeutic interventions such as the upcoming gene therapy by the uk cfgt consortium. methods: patients aged 10 years and over, presenting with an exacerbation requiring intravenous antibiotics, were recruited. lci was assessed in triplicate by multiple breath washout of 0.2% sulphur hexafluoride (sf 6 ), using a novel gas analyser (innocor, denmark) and custom-built software within the first 24 hours of starting treatment. the test was repeated at the end of parenteral antibiotic therapy and again 4-10 weeks later when clinically stable. patients were recruited at three sites and assessed using standardised equipment and a data analysis protocol. results: paired lci measurements were available in 33 patients, mean age 24 (range 11-44) years, 18 male. in a further 4 patients, technically acceptable washouts could not be obtained at one or other time point. mean (sd) lci improved from 14.7 (2.9) to 13.9 (2.4), p<0.005. in 11/33 patients there was an improvement in lci of >10% after antibiotics. 2 patients showed a >10% deterioration in lci. mean fev 1 was significantly greater after treatment (2.34 v 2.11 l/s, p<0.05, n=18). there was a weak correlation between percent change in fev 1 and lci (r 2 =0.33, p<0.05). 26 patients also had lci measured at the follow up. mean (sd) lci at this visit was 13.9 (2.5), p<0.05 vs visit 1, no significant difference vs visit 2. conclusions: lci can be used to demonstrate changes in the lung after antibiotic therapy. this is the first study in cf to demonstrate a lon-gitudinal improvement after intervention in a marker of ventilation heterogeneity, and offers different but complimentary information to spirometry. the higher sensitivity of lci may make possible measurement of improvement in mild patients after novel therapies such as gene therapy. this work was funded by the cystic fibrosis trust. aim: inhaled hypertonic saline positively affects sputum production in patients with cystic fibrosis (cf) by improving mucociliary clearance. this study was conducted to compare the aerosol delivery performance of various nebulizers upon aerosolization of hypertonic saline (hyper-sal™) 3.5% and 7%, available as preservative-free, sterile 4 ml solution in single dose, patient friendly blow-fill seal vials. method: 4 ml hypertonic saline (hyper-sal™) 7% was nebulized by three jet nebulizers the pari lc plus®, lc star® and hudson rci micro mist as well as the eflow® for hypertonic saline (hs) generating aerosols via a perforated vibrating membrane. hs 3.5% was characterized in the eflow®hs. droplet size distribution patterns were assessed by laser diffraction at a flow rate of 15 l/min and aerosol delivery performance by breath simulation tests mimicking a sinusoidal breathing pattern. the delivered dose (dd) was determined by gravimetric analysis of the inhalation filters. inhalation and exhalation filters were changed after 2 min and the drug delivery rate (ddr) was calculated as nacl found on inhalation filters per min. respirable drug delivery rate (rddr; ddr x respirable fraction <5 µm) indicates how much nacl can reach the lungs per minute. results: aerosol characteristics of the different nebulizers after aerosolization of 4 ml hs 7% are summarized in the table. data for hs 3.5% nebulized by the eflow®hs were as follows: dd= 61.1%, ddr= 9.3 %, nebulization time = 6.6 min and comparable to data for hs 7%. the lower recoveries for the jet nebulizers are probably associated with increased evaporation and longer nebulization times. conclusion: about 18 -59% of the charged sodium chloride from 4 ml hs was delivered and 57% -76% of these droplets were in a respirable size range <5 µm resulting in vitro respirable doses of about 10% -40% of the label claim. ddr and rddr are about 3-to 10-fold higher for eflow®hs indicating a much higher delivery efficiency compared to conventional jet nebulizers. further studies will be needed to demonstrate that about 2 ml hs may be sufficient to deliver a comparable dose to the lungs using an eflow®hs compared with jet nebulizers. this bears the potential to further reduce nebulization time and increase acceptance by patients. objectives: individuals with cf frequently have complicated and time consuming treatment regimens consisting of inhaled antibiotics, mucolytics, and airway clearance. patients and clinicians may have difficulty determining which specific therapies are effective for any individual patient or whether a particular treatment is causing adverse effects. this study assessed the utility of home spirometry for providing an objective measure of pulmonary status in between scheduled clinic visits. methods: consecutive adults with cf were recruited at a single academic medical center and were asked to measure fev1 twice daily for six months using the piko-1 meter (nspire health™, llc). 35 subjects agreed to participate and 24 completed the study. subjects were given diaries to record changes in respiratory symptoms and changes in medications during the study period. subjects were evaluated in clinic at least every three months and fev 1 values obtained in the pulmonary function lab were compared to home fev 1 from the same day using linear regression and bland-altman analyses. graphical displays of home fev 1 measures were visually inspected for changing trends and these were compared with medication changes. results: the mean fev 1 measured at home was 2.14±0.2l. the mean fev 1 in the pulmonary lab was 2.35±0.22l. the home fev 1 was significantly correlated with the clinic value from the same day, r 2 =0.94, p<0.001. fev 1 measures fell within the limits of agreement (±2 sd) by bland-altman plot. in 7 of 24 subjects (29.2%) there was at least one sustained change in fev 1 that correlated with either stopping or starting a particular treatment. the figure shows two significant changes in fev 1 in one patient related to medication changes. there is an increase in fev 1 at the initiation of aerosolized 7% saline and a fall in fev 1 that corresponded to inhaled tobramycin use. the improvement on hypertonic saline increased this patient's enthusiasm for this treatment. conclusions: home fev 1 monitoring is feasible and appears to provide accurate measurement of pulmonary function. it can provide clinicians with objective data to tailor treatments without necessitating a clinic visit. feedback from home monitoring may improve medication adherence and may allow treatments to be individually tailored based on patient responses to the medications. 1 1. respiratory unit, royal hospital for sick children, glasgow, united kingdom; 2. diagnostic imaging, nhs greater glasgow & clyde, glasgow, united kingdom; 3. medical statistics, royal hospital for sick children, glasgow, united kingdom; 4. medical physics department, gartnavel hospital, glasgow, united kingdom background: there have been calls for caution regarding the suggested use of regular chest computed tomography (ct) scans for monitoring disease progression in cf 1 . consideration of the associations of higher ionizing radiation exposure must be made before regular chest cts are performed on the wider cf population. aim: we tested the hypothesis that higher ionizing radiation exposure from chest radiographs (cxr) and chest cts would be associated with markers of severity including gender, genotype (class i/ii or iii-v), height sds, weight sds, bmi, fev 1 sds, length of hospital admission (days), scottish index of multiple deprivation (simd) 2 scores and pa/bc microbiological infection (chronic or intermittent colonisation with pseudomonas aeruginosa and/or burkholderia complex). methods: effective doses of ionizing radiation (msv) from cxrs and chest cts (including hr scans) were determined for all cf children at our centre between 1st january 2003 and 11th july 2006 (184 weeks). the most recent anthropometric and lung function data to july 2006 were used. simd scores report a data zone that encompasses ~750 individuals and comprises 31 indicators in six domains (current income, employment, health, education, housing and access). results: 134 cf patients (67 male:67 female) underwent 1853 radiation exposure procedures (mean of 3.9 procedures per patient per year). the average effective doses were calculated for 1806 of the 1853 procedures (97.5%) including 1591 cxrs and 35 chest cts. lung function data were available on 89 patients (66%). univariate analysis revealed that higher ionizing radiation exposure was significantly associated with fev 1 sds (p<0.001), length of stay (p<0.001) and pa/bc infection (p<0.001). the regression line indicates that for each drop of two fev 1 sds, each child receives an extra 3msv in radiation exposure from cxrs and chest cts alone. similarly, each child receives an extra 2msv in radiation exposure from cxrs and chest cts for every 100 days of hospital admission. multivariate analysis with backward selection demonstrated that length of hospital admission (p<0.001) and pa/bc infection (p=0.05) were associated with higher ionizing radiation exposure. discussion: in cf, higher ionizing radiation exposure from cxrs and chest cts in childhood are associated with poor lung function, pa/bc infection and length of hospital admission but not other indicators of severity such as gender, severe genotype, anthropometric measures or socio-economic status. using effective doses of ionizing radiation as an outcome marker in cf is an alternative means of assessing the impact of the disease and its management on the child. background: exhaled volatile compounds have attracted attention as potential non-invasive biomarkers of chronic lung disease. early sustained airway inflammation and bacterial infection are characteristic features of pulmonary involvement in cystic fibrosis (cf). the cellular response is dominated by neutrophils, which have been shown to generate oxygen-and halogen-derived radicals as part of their host defense armamentarium. aims: to gather pilot data on the pulmonary output or uptake of selected organic trace gases in cf patients and healthy controls; and to explore the impact of pulmonary exacerbations and antibiotic treatment. methods: samples of ambient air and exhaled breath were collected from 30 children, adolescents and young adults with cf and from 39 healthy controls. the patients' age ranged from 9-29 years and their fev 1 between 21-114 % predicted, 27 were pseudomonas positive. twelve patients were studied at the start of i.v. antibiotic treatment for a pulmonary exacerbation; repeated sampling after the course was performed in 7 subjects. all samples were analyzed on a customized gas chromatography system with flame ionization and electron capture detectors. after determining the mixing ratios of a variety of volatile organic compounds, alveolar gradients were calculated from the corresponding exhaled and ambient values. results: compared to healthy control subjects, cf patients showed a significantly lower respiratory output of methanol, ethanol, acetaldehyde and dimethylsulfide but a higher net release of benzene, methylchloride, trichloromethane and trichloroethene. these differences were most pronounced in patients with acute exacerbations. a relevant scatter and overlap between groups was noted for ethane, pentane, acetone, isoprene, toluene, and tetrachloromethane. after antibiotic treatment, the output of benzene, methylchloride and trichloromethane decreased by 40-250%. conclusion: although important pathways remain incompletely understood, the local biochemical milieu in cf lungs may be informative and accessible through non-invasive breath testing. in this context, the diagnostic potential of exhaled organic traces gases (especially oxygenated, halogenated and aromatic compounds) should be further explored. supported by: german research foundation (dfg ba 1895/3-2). toy, e. 2 ; weiner, j. 2 ; sacco, p. 1 ; duh, m.s. 2 1. health economics, novartis pharmaceuticals, east hanover, nj, usa; 2. analysis group, inc., boston, ma, usa objective: to review up-to-date economic outcomes data in patients with cystic fibrosis (cf), and in particular, costs related to respiratory infection by pseudomonas aeruginosa (pa), which is the leading cause of morbidity and mortality in cf patients. methods: a systematic search of the medline database from 1990-2007 was conducted to identify major and review articles that contained the terms "cystic fibrosis" and "cost" and which were published in english language journals. selected conference abstracts were searched, and additional articles were identified through bibliographies of retrieved articles. recent articles that contained economic data on antibiotic and mucolytic therapies were selected for in-depth review. cost estimates were converted to 2006 us dollars for comparability. results: approximately 300 articles fulfilled the initial search criteria, and 27 were selected for in-depth review. articles were divided into four categories: economic impact of inhaled tobramycin (2 articles), the effect of home-vs. hos-pital-based antibiotic therapies for pulmonary exacerbations (4 articles), economic impact of recombinant human deoxyribonuclease (rhdnase) (10 articles), and cost-of-illness for cf (11 articles). inhaled tobramycin (actual or recommended dose of 300 mg/5ml) has been associated with reductions in health care costs; these cost savings offset between 37% and 57% of the cost of this therapy. antibiotic therapy for pulmonary exacerbations generally resulted in lower health care costs when administered in a home setting rather than a hospital setting. use of rhdnase led to reduction in health care costs, with higher reductions observed in patients with higher levels of use; the cost savings offset between 17% and 38% of the drug cost. cost-of-illness studies have been conducted in 7 different countries; the economic estimates varied widely across studies due to differences in treatment patterns, health systems, methodologies, and patient populations. most cost-of-illness studies were retrospective observational studies from the perspective of a hospital or third-party payer, and the cost of cf ranged from $9,000 to $64,000 per patient per year. the largest cost categories included hospitalizations, out-patient visits, rhdnase and antibiotics, while disease severity and presence of pa infection were common determinants of cost. cost-of-illness studies have underestimated societal costs because they have rarely considered patient time costs and none have considered indirect costs (e.g., burden on lost productivity or informal caregivers). conclusions: studies show that inhaled tobramycin and rhdnase can partially offset medical costs; home-based iv antibiotic therapy is likely to reduce costs; and direct costs associated with cf can be high but vary widely across countries and analytical methodologies. areas for future research include direct comparisons of inhaled antibiotic therapies, examination of the relationship between treatment adherence and economic outcomes, and estimation of societal cost-of illness. this study was sponsored by novartis pharmaceuticals corporation, east hanover, nj. background: sputum interleukin 8 (il8) and myeloperoxidase (mpo) are advocated as markers for infective exacerbations in cf. we have previously shown that the neutrophil protein, calprotectin, is a sputum biomarker of disease activity in cross sectional and longitudinal studies. in the present study we evaluate longitudinal measurement of sputum and serum calprotectin during infective exacerbation. methods: sputum and venous blood samples were taken at the beginning (visit 1) and end of a course of iv antibiotics (visit2), and at time of clinical stability following the infective episode (visit 3). processed sputum supernatant was analysed by elisa for calprotectin, il8 and mpo. serum calprotectin was assayed by elisa. results: 36 paired sputum samples samples were available for visits 1 and 2 and 25 paired samples for visits 2 and 3. all data are presented as mean(sem). sputum calprotectin decreased from 1123(134) to 852(136) µg/ml, p<0.05, from visit 1 to 2. there was a non-significant increase to 1052(159) µg/ml at visit 3. sputum il8 did not change from visit 1 to 2 [13.3(1.5) 6(4.2) ]. serum calprotectin decreased from 51(9.5) to 16.5 (2.5) ng/ml, p<0.0001 (n=35 paired samples) from visit 1 to 2. at visit 3 there was an increase to 18(2) ng/ml, p<0.05 (n=28). discussion: calprotectin appears a valuable marker both in sputum and serum for tracking changes in lung inflammation during an exacerbation of cf and may be more sensitive to change than il8 or mpo. on stopping antibiotic therapy sputum and serum calprotectin increase implying that this marker may be sensitive to small sub-clinical changes in lung inflammation. these data suggest that sputum and serum calprotectin might be employed to assess responses to drug therapies in cf such as gene therapy. this research was funded by the cf trust uk. chronic lung disorders are usually associated with a hypoxia driven increase in red cell mass. however, patients with cystic fibrosis often have normal or decreased haemoglobin levels. the present prospective observational study in cystic fibrosis (cf) patients was performed to determine which factors were involved in alterations in the hematopoetic response to corresponding arterial oxygen pressure. sixty adult patients (age 21-51) with stable cf were included. they all had vitamin a, d, e and k but no vitamin b12 supplementation. 25 patients were on oral fe2+ (100mg/day). resting arterial blood gases, lung function, complete blood counts, parameters of iron status, crp, sputum microbiology and serum erythropoietin were measured at recruitment and after 3 and 6 months. patients had varying degrees of pulmonary functional impairment and 9% were hypoxemic (arterial oxygen pressure < 60 mm hg). low-grade systemic inflammation (crp > 0.5 mg/dl) was present in 40% of the patients, who all had bacterial colonization. none of the patient had erythrocytosis and 12 patients had anemia . there was no significant difference in iron status between patients with or without chronic iron supplementation and erythropoietin levels were normal. during the 6 months observation period no significant changes occurred. the patients exhibited an impaired erythropoietic response to hypoxemia with normal or low hematocrit in spite of chronic lung disease. linear multivariate regression analysis (table) revealed crp and iron levels but neither iron substitution, nor erythropoietin levels nor lung function parameters as independent determinant of haemoglobin levels. cf may be associated with anemia of variable severity as expression of the chronic inflammation present in these patients. the therapeutic consequences are to treat the underlying inflammation rather than to supplement iron. variables included in initial model: background improvement in the treatment and survival of females with cf has led to many leading a normal adult lifestyle, and some can now expect to raise a family. however, pregnancy can have a profound impact on wellbeing, and caring for a young child can affect compliance with cf treatment. to look at this further, we surveyed the experience of pregnancy and motherhood in cf women attending our large adult cf centre (230 patients). methods we reviewed the records of 10 cf women who chose to remain pregnant (12 babies [7 male] from 11 pregnancies), looking at changes in lung function, bmi, respiratory exacerbations during pregnancy, clinic attendance, and oral and iv antibiotic therapy (home and inpatient care) during pregnancy and also in the first year post delivery. we devised a questionnaire to examine how patients coped with pregnancy and the demands of motherhood, focussing on changes in compliance with therapy, worries regarding the effect of cf treatments on the baby, and support given by the cf team. results at conception mean fev1 was 82% predicted (range 61 to 107) and mean bmi 21 (range 18 to 26).there were no miscarriages or neonatal mortalities and all children remain well, but the sickest mother died at 4 years. mean fev1 following pregnancy was 73% [range 51 to 113] (p=0.01), with 1 mother losing 21% by one year post delivery. at one year, bmi was unchanged (mean 21, range 16 to 27). rates of respiratory exacerbations and iv antibiotics were similar pre and during pregnancy, although during the first year post delivery 2 mothers avoided hospital based treatment preferring home care. six mothers' clinic attendances increased during and post pregnancy (1 attended outpatients 17 times in the first year post delivery). nine mothers worried about the effects of cf medication on their unborn child: the remaining mother was in her 2nd pregnancy. all managed their health differently during pregnancy: whilst 6 increased compliance, worked harder to stay healthy, and increased clinic attendances; 4 avoided clinic, postponed treatment, avoided any medication in the first trimester, and reduced oral antibiotic use to prevent harm to their unborn child. following delivery, 7 mothers spent less time on their own health due to motherhood demands, and 8 missed treatments during the first year: 4 missed all except iv antibiotics. seven mothers ceased chest clearance and nebuliser therapy, and whilst 4 missed oral therapies only 1 avoided iv therapy. one mother who returned to work early felt this had contributed to her post natal depression. nine mothers indicated that cf team support was adequate, but the remaining mother felt that she should have been offered more frequent home visits post delivery due to lack of family support. conclusions the experience of pregnancy in our centre has positive outcomes for most mothers. however, compliance with treatment was affected and a number of mothers lost significant pulmonary function due to pregnancy. our study has shown that the demands of motherhood especially in the first year challenge the management of their own health. we offer a proactive service to women considering pregnancy, with early participation of the multidisciplinary team, and have increased home visit frequency to during the first year post-delivery. geller, d.e.; kesser, k.c. research, nemours children's clinic, orlando, fl, usa background: alpha-1 antiprotease (α-1ap) is being developed as an inhaled drug in cf to inactivate excess elastase in the lungs. it is estimated that tens of milligrams of α-1ap need to be deposited in the lungs to be effective. delivering a high dose with conventional jet nebulizers may be too time-consuming. we speculated that slowing and prolonging inspiration could optimize α-1ap delivery. the i-neb® adaptive aerosol delivery (aad® system, respironics) is a portable, electronic, battery-powered, mesh nebulizer that uses the aad algorithms to deliver drug in the first portion of inspiration. interchangeable mouthpieces allow the i-neb to be used in a conventional tidal breathing mode (tbm) or in the target inhalation mode (tim) that guides the patient to inhale very slowly. the low residual volume after nebulization reduces drug waste and optimizes efficiency. we compared delivery efficiency of α-1ap in vitro with the i-neb in the tbm vs. 2 tim modes. methods: we studied 3 new i-neb devices using tbm (tidal volume [vt] 0.5l, respiratory rate [rr]=15, i-time=2 seconds), tim-6 (vt 1.6l, rr=6, i-time=6 sec), and tim-9 (vt 1.6l, rr=4, i-time=9 sec) using a breath simulator. nebulizers were filled with 0.5 ml α-1ap (50 mg/ml). nebulized drug was captured on a filter and measured via spectrophotometer (inhaled dose), and particle size was measured with an insitec laser system. results: median particle sizes for the 3 modes were similar (4.4-4.8 µm) . the dose emitted from the i-neb was very high, and similar between modes (82-90% of nominal dose). predicted lung doses for these experiments were calculated from a previous scintigraphy study (nikander 2006 ) that showed 63% of the emitted dose deposited with tbm and 73% with tim. predicted lung dose of α-1ap (as % of nominal) and delivery times were tbm: 56.6% in 7.5 min, tim-6: 59.9% in 4.4 min, and tim-9: 64.5% in 2.5 min. discussion: the i-neb has a very high predicted lung dose with both tbm and tim. however, tim reduces the time of administration to as little as 1/3 that of tidal breathing. if larger predicted lung dose is necessary to see a clinical effect, either the initial drug volume or drug concentration may be increased. we conclude that slow, deep, controlled inspirations using i-neb is a very efficient method to deliver α-1ap, and is faster than tidal breathing. we also speculate that tim has the additional potential advantage of better distribution of drug in the lungs because of slower flow rate and particle velocity. objective: early inflammation was observed in infants with cf regardless the occurrence of bacterial infection. lung function (lf) is a tool to assess bronchial inflammation consequences. few data are available on the pronostic value of impaired lf in infancy. methods: in infants, airflow obstruction (t ptef/te , curve shapes, tidal volume, functional residual capacity (frc, nitrogen washout technique) were assessed with the sensor medics 2600 and interpreted with stocks reference values. at 6y, fev 1 and pulmonary flows (spiro 2000, nova medical sc, zapletal reference values) and frc he , were measured. symptomatic children were defined when cough, wheeze or rattle were recurrent. all symptomatic infants have been treated with inhaled steroids as any infantile wheezer. results: 30 screened infants (11 ∆f508/∆f508, 14∆f508/other, 5 other/other) were investigated at mean age 16mo and 6 years. 16 (54%) had respiratory symptoms (rs); the mean tidal volume was normal (9.5ml/kg), 19 (64%) had a marked obstructive curve shape, 9 (30%) a decreased t ptef/te , and 7 (23%) hyperinflation. then, 7 (23%) had normal lf, 16 (53%) ao, 3 (10%) hyperinflation and 4 (13%) both. at age 6, 50% had rs, and lf with mild alteration as mean flows ≥ 95%, and frc he = 97.7%. accurate analyses showed that 10 (33%) had normal lf, 9 (30%) ao and 11 (47%) small airway diseases (only distal ao and/or hyperinflation). frc in infancy and at age 6 were correlated (r=0.579; p=0.03). conversely, no correlation between ao parameters in infancy and at age 6 was found. the lf alteration was independent of the genotype, bacterial colonization and recurrent symptoms at both ages. conclusion: ao in infancy is frequent as found in half of the screened cf infants. the lack of correlation with the ao observed at age 6 suggested that ao in infants might be due to a non specific inflammation (i.e viral), transient and then relevant from a treatment. hyperinflation, although mild, is conversely an early marker of the cf disease as persistent between infancy and age 6. we describe the efficacy of a novel system using an electronic real-time distant home monitoring of symptoms and spirometry in cf patients with the aim of early detection of p exs patients and methods: this is a 6 month prospective observational study. all patients were provided with a set consisted of a mobile phone-palm pc (the x daii, o2-uk) and with a spirometer (vitalograph) with an attachment to the pc. the pc contained a purpose-built software on which cf symptoms and spirometric values were recorded once daily. these were then sent on a real time to a computer server where the data were plotted on a time-magnitude graph. the site was accessed by a password-permitted cf physicians. patients were asked to score four symptoms (cough, sputum, breathlessness and fatigue) once a day and to record at least three attempts of spirometry. these were accepted by the software only if they met the ats criteria. preset criteria for p exs were stipulated. when the recordings met the criteria for p exs patients were invited to come to the cf clinic for review and were managed by either watchful waiting, oral or iv antibiotics. a prodrome phase is deemed to be present when a patient had an increase in one or more symptom for one week prior to the p ex. twenty one evaluable patients were included in the study. in 19 (90%) there were an increase in one or more than one recorded symptoms throughout the study. a total of 50 p exs median 1.2 per patient were detected within the 6 months study period. in 21(42%) p exs, symptom increase and/or decline in fev1 met the preset criteria for p ex and in 29 cases patients presented despite that symptom changes were not sufficient to meet the pre-set definition of p ex. prodromal phase prior to p exs were seen in 36 (72%) patients. adherence and technical issues were the main two problems throughout the study. distant daily monitoring of patients symptom is easy and sensitive. it can be used to monitor symptoms and early detect p exs. the vast majority of our patients have an increase in their symptoms even when they perceive their disease to be stable. a prodormal phase preceded p ex is seen in the majority of patients. the pre-set criteria for defining p ex may have been too stringent. the research was funded by o2 telecommunication, uk the monitoring system used in the study. a combination of a mobile phone-palm computer (x daii, o2 uk) and a spirometer (vitalograph uk) .. the aim of this research was to examine whether systemic inflammation is present in adult patients with cf during their stable status. patients and methods: blood samples were obtained from a total of 17 cf patients 6 female, mean age 23, range(18-26)11 males mean 23.5 range15-36 and from 32 control subjects, 20 female, mean age 21.3, range(21-40)12males,mean age27.1yrs, range(21-33) all cf patients were included when they were at least 2 weeks away from any pulmonary exacerbations. all control subjects were not known to have any pulmonary or systemic disease. venous blood samples were taken and spun at3000 rpm.separated sera was aliquted and frozen at -70 degrees c. they were analysed in batches for 6 cytokines il-1b,il-6,il-8, il-10, ilp70,and tnf-alpa using a cba human inflammatory kit(bd bioscience). whitney-u test was used to compare values of serum cytokines between stable cf patients and control subjects.. serum il-6 and il-8 were raised in cf patients compared to control subjects. the mean value for il-6 was 8.2pg/ml (sd 2.0) for patients and 2.3pg/ml (sd 0.3) for controls, p<0.0001. similarly, the mean value of serum il8 was 29.6pg/ml (sd 6.7) in cf patients compared to 8.6pg/ml (sd 0.64) in the control group, p<0.0001 (figure below). there was no difference between cf patients and control with regards to serum il-1b, il-10 and il-12p70. conclusion: serum il-6 and il-8 were raised in cf patients compared to an agematched control subjects. inflammatory markers were raised in the sera of cf patients even during disease stability. even during disease stability, the lungs remain an inflamed organ which could explain the decline in lung function tests and the countinued daily systemic symptoms experienced by patients with cf. serum il6 and il8 in cf patients with stable disease compared to the control group (please see abstract body) we have shown that even during disease stability, an increase in inflammatory markers are present in the sera of cf patients compared to an agematched control group. this longitudinal study examines changes in serum inflammatory markers during p exs. we investigated 22 cf patients 6 female, mean age 23, range15-36 years. their mean baseline fev1 was 62.8% of predicted (range 27.7-99 %). a baseline venous blood sample was withdrawn and sera were stored at -70 degree c from 22 patients during disease stability. out of those there 17 experienced p exs. blood samples were obtained at the beginning and at the end of the p exs.the following inflammatory markers were analysed: white blood counts, c -reactive protein (crp), il-6, il-8, il-10, il-1b, il-12p70 and tnf-α. patients recorded their symptoms and their fev1 on a daily basis on an electronic diary and the data were beamed daily to a research centre where data were analysed. p ex was defined as 1). an increase in two symptoms for 3 successive days, or 2). an increase in 2 symptoms and 10 % reduced fev1 from baseline over 3 successive days or 3). 10% decline in fev1 over 3 successive days or 4).when a patient felt that he/she was going through a p ex. paired t tests were used to analyse changes in inflammatory markers. there was no change in the number of circulating neutrophils, lymphocytes or eosinophiles at the start compared with the end of p ex. there was an increase in mean crp values at the start of p ex and reduction at the end of p ex, but this did not attain statistical significance. mean (sd) of serum il6 increased from 8.8pg/ml (2.8) at baseline to 16.18pg/ml (4.0) at the start of p ex (p=0.007) and returned to base line 8.88pg/ml (2.5) at the end of p exs (p=0.012 compared to values at the start of p ex). (figure 1) there were no changes in the values of serum il-8, il-10, il-1b, il12p70 or tnf-α at the start and at the end of p exs. serum il6 increased at the start of p ex and returned at the baseline at the end of p ex. crp was the closest inflammatory marker to follow that pattern. none of the other inflammatory markers changed with the diagnosis of p ex's. this and other studies call for the price of analysis of il-6 to be reduced to become available in routine clinical practice. background. abpa can be a therapeutic challenge in children with cf. aim was to describe the tolerability of voriconazole and nebulised liposomal amphotericin in the treatment of abpa in children with cf. methods. we performed a retrospective case note review of voriconazole and nebulised liposomal amphotericin use, in the treatment of abpa in children with cf, at our centre. results. 7 children diagnosed with abpa according to the current criteria, failed to improve despite previous treatment with itraconazole and steroids, and received voriconazole as a second line agent. the dose of voriconazole was as recommended by the manufacturer's summary of product characteristics and the duration of treatment was two weeks. significant side effects noted in 4 children. observed side effects included visual hallucinations (n=1), photopsia (n=2), sleep disturbances (n=2), severe headache (n=2), influenza like symptoms (n=1), gastrointestinal side effects (n=1) and elevation of alkaline phosphatase (n=1). of these 4 children, one child tolerated voriconazole when concomitant treatment with omeprazole was withdrawn. a second course of voriconazole, given at a lower dose and without the loading dose was tolerated by two children. voriconazole blood levels were not measured in any patient. we used nebulised liposomal amphotericin in 4 of these 7 children. one child received amphotericin as a second line agent when voriconazole had to be stopped because of significant side effects. 3 children had active abpa despite treatment with first line (itraconazole and steroids) and second line (voriconazole) agents and received amphotericin as a third line agent. amphotericin was used at a starting dose of 2mg nebulised twice daily, with weekly increments as tolerated, up to a maximum dose of 15mg twice daily for four weeks. the starting dose and all subsequent increments in dose were supervised. all children received salbutamol immediately before nebulised amphotericin. the standard formulation available for intravenous infusion was made up into single doses for nebulisation by the pharmacy. this was stored at 4 0 c and diluted with water for injection for use with the nebuliser. the only side effect, experienced by all 4 children was a dry irritant cough at the start of nebulisation that settled soon without need for discontinuation of the medication. improvement in clinical symptoms, lung function and total serum ige was noted in all 7 children. the improvement in fev1 and fvc, expressed as mean (range) of percentage predicted were 18 and 20 respectively. the mean (range) fall in serum total ige and aspergillus specific ige were 1395ku/l (240-3299) and 11 mg/l (4-18) respectively. conclusion. voriconazole use was associated with significant side effects. avoidance of drugs that may potentially result in interaction is important. omitting a loading dose and using a lower maintenance dose may help selected patients tolerate voriconazole. nebulised liposomal amphotericin was tolerated by our children with cf and abpa who did not tolerate or responded poorly to voriconazole. recent studies have shown that high resolution computed tomography (hrct) is a more accurate method of evaluating early lung disease in cystic fibrosis (cf) than pulmonary function tests (pfts) or chest x-rays. since young children can not perform the breathing maneuvers required for high quality hrct, lung volume control is necessary. positive pressure facemask ventilation using chloral hydrate allows full inspiration and end exhalation images, but requires special expertise. controlled ventilation can also be produced with general anesthesia (ga). ga has not been reported in children undergoing hrct for cf research. the advantages of ga over sedation include rapid onset, faster recovery and reliability of scan completion. the undesirable effects of sedation are unpredictability and the higher failure rates. additionally, failed sedation contributed significantly to parental dissatisfaction. (malviya, et al., 2000) . the primary outcome of our study was to examine the safety of using ga for hrct scans and the quality of images obtained. a secondary outcome was parental satisfaction. methods: participants were recruited from a larger nih/cffti funded multi-site randomized clinical trial focusing on behavior and nutrition treatment to improve growth in preschoolers with cf. hrct in subjects 2 to 5 years old (mean age 38 ±10 months) was performed with ga. parents accompanied the child during induction and left after the child was asleep prior to the intravenous (iv) being placed. after induction of anesthesia, a proseal®, specialized laryngeal mask airway (lma), was placed which allowed ventilation with higher pressures and the ability to suction the stomach. volumetric thin section ct scans were obtained. ga using inhalation (sevoflurane) and/or iv (propofol) agents was used and 6 out of 7 families who were eligible (85% recruitment rate) participated in the sub-study. results: the quality of the hrct scans in all 6 subjects were optimal. there were no anesthetic complications upon emergence and none of the patients experienced any side effects after follow-up phone calls at 24 hours. parents also expressed that the clinical interpretation of the hrct scan provided beneficial information about their child's current lung disease status. the mean time from completion of scan to discharge was 38.8±15 minutes. conclusions: the use of ga in radiology departments is becoming more common in pediatric centers. our initial findings of this study demonstrate that ga can be safely administered for children with cf. in addition to very good quality scans being obtained, parental satisfaction was also noted. the use of the proseal® lma for hrct may improve the image quality and aid in the diagnosis of early lung disease in preschoolers with cf. supported by nih grant r01 dk054915 and a cf foundation clinical research grant. whey protein is rich in sulfhydryl (sh) groups and is recognized for its ability to increase glutathione and reduce oxidative stress. hyperbaric pressure treatment of whey protein increases its digestibility, promotes the release of novel peptides for absorption, increases intracellular glutathione in healthy subjects (zavorsky et al, int j vit nutr res, 2007) , and reduces in vitro production of il-8 (vilela et al, inter immnuopharmacol, 2006) . we present here the initial results of a pilot study of supplementation with pressurized whey in cf patients. children and adults with cf were supplemented with hyperbaric whey protein for 1 month (20 gm/day in patients less than 18 years of age, 40 gm/day in older patients). anthropometric measures, pulmonary function, serum c-reactive protein (crp) and plasma lipid peroxides were measured before and after supplementation. the results of the first 11 patients to have completed the study are reported, with further data to be presented. in 7/11 subjects weight increased and in 7/11 subjects lean body mass increased. there were no significant changes in fev1 or fev/fvc ratio. in 7/11 patients the crp fell (4/7 by more than 50%) and in 2/11 the crp increased. in the remaining two patients, the crp was undetectable at baseline and did not change at follow-up. plasma lipid peroxides decreased in the three patients in whom it could be measured (2/3 to undetectable levels). these preliminary results suggest that supplementation with pressurized whey protein can reduce systemic inflammation in cf patients and has the potential to beneficially modulate the inflammatory response in cf patients. supported by the breathe initiative of the canadian cf foundation with pseudomonas aeruginosa. soon after its introduction, the mesh technology based pari eflow® nebulizer gained preference over jet nebulization in the cf community because of its short nebulization time, portability and noiseless operation. concerns were raised about the durability of the device due to expected vulnerability of the mesh. the aim of this study was to compare the particle size distributions in the aerosol, delivered doses and output rates from the eflow and the pari lc plus®(lc+)with paritur-boboy compressor before and after use in daily practice. lc+ nebulizers in use by the adult patients taking part in the study were taken in for testing and new eflows, cleaning instructions and a disinfection device were provided. after 6 months use (3 cycles of 2x28 doses tsi: 300 mg/5 ml), the eflows were collected by the local cf center. particle size distributions were measured at a constant flow rate of 30 l/min with a laser diffraction apparatus. because of uncertainty regarding the cleaning procedure after last use, 8 additional e-flows were collected from the same patients after another period of 6 months use. conclusions: new eflows produce slightly larger droplets in a narrower size range than new lc+ nebulizers. after use in daily practice, droplet size distribution changes for both devices. output rate (delivered dose per minute) decreases for both devices. for used eflows delivered dose is lower than for used lc+ nebulizers, because the eflow is programmed to switch off after 10 min, even when nebulization is still incomplete (9 out of 21 tested eflows). this may result in a considerable reduction of delivered dose. however, when a mesh is replaced, performance is the same as that of a new device, suggesting that the changes in performance originate in the vibrating mesh. for daily practice these results indicate: (1) the performance of both nebulizers changes during use but the changes are smaller for eflow than for lc+ (2) changes in droplet size distribution are relatively small for both devices;in contrast with output rate and delivered dose (3) proper functioning of eflow can be improved by timely replacement of the mesh, ultimately when eflow switches off after 10 minutes. without replacement, the delivered dose is unpredictable. mean values for size distribution, delivered dose, nebulization time and output rate (x 50 is volume median diameter) backgrounds: lung surfactant protein a (sp-a) belongs to the family of collagen containing, c-type lectins, called collectins. sp-a plays a critical role in the innate immune defense of the lung. sp-a binds calcium dependent to carbohydrate structures on pathogens via its carbohydrate-binding domain (crd). cystic fibrosis (cf) lung disease is characterized by chronic inflammatory and proteolytic processes in the airways, leading to destruction of the lungs and early death. aims: we hypothesized that sp-a might have a reduced function in cf. this might result from sp-a degradation by neutrophil proteases, an altered macromolecular organization of sp-a, or inhibition of crd-binding by airway components. methods: we studied bronchoalveolar lavage fluid (balf) from 31 clinically stable patients with cf and from 13 children suffering from bronchitis. balf samples were purified by carbohydrate-binding assay on fucose attached to sepharose resin with and without calcium. in the absence of calcium sp-a cannot bind via its crd. each fraction was analysed for sp-a and igg by immunoblotting. macromolecular organization of sp-a was determined by gel chromatography. results: only 49 % of total sp-a of cf balf bound to the fucose column, whereas 92 % bound from the bronchitis balf. the structural organization of the sp-a oligomers did not differ between the non-binding and the binding fractions of both groups. 4 of the cf balf had sp-a at 27 kd that did not bind to fucose. these balfs had a high percentage of neutrophils (> 80%). in vitro human leucocyte elastase decreased sp-a to less than 25% of its initial levels and sp-a fragments at 27 kd were detected. conclusions: neutrophil proteases degrade sp-a in cf lungs. the carbohydrate dependent binding of sp-a is diminished due to a reduced function of the crd. these alterations contribute to the reduced ability in cf to remove specific pathogens from the lungs. chiron, r. 1,6 ; murris-espin, m. 2, 6 ; crampette, l. 3,6 ; varrin, m. 4 ; didier, a. 2, 6 ; wallaert, b. 5, 6 ; chanez, p. 1, 6 ; leroy, s. 5, 6 1. crcm, chu, montpellier, france; 2. crcm, chu, toulouse, france; 3. orl, chu, montpellier, france; 4. iurc, chu, montpellier, france; 5. crcm, chu, lille, france; montpellier, france in cystic fibrosis (cf) patients, upper airways involvement is frequent but its impact and relationships on the course and severity of the disease has been rarely investigated. objective: to relate upper airways involvement with lower airways parameters and quality of life in 91 cf patients. methods: clinical and radiological prospective study of subjects with cf at 3 french cf centres. clinical rhino-sinusitis (rs) was defined by the presence of more than 3 episodes of nasal symptoms during the last year, current nasal obstruction or rhinorrhea. nasal investigations included: anterior rhinoscopy, sinus ct scan analysis and rs quality of life questionnaire. lower airways were assessed using a thoracic ct scan (bhalla score), spirometry and sputum culture and a cf quality of life questionnaire (cfq14+). results: 91 (47f/44m) patients aged 25.4 (14-71) years old were enrolled. endoscopy demonstrated polyposis in 12% and nasal hyper secretions in 67.5% of the patients. spirometry, bhalla score, sputum colonization did not discriminate patients with (n=61) or without rs (n=30), and regardless the presence of polyps. cfq14+ was significantly lower in patients with of rs (p=0.04). conclusions: rs involvement altered quality of life in cf patients, although, they do not have a more severe pulmonary disease. a specific attention and treatment to rs should be paid in cf patients. the impact on different outcomes deserves to be examined in longitudinal studies. as part of the uk cystic fibrosis gene therapy consortium "tracking study", ebc was measured (ecoscreen, jaeger) in adult and paediatric patients presenting with an infective exacerbation requiring iv antibiotics. exacerbation was defined by pre-determined criteria. ebc was repeated at the end of parenteral antibiotic therapy and again 2-4 weeks later. patients were recruited at three sites and assessed using standardised equipment. samples were obtained according to ers guidelines. exhaled breath ph was assayed (shindengen ph meter, camlab, uk) immediately after collection without a de-aeration step. results: paired ebc measurements at the start and end of antibiotic therapy were available in 40 patients. mean age was 24yrs (range 11-44). data from 6 patients were excluded because at least one ph measurement was >7, suggesting salivary contamination. for the remaining 34 patients mean ebc (sd) ph increased from 5.9 (0.5) to 6.1 (0.3), p<0.01. of these patients, 28 also had measurements available at a third visit, 2-4 weeks after completion of antibiotics. 2 were excluded because of salivary contamination. no difference in mean(sd) ebc ph was found (6.1 (0.4) vs. 6.1 (0.4)) conclusions: ebc ph increases after treatment with antibiotics, and may offer a non-invasive means of assessing airways inflammation in chest exacerbations. further work is required to follow the longitudinal change in ebc ph and other inflammatory markers in clinically stable patients, as ebc may be a useful tool in assessing response to novel treatments, such as gene therapy. this work was funded by the cystic fibrosis trust. background: children with cystic fibrosis (cf) undergo multiple procedures exposing them to ionizing radiation in hospital. some specialist centres have advocated regular chest computed tomography (ct) as a means of better quantifying lung pathology. the clinical benefit of this approach is unproven and previous calculations of risk have failed to account for other ionizing radiation exposure procedures 1 . aim: to quantify the exposure of ionizing radiation in the pediatric cf population of a tertiary level children's hospital. to determine the potential difference in exposure of introducing biannual chest ct scans. methods: effective doses of ionizing radiation from chest (cxr) and abdominal (axr) radiographs, fluoroscopy and cts (sinuses and chest including hr scans) were determined for cf children at our centre between 1st january 2003 and 11th july 2006 (184 weeks). the maximal effect was tested i.e. introduction of biannual chest ct scans from age 2 years abolishing the requirement for cxrs and the assumption was made that axrs, sinus cts and fluoroscopy rates were unchanged (median exposure values calculated for age groups 0-<1year, 1-<2year, 2-<4year, 4-<8year, 8-<12year, >12year) . results: cf patients (n=134, gender ratio 1:1) underwent a mean of 13.8 ionizing radiation exposure procedures per patient over the study period (total of 1853 procedures). the average effective ionizing radiation doses (msv) were calculated for 1806 (97.5%) procedures. each cf child received a mean (median) effective radiation dose of 1.47 (0.04) msv/year with a range of 0.004 -25.76msv/year. the reason for the skewed distribution was identified by analysing the proportions of the procedures to the total effective dose in this population: fluoroscopy 83.2%, chest ct 7.1%, sinus ct 6.3%, cxrs 2.5%, axrs 0.7% and hrct 0.2%. it was calculated that these patients undergo 3.4 cxrs per patient per year. we calculated that an average patient diagnosed by newborn screening could expect to receive 1.24msv from ionizing radiation procedures between birth and age 16. biannual ct chest scans in addition to median exposures for each age group from axrs, sinus cts and fluoroscopy would potentially result in 14.34 msv by age 16, an 11 fold increase. discussion: there is a 1 in 100 lifetime risk of developing a solid or haematological cancer with each exposure to 100 msv of ionizing radiation 2 . although fluoroscopy forms the most significant contribution to a pediatric cf patient's ionizing radiation exposure in our centre, introducing biannual chest cts would markedly increase exposure. as cf survival improves, cf physicians must be cognisant of the potential health cost of survival. references : previous studies have used generic measures of hrqol, which are not sensitive to important changes that may occur as a result of treatment. this study assesses the impact of iv antibiotic treatment for a pulmonary exacerbation on both generic and disease-specific hrqol of children and adolescents with cf. method: participants included 45 patients (m age = 13.4 years) from the cincinnati children's hospital and university of florida cf centers. fiftyfour percent of the sample were male. patients completed two hrqol measures, the pedsql™ (generic) and cfq-r child or teen/adult version (cf-specific), which have excellent reliability and validity. these measures were completed within 48 hours of hospital admission (pre) and 48 hours of discontinuation of iv antibiotics (post). note that approximately 32% of patients went home on iv antibiotics and 68% remained in the hospital for approximately 2 weeks. results: significant improvements were found in pulmonary functioning (fev1 % predicted) pre to post iv antibiotic treatment (paired t (44) = -6.9, p < .0001), with an average of 14% increase in fev1 % predicted. paired t-tests were conducted to examine changes in hrqol scores, using holm's procedure to correct for multiple comparisons. significant improvements were found on the cfq-r respiratory (paired t (45) = -5.3, p < .0001) and weight scales (paired t (23) = -4.1, p < .0001). trends were also noted for improved emotional functioning (paired t (45) = -1.8, p = .08) and vitality (paired t (23) = -1.9, p = .07), and worse treatment burden (paired t (45) = 1.7, p = .09). on the pedsql, only emotional functioning improved significantly (paired t (44) = -2.6, p < .05). conclusions: results of this study confirm the effectiveness of iv antibiotics for the treatment of pulmonary exacerbations, with significant improvements found for both pulmonary functioning and hrqol. these results also highlighted the importance of using a disease-specific hrqol instrument, which proved to be more sensitive than the generic measure. on the cfq-r, significant improvements were observed in respiratory symptoms and weight, aspects of functioning that are not assessed on generic measures. fukushima, l.k.; hsu, e.; woo, m.s. division of pediatric pulmonology, childrens hospital los angeles, los angeles, ca, usa do hispanic and caucasian cf patients share the same frequency of pulmonary exacerbations? increased frequency of pulmonary exacerbations is linked to increased risk for cf morbidity and mortality. we have previously reported that pediatric hispanic cf patients have greater mortality than our caucasian cf patients who attend the same accredited cf center. we performed a retrospective review of cf pulmonary exacerbations (defined as physician prescription of intravenous or oral antibiotic therapy) in our cf patients who routinely attended the cf clinic during the period of january 1, 2005 through december 31, 2006. data collected included demographics, number of intravenous antibiotic courses and number of oral antibiotic courses. unpaired student's t-test was used to compare age of the groups and chi-square was used to compare use of intravenous and oral antibiotics between hispanic and caucasian cf patients as well as between males and females. during the study period, there was a total of 197 cf patients (106 males:91 females; mean age 10.8 ± 5.7 years). of the 197 patients, 82 (42%) were classified as hispanic (46 males:36 females; mean age 11.0 ± 5.3 years) and 99 (50%)were classified as caucasian (49 males:50 females; mean age 10.8 ± 6.2 years; p=0.42, ns). hispanic cf patients (51 patients had 299 episodes; 26 males:25 females) had significantly greater number of pulmonary exacerbations (received treatment with intravenous and/or oral antibiotics) than caucasian cf patients (35 patients had 182 episodes; p = 0.0006). hispanic cf patients who required intravenous antibiotic therapy were significantly younger (12.3 ± 5.1 years vs 15.0 ± 5.9 years; p<0.00001) than the caucasian cf patients who were treated with intravenous antibiotics. there was no significant difference in age between hispanic and caucasian cf patients who received oral antibiotic courses (11.2 ± 5.3 years vs 13.1 ± 6.4 years; p=0.08, ns). gender did not have a significant impact on pulmonary exacerbation risk in our population (hispanic iv use by gender p=0.82; hispanic oral use by gender p=0.12; caucasian iv use by gender p=0.38; caucasian oral use by gender p=0.37). we conclude that hispanic cf patients had increased incidence of pulmonary exacerbations than caucasian cf patients who attend the same cf clinic. hispanic patients who were treated with intravenous antibiotics were younger than the caucasian cf patients. we speculate that hispanic ethnicity has a major impact on the incidence of pulmonary exacerbations. other factors, such as modifier genes, environmental exposures, or inflammatory responses, may play a role in the increased risk of pulmonary exacerbations in the hispanic cf population. introduction: one of the main goals of the multidisciplinary team of the cf association of minas gerais, brazil is to find strategies to preserve lung tissue from the bacterial exacerbation in cystic fibrosis patients. objective: to investigate clues on onset of bacterial exacerbation. to our knowledge, no studies were found regarding the identification of the early warning signs of bacterial exacerbation with cf patients. we wanted to know whether cf patients present early warning signs before onset of fever and/or breathing difficulties. methods: telephone interviews were carried out with the 330 cf patients registered in the association, by a physiotherapist academic who did not know the patients and just ask two questions. the first question was about the use of antibiotics during the year of 2006 and the second was related to early signs before the onset of fever. results: from the 330 patients, only 207 patients replied. patients who were using antibiotics therapy during 2006 and had fever: 117/207 patients who were using antibiotics and did not have fever: 37/207 patients who had fever and did not use antibiotics: 9/207 patients who did not have fever and did not use antibiotics: 44/207 the results revealed the following warning signs and also that some patients had more than one sign. tiredness, quietness, slowness (51/117); increased cough (27/117); lack of appetite (19/117); sticky sputum (13/117); sleepiness (12/117); headache (14/117); increased excitement, nervousness (3/117); crybaby (11/117); dry mouth (1/117); clammy (1/117); difficult to walk (1/117); gastric upset (5/117); itchy throat (6/117); abdominal pain (7/117) ; itchy eyes (10/117); breathing difficulties (7/117); tremulous (3/117); paleness (3/117); burning ear (1/117); chest pain (1/117); hissing (1/117) ; and syncope(1/117). conclusions: it is helpful to know these early warning sings. we assume that the earlier the diagnosis of the exacerbation is made, the easier it is to resolve the problem and the less destruction of the lung tissues. reference: lung line, national jewish center for immunology and respiratory medicine 1988. background: cystic fibrosis patients are experiencing increasing survival and are being treated with more chronic therapies. how these changes are reflected in day to day clinical symptoms has not been evaluated. objective: we examined data from the epidemiologic study of cystic fibrosis, a large longitudinal observational study, to characterize the change in respiratory symptoms over time. for each year between 1995 and 2005 data from patients with at least one visit recorded as occurring while clinically stable were included. data from all visits, including sick as well as stable, were used for each year in which at least one clinically stable visit was recorded. patients were separated into age groups less than 6, 6 to 12, 13 to 17, and 18 or older. note that in 2003 data collection changed, potentially confounding the results. results: the average number of patients per year was 16393. the percent of patients with no reported cough progressively increased over time (table) . results were similar for pateints with no reported sputum production at clinically stable visits. when sick visits were added there were similar results. the frequency of cough reported at greater than 90 percent of visits progressively decreased over time (table) . again the results for sputum production were similar. these findings were seen in all age groups although older patients are more likely to experience these symptoms. conclusions: these data suggest that cf patients are experiencing fewer respiratory symptoms of cough and sputum production over the last decade. this improvement has occurred in patients with either intermittent or chronic symptoms. how clinical care has impacted on these symptoms has yet to be determined. also, more people are being diagnosed with cf at an earlier age or with milder disease, potentially resulting in the entire cf population having fewer symptoms. lung disease in children with cf begins in early childhood, with intermittent and then chronic infection, associated with an exaggerated inflammatory response. the extent to which inflammation contributes to bronchiectasis has not yet been identified. the aim of this study was examine relationships between inflammation and early lung damage methods: twenty eight children with cf were assessed at diagnosis and annually thereafter with a bronchoalveolar lavage (bal). bal fluid was used to assess micro-organisms and inflammation, including total and differential cell counts. a three slice hrct technique (inspiration and expiration) was performed under general anesthesia at age 3-6y, immediately preceding the annual bal. hrct scans were scored by an independent and experienced radiologist using a modification of the brody score. results. twelve children (43%) had evidence of bronchiectasis on hrct. inflammation present in bal at 12 months of age predicted bronchiectasis at 3-6 y. total cell count at 12 mo, predominantly neutrophils, was higher in those who developed bronchiectasis compared to those who did not (0.81 vs 0.43 x10 6 cells/ml bal; p=0.047). the total number of infections detected in bal was not related to bronchectasis. however, children with bronchiectasis had a higher incidence of pseudomonas aeruginosa within the first 3 years of life (50% vs. 25%). conclusions: bronchiectasis is evident within the first five years of life and appears to be associated with an exaggerated inflammatory response and the early acquisition of pseudomonas aeruginosa. supported by: cystic fibrosis foundation (usa), national health and medical research council (australia). fortunately the survival of patients with cystic fibrosis has improved remarkably over the last few decades. many patients can life quite "normal". therefore family planning becomes more important. several reports docu-mented the good maternal and fetal outcome in pregnant women with cystic fibrosis. besides the data from the french cf registry no longterm data about the impact of paternity of male patients with cystic fibrosis are available until now. in a retrospective analysis we gathered data about all our male patients who became father while being followed-up in our cystic fibrosis center for adults. from 43 male patients, 6 patients became father between 1999 and 2006. patient no. 1 became father twice (second time father of twins); patients no. 6 became also father of twins .we looked at fev1 % 1 year prior to childbirth, perinatal and 1 and 5 years after the childbirth. for the fev1% one year prior to birth as well as for fev1% 1 and 5 years after birth, we calculated the difference to the fev1% at time of birth. between 1999 and 2006, 7 male adult patients with cystic fibrosis became fathers of a total of 9 children, all by the assistance of reproductive techniques. their fev1% are shown in table 1. although the male patients are not directly influenced by a pregenancy (as the pregnant women are by growth of the uterus), there seems to be more than ususal differences in this lung function parameter. there were also more fluctuations in fev1 per year. the biggest differences in fev1% were seen 1 year after birth of their children. further investigations in a larger group (in cooperation with other centers) are planned; especially with a focus on the frequency of infections and quality of life compared to male patients without children. intravenous (i.v) aminoglycosides are widely used in cystic fibrosis (cf) patients as treatment for pulmonary exacerbations. in an effort to reduce undesiderable effects of these antibiotics it is recommended to measure drug serum levels. recent experience suggests that tobramycin (to) may be administered as a single daily dose with equal efficacy as multiple doses but less risk of nephrotoxicity. aim: we evaluated the efficacy of once-daily i.v to treatment to improve pulmonary function expressed as forced expiratory volume in one second (fev 1 ) in 15 cf patients with pulmonary exacerbations compared to the same group of patients who were previously treated with to in multiple daily i.v doses, as historic control. we evaluated baseline and peak drug serum levels of to administered once daily compared to multiple dose levels. methods: all patients were treated with i.v to in combination with a beta-lactam antibiotic for two weeks. respiratory exacerbation was defined according to cff criteria. all cf patients were colonized by non-fermentative gram-negative bacteria. serum concentrations of to were measured (fluorescent polarisation immunoassays) before the fifth infusion (basal) and 30 minutes after the fifth infusion (peak). reference to blood levels were considered to be ≤ 2 mg/l (basal) and <12 mg/l (peak) for multiple daily doses, and ≤ 1 mg/l (basal) and 20-30 mg/l (peak) for single daily dose. treatment efficacy was evaluated on the basis of improved clinical condition and increased fev 1 values compared to the beginning of therapy. results: 15 patients with respiratory exacerbation were evaluated, with a mean (± ds) age of 27.05 years (± 6.09; median 25.83, range 16.8-39.4) . 14 out of 15 patients were chronically colonized by pseudomonas aeruginosa. the total mean (± sd) daily dose of to administered in multiple doses was 410 (± 68.66) mg whereas the mean single dose was 406.6 (± 37.1) mg. of those patients given multiple doses of to, only one patient (6.6%) had basal to values outside the range and 4 patients (26.6%) had peak values outside the range. of those patients given a single daily dose, no patients had basal to values outside the range whereas 12 out of 15 (80%) had peak values of less than 20 mg/l and 3 patients out of 15 (20%) had peaks between 20 and 30 mg/l. the mean (± sd) increase in fev 1 was 5.6% (± 9.09) in patients treated with a single dose and 7% (± 9.41) in those treated with multiple doses. the mean (± sd) period between fev 1 measurements before and after exacerbation was 59.53 days (± 53.4, median 37, range 6-201) in case of monodose therapy, and 36.93 (± 24.79, median 29, range 17-105) in case of therapy with multiple doses. conclusions: both single and multiple-dose i.v to therapy caused a comparable fev 1 increase and were effective in resolving the respiratory exacerbation. although clinical improvement was observed, the mean i.v to dose of 400 mg/day in patients treated with once-daily i.v to determined peak values between 20 and 30 mg/l in 20% of subjects. intravenously administered aminoglycosides are efficacious for the treatment of pulmonary infections of cystic fibrosis (cf) patients, with the principal adverse effects of these molecules being acoustic nerve damage and nephropathy. damage to the eighth cranial nerve varies from 1.8 to 32% in cf patients treated with multiple daily doses of aminoglycosides. recent experience suggests that tobramycin can be administered as a single daily dose, resulting in less nephrotoxicity but equal efficacy. patients and methods: we evaluated the prevalence of oto-and nephrotoxicity due to aminoglycosides administered to cf patients followed at the tuscan regional center where we are currently following 192 patients. cochlear damage was evaluated using tonal audiometry (amplifon amplaid 171 s). renal damage was evaluated by measuring patients' creatinine clearance. results: 96 (50%) patients (52 males and 44 females) between the ages of 9 and 43 (mean age 22.5 years, median 21, sd 9) were tested with audiometry. all these patients had been repeatedly treated with multiple daily doses of intravenous aminoglycosides. 15 (7.8%) (5 males and 10 females) out of 192 patients between 3 and 45 years (mean age 24.8, median 24.2, sd 13.1) had their creatinine clearance measured. we found that 7 patients (7.2%) had an abnormal audiometry, typically attributable to aminoglycoside ototoxicity (high frequency deficit). two patients required a hearing aid. one patient with normal cochlear function had labyrinth deficit. one out of 15 patients tested had pathological creatinine clearance values. conclusions: our data indicate that 8 (8.3%) of our patients had damage to the eighth cranial nerve due to repeated multiple daily doses of aminoglycosides. we recommend that cf patients under aminoglycoside therapy be routinely given audiometric and creatinine clearance exams in an attempt to reduce the incidence of undesirable side effects of these drugs. davies, j. 1, 6 ; voase, n. 1, 6 ; dewar, m. 2,6 ; mullard, k. 1,6 ; gammie, f. 1, 6 ; saunders, c. 1, 6 ; horsley, a. 2, 6 ; gray, r. 2, 6 ; macleod, k. 3, 6 ; somerton, l. 1,6 ; higgins, t. 1,6 ; donovan, j. 1, 6 ; cornish, n. 1, 6 ; hansell, d. 4, 6 ; aziz, z. 4, 6 ; ashby, d. 5 ; geddes, d. 1, 6 ; greening, a. 2, 6 ; cunningham, s. 3,6 ; innes, a. 2,6 ; alton, e. 1, 6 1. gene therapy, imperial college, london, united kingdom; 2. western general hospital, edinburgh, united kingdom; 3. royal hospital for sick children, edinburgh, united kingdom; 4. royal brompton hospital, london, united kingdom; 5. queen mary, university of london, london, united kingdom; 6. uk cf gene therapy consortium, edinburgh, london, oxford, united kingdom the improving clinical status of patients with cf and the slow rate of decline of conventional outcome measures such as fev 1 necessitate the development of clinically-relevant surrogate outcome assays for use in clinical trials. in our forthcoming clinical trial of cftr gene therapy, the uk cf gene therapy consortium will use both established, clinically-available assays and more novel measures designed specifically for this purpose. both groups of assays are being subjected to rigorous testing prior to use to establish their utility as disease biomarkers. in this study, we examined their performance in the context of an infective exacerbation treated with iv antibiotics. this abstract will report the response of established, clinically-available assays; available data from novel assays will be reported separately. children (12 years and above) and adults with cf were recruited from three centres at the time of a clinician-defined infective exacerbation requiring iv antibiotic treatment. exclusion criteria included fev 1 <30% predicted and requirement for supplemental oxygen. a panel of assays was performed at the start and end of treatment, which was most commonly 14 days. data are presented as mean (sd) or median (range) and differences compared with either parametric or non-parametric paired analysis as appropriate. clinical assays included spirometry, sputum microbiology, sputum cell count and differential, serum inflammatory markers (crp, white blood cell (wbc) count,) and chest ct. patients also completed a symptom score sheet. to date, 40 patients (mean age 24.2 [7.5] years, 21 male) have paired data available from the start and end of the course of ivs. at baseline, 24 were infected with p. aeruginosa, 8 with b. cepacia complex organisms and 14 with s. aureus (2 mrsa). significant changes from baseline were observed in fev 1 (53.3[15.1] the clinical response to any novel intervention, for example, cftr gene therapy is difficult to predict. prior testing of experimental assays in a study such as this provides data on the variability of the measurements within the disease population and the degree of change observed with an intervention known to lead to clinical benefit. this should aid the design of rational, powered clinical trials. funded by the cf trust influenza can worsen cf and lead to serious complications and mortality. influenza vaccine is safe and effective in cf patients. in france, annual influenza vaccination is recommended for all cf patients aged over 6 months and for healthcare workers in regular and prolonged contact with high-risk patients. objective: to estimate the influenza vaccination coverage rate for 2005-2006 season in cf patients and healthcare workers in cf care centres of the great south region of france. methods: a multicentric observational study between february and september 2006 was conducted. healthcare workers were contacted by telephone and were questioned about their 2005-2006 influenza vaccination status, and the reason for not getting vaccinated. for patients aged over 6 months whose vaccination booklet was available, the physician filled in a written questionnaire. results: a total of 128 professionals were interviewed. the survey included 33.6% of doctors, 27.3% of nurses, 9.4% of dieticians, 9.4% of physiotherapists, 9.4% of social workers and 4.7% of psychologists. the overall influenza vc rate was 59.4% and varied considerably according to profession: 81.4% of doctors, 66.7% of physiotherapists, 58.3% of dieticians, 50% of psychologists, 48.6% of nurses and 16.7% of social workers. the overall influenza vc rate in cf patients was 80.6% (86.5% in children (age < 18 years old) versus 70.2% in adults; p<0.05). receiving a voucher for free vaccination from the national insurance increased the influenza vc rate. for healthcare workers, the main reason for non vaccination was that flu was considered as a benign disease (useless vaccine); for patients, it was a lack of time. conclusion methods: adult patients with cf were studied. patients completed a standardised questionnaire. the questionnaire identified which changes and duration of symptoms would cause them to contact the cf team and/or change treatment. results: all patients would contact the cf team for haemoptysis but 40% would wait for more than 24 hours and 20% would wait for more than 48 hours before doing so. up to 60% of patients would wait a week or more before contacting the cf team because of sputum purulence. 20% of patients would not contact the cf team if they developed new chest pain. conclusion: there appears to be a disconnect between recognizing a change in symptoms and the length of time before acting on the change. this has major implications for the delivery of cf care. supported by cystic fibrosis association of ireland. aim: to determine the efficacy of standardized desensitization protocols in treating antibiotic allergies in adults with cystic fibrosis (cf). background: dependence on high dose and multiple combination intravenous (iv) antibiotics to treat pulmonary exacerbations in adult cf patients has resulted in an increased frequency of allergic reactions when compared with the general population. (1, 2) strategies to address these allergies are essential to maintain effective antibiotic treatment in this population. antibiotic desensitization is the process by which one induces immune tolerance by incremental exposure to the antibiotic, which may induce stabilization of mast cells. the mechanism remains undetermined but may be mediated via ige or memory t-cells. prior to the study period, 9 standardized antibiotic desensitization protocols were developed by an allergist/immunologist based on published reports and were approved through the hospital's pharmacy and therapeutics committee. methods: the toronto cf database was accessed to generate a list of hospital admissions for iv antibiotics during the 5-year study period (1999) (2000) (2001) (2002) (2003) (2004) . patients who underwent desensitization were identified and each of their case notes underwent retrospective analysis. data were collected with respect to age, organism requiring antibiotic therapy, antibiotic allergy requiring desensitization, reactions during desensitization, reactions post desensitization, treatments required to treat these reactions, and the completeness of antibiotic therapy post desensitization. statistical analyses were performed using graph pad prism®. results: during the study period the cf unit dispensed and initiated approximately 600 courses of iv antibiotics. a total of 63 desensitizations were performed in 22 patients (17 female). although some patients were desensitized to only one drug or to the same drug more than once, there were 43 patient-drug combinations with a median of 2 (+/= 1.2) unique combinations per patient. of the 63 desensitizations protocols initiated, 7 patients developed documented reaction to the antibiotic during desensitization however only 1 desensitization protocol was not completed due to an adverse event. there were no episodes of anaphylaxis either during or after desensitization. a total of 9 patients developed documented reactions during the subsequent antibiotic course -5 required termination of the antibiotic and 4 completed the course of iv antibiotics with management of allergic type reactions with antihistamines. conclusions: standardized desensitization protocols were initiated in 22 patients during the study period. the success rate for desensitizations in these patients was 79% however when the data is analyzed for true allergic phenomenon, the success rate rises to 90%. this demonstrates the efficacy of these standardized protocols in the treatment of ige-mediated allergic reactions to antibiotics in the adult cf patient. references: 1. burrows, j., toon, m., bell, s., (2003) . antibiotic desensitization in adults with cystic fibrosis. respirology, 8: 359-364. 2. ramesh, s. (2002) . antibiotic hypersensitivity in patients with cf. clin rev allerg immun, 23: 123-139. surgery for symptomatic disease. there is a long-held suspicion that poorly-controlled chronic sinusitis negatively impacts the respiratory status of cf patients, but whether sinus surgery improves the clinical course of cf lung disease is unclear. aim: assess the impact of functional endoscopic sinus surgery (fess) on respiratory exacerbation rate and lung function in adult cf patients methods: this is a single-center, retrospective study based on chart review. during the study period, 1999-2006, 40 adult patients underwent 55 separate fess procedures. this abstract contains results so far from the analysis of 26 patients that underwent a total of 37 surgeries. primary outcomes were 1-year change pre to post fess in pulmonary function parameters and pulmonary exacerbation rate. results: at the time of surgery, the mean fev1 was 62% of predicted (ppd), the mean fvc 79.6 ppd, and the median age was 24.0 years (range 18.0 -46.0). 56.1% of patients were female. the median number of iv antibiotic courses for respiratory exacerbations in the 12 months prior to fess was 1 (range 0-5) compared to 2 (range 0-5) in the year after surgery (p = .04). further,there were fewer days on intravenous (iv) antibiotics for respiratory exacerbations in the 12 months prior to fess than in the 12month post-operative period (median 14 vs. 23 days), but this difference was not statistically significant (p = .09). the use of oral antibiotics for respiratory flares was comparable in the 12 months before and after fess (median 122 vs. 104 days, respectively, p = 0.53). the median number of hospital admissions and median number of days hospitalized for respiratory exacerbations did not differ in the 12 months pre vs. post-fess (p = 0.70 and p = 0.77, respectively). the mean fev1 and fvc in the 12 months pre and post surgery did not differ significantly (61.9 vs. 57.7 ppd, p = 0.13, and 77.4 vs. 76.0 ppd, p = 0.11). the linear rate of change in fev1 and fvc did not differ significantly in the 12 months prior to and following fess (-.01 l/month vs. +.01 l/month, p = .16 and -.01 l/month vs. +.02 l/month, p = 0.11, respectively). there were no significant differences (p > 0.10) for any of the above comparisons when the analysis was limited to 6 months before and after fess. there were no significant differences (p > 0.10) in antibiotic use and hospitalizations when the analysis was limited to the 29 surgeries in which patients were experiencing both respiratory and sinus symptoms. this latter group had lower fev1 and fvc ppd in the 12 month post-fess period (59.9 vs. 56.7 ppd, p = .001, and 75.4 vs. 72.9 ppd, p = .03, respectively). however, the rate of decline in fev1 and fvc in the 12 months before and after surgery did not differ significantly ( p = 0.17 and p = 0.18, respectively). conclusion: fess did not significantly affect the rate of respiratory exacerbations or rate of decline in pulmonary function in adults with cystic fibrosis. cumulative decline in lung function as measured by fev 1 in cystic fibrosis (cf) pulmonary exacerbation is a major cause of cf-related morbidity and mortality. high-dose extended interval aminoglycoside (hdei ag) use may be more effective in improving lung function than traditional multiple daily dosing in cf-related pulmonary exacerbation. intermittent dosing of beta-lactam antibiotics are often used with hdei ag in patients with cf pulmonary exacerbation, which does not optimize betalactam pharmacodynamics. the primary purpose of this study is to determine the effect of hdei ag plus continuous infusion beta-lactam (cibl) on patients' return to best baseline fev 1 from the previous 12 months. this study was conducted at university of kentucky healthcare. this was a concurrent observational trial with patients serving as their own historic controls via review of the medical record. all pediatric and adult patients with cf respiratory disease hospitalized with an acute pulmonary exacerbation between november 1, 2005 and may 1, 2007, with pseudomonas aeruginosa in their respiratory culture and were followed for at least one year were included. excluded were patients with known hearing disability or renal insufficiency or an inability to reliably perform spirometry (ie.less than six years old). patients served as their own historic controls via conventional treatment (control) versus hdei ag plus cibl (treatment). the primary endpoint was to determine the percent of patient courses that return to best baseline fev 1 in the last twelve months after treatment with hdei ag plus cibl. secondary endpoints included determination of patient courses that returned to mean baseline fev 1 in the last twelve months after study treatments, and description of beta-lactam dosing characteristics. thirteen patients were included in the pilot analysis which totaled 21 patient courses (mean 1.6 courses per patient). average patient age was 24 ± 11.1 years with a mean fev 1 of 42%; 61.5 % were female. after hdei ag plus cibl no patients showed a return to their best baseline fev 1 in the previous 12 months with the exception of one patient whose value was unchanged. average overall percent change in best baseline fev 1 group was -11.0 ± 7.8 at follow up. 62% of patient courses exhibited a return to their mean baseline fev 1 . average overall percent change in mean fev 1 baseline was 1.8 ± 6.8 at follow up. five of the 8 (62.5%) study patients who had serum beta-lactam concentrations obtained were able to achieve at least four times the mic of the target organism. more data will be collected on subsequent courses and follow up lung function. our preliminary data suggest that our patients lost 11% of their best fev 1 with an acute exacerbation and returned to 1% above mean baseline after treatment with hdei ag and cibl therapy. this evidence suggests that this might be an alternative to intermittent beta-lactam therapy. follow up data on remaining patients may provide sufficient evidence to validate this hypothesis. respiratory exacerbation in cystic fibrosis patients is characterized by increased sputum that may become more purulent. the detection of the exacerbation is based mainly on clinical subjective parameters. fev1 measurements are accepted as gold standard to be used for the life-time of the patient but do not adapt fast enough after resolution of an exacerbation. we compared parameters that are affected by the uneven distribution of ventilation in the lung due to increase sputum. methods: a zen body plethysmograph was used to measure tlc, fev1 and dlco (co/ch3 mixture). tlc was also calculated by the volume of ch3, the angle of the slope of phase iii was calculated in 20 cf patients at the beginning of the respiratory exacerbation and after 2 weeks of antibiotic treatment. patients were 17 -30 years old (12m/8f). results: fev1 changed by 9.8+7.41% at the end of the antibiotic treatment, the tlc(pleth) however was stable and changed by only 0.9+8.7%. the rv/tlc decreased by 23.8+ 23.4% and the tlc(gas) was increased by 5+36.6%. the difference between tlc (pleth) and tlc (gas) before and after the antibiotic treatment decreased from 24.7+13.6% to 10.2+7.1 %. conclusion: the changes in the parameters affected by the unevenness of distribution of ventilation (m/p reflecting the increased sputum in the airway) are more pronounced than fev1 and may be used as a more sensitive parameter for assessing the course of respiratory exacerbation. fibreoptic bronchoscopy was performed in 78 children aged 6-16 years: 27 with cf, 16 with non-cf bronchiectasis (bx), 24 with asthma, and 11 control children without lower respiratory disease. endobronchial biopsies were taken and stained with haematoxylin and eosin. asm content, myocyte number and size were quantified using stereology. results: the volume fraction of asm in subepithelial tissue (median; iqr) was 0.04 (0.02-0.05) in controls. by comparison, it was 0.12 (0.6-0.21, p<0.01) in cf, 0.16 (0.04-0.21, p<0.01) in bx, and 0.27 (0.12-0.49, p<0.0001) in asthma. myocyte number (cells per mm 2 of reticular basement membrane) was 1944 (1596-6318) in controls, 4504 (2838-8962, ns) in cf, 4971 (3476-10057, ns) in bx, and 8204 (5270-11749, p<0 .05) in asthma. myocyte size (um 3 , mean; sd) was 1948 (386) in controls, 3364 (890, p<0.01) in cf, 2857 (873, p<0.05) in bx, and 3249 (801, p<0.01) in asthma. in cf, the volume fraction of asm in subepithelial tissue was related to myocyte number (r=0.88, p=0.001), but not to myocyte size. conclusions: asm remodeling occurs in cf children. this, however, is not disease-specific and is also found in non-cf bronchiectasis and in asthma. both myocyte hypertrophy and hyperplasia contribute to increase in asm in cf. support: ers long-term fellowship and swiss national foundation grant to nr purpose: ct scans are increasingly being ordered on a routine basis in patients with cystic fibrosis. while prior studies have investigated their clinical utility in children, there is limited and conflicting information on their utility in adults. the purpose of this study was to determine the relationship between clinical disease severity as assessed by spirometry and ct findings as well as initial ct score ability to predict change in lung function in a nonselected group of adult cf patients. methods: a retrospective review of 45 cf patients (mean age 30.2 +/-8.5, male 62%) who had undergone a routine chest ct was performed. the ct scans were scored using the brody method by a blinded reader with the degree of bronchiectasis, mucous plugging, peribronchial thickening, parenchymal disease, and air trapping assessed individually and a composite score generated. the ct metrics were first compared with spirometric test that were temporally related to the date of the ct scan, and then with future spirometric tests. results: for the cohort the patients had a wide range of severity of disease with the average fev1% 42.9 +/-21.9, and the average fvc% was 63.5 +/-20.7. no significant correlation was found between initial fev1% and total ct score (0.051 p value 0.739), or initial fvc% and total ct score (-0.082 p value 0.594). for ct metrics and future spirometry, the average time from ct to pft's was 671 days +/-528 with median of 484 days. predictors for a significant decline in pft's were a decreasing bmi and male sex (p-values 0.015 and 0.035 respectively). further, for patients with a low fev1% (<40% at baseline), the fvc% change over time was associated with total ct score, male gender, and bmi (p-values 0.006, 0.007, and 0.027 respectively) conclusions: in adult cf patients, ct scan findings did not correlate with lung function. however in longitudinal analysis, bmi and gender had an effect on fvc change. for patients with low lung function, total ct score did have an association with change in fvc along with bmi and gender. pulmonary exacerbations (pes) are episodes of worsening of respiratory symptoms that commonly occur in patients with cystic fibrosis (cf). in clinical trials of new therapies in cf, pulmonary exacerbation is widely used as a primary or secondary endpoint. despite its importance, no single clearly agreed upon definition exists. some are based on patient reported symptoms while others require hospitalization or antibiotic prescription. use of different definitions makes it difficult to compare the results across studies and also to plan future studies. moreover, the rate of pes (proportion of patients with at least one exacerbation over a given time-period) is affected by baseline patient characteristics such as age, fev 1 and medication use at the start of the clinical trial; factors which also contribute to the difficulty in comparing trials. using data from the cf therapeutics development network (tdn) data bank, we examined several pe definitions and baseline characteristics for their effect on pe rates. we find that pulmonary exacerbation rates vary substantially depending on both the pe definition and the baseline characteristics. for example, in one study, the proportion of subjects hospitalized and/or prescribed antibiotics during the 6 month treatment period was 29%, while the proportion who met a symptoms based criteria for pe during the same period was 65%. we also examined the implications of varying control group exacerbation rates on sample size requirements for clinical trials and find that the control group pe rate has a large impact. for illustration, we assume a two group study with two-sided significance level 0.05 and 80% power. if the control group rate is 30%, then approximately 200 subjects per group are required to detect a relative rate reduction of 40% (corresponding to a treatment group rate of 18%). however, if the control group rate is 65%, then only approximately 70 subjects per group are required to detect the same relative rate reduction of 40% (corresponding to a treatment group rate of 39%). thus, a pe definition that selects for a greater pe rate in the control group will require fewer study subjects to detect the same relative rate reduction given that all other variables remain equal. sample size requirements can be further reduced by analyzing the number of events or the time to first event instead of the proportion of subjects who have at least one pe during a given time period. supported by cystic fibrosis foundation therapeutics, inc. (ram-sey03y0), the national center for research resources (ncrr mo1-rr00037) and csl behring. briglia, h.; hilliard, j.; chmiel, j.f.; krenicky, j.; konstan, m.w. pediatrics, case western reserve university, cleveland, oh, usa cystic fibrosis (cf) is characterized by a vicious cycle of obstruction, infection and inflammation. the inflammatory response, which is profound and excessive relative to the burden of bacteria, is characterized by a massive neutrophil (pmn) influx. cf patients often experience intermittent declines in lung function associated with increases in both bacterial burden and pmn influx. these pulmonary exacerbations require treatment with antibiotics. there are substantive issues in the identification of pulmonary exacerbations and the assessment of therapies. a marker which indicates the inflammatory state of the lung would be useful to identify infective/inflammatory exacerbations, as opposed to worsening due to pulmonary vascular disease, or simply upper airway infection (cold), and might guide therapy for intensity and duration. g-csf and gro-α are cytokines produced in the lung by a variety of cells, including macrophages and epithelial cells. g-csf is involved in the proliferation, differentiation and activation of pmn precursors, while gro-α has known chemotactic and activating effects on pmns. transfer of these cytokines through the basolateral membrane into the bloodstream results in increased pmn production and activation in the bone marrow. the plasma concentrations of g-csf and gro-α might serve as potential biomarkers for a pulmonary exacerbation. in this pilot study we measured g-csf and gro-α in the plasma of healthy volunteers (n=3), clinically stable cf patients (n=23) and cf patients experiencing a pulmonary exacerbation, both before (n=14) and following (n=10) 10-14 days of antibiotic (abx) therapy. cytokines were measured using commercially available elisa kits (r&d systems, minneapolis, mn) . t-tests were performed on natural log transformed cytokine data (sigmastat v3.5, systat software inc., san jose, ca). ᭹ the lack of a significant difference between g-csf and gro-α plasma levels in healthy and clinically stable cf patients suggests that these cytokines are not indicators of chronic inflammation. ᭹ both cytokines are increased during pulmonary exacerbations in patients with cf and decrease to baseline values following antibiotic therapy, suggesting that these cytokines might serve as potential biomarkers for cf pulmonary exacerbations. ᭹ although not statistically significant, there was a trend towards an inverse correlation between both cytokines and fev 1 during pulmonary exacerbations in this small study (data not shown). ᭹ further studies are warranted to increase our understanding of g-csf and gro-α as potential biomarkers of pulmonary exacerbation in cystic fibrosis. this work was supported in part by the cystic fibrosis foundation through an institutional research development grant. pletcher, s.d. 1 ; koff, j.l. 2 ; kleinhenz, m. 2 1. otolaryngology, university of california, san francisco, san francisco, ca, usa; 2. medicine, university of california, san francisco, san francisco, ca, usa introduction: while sinus disease is common in adult patients with cystic fibrosis (cf), the severity of sinus symptoms and relationship of sinus disease to other manifestations of cf are relatively unknown. objectives: to evaluate the severity of sinus symptoms in adult patients with cf and correlate these findings with other cf manifestations. methods: twenty-six consecutive adult patients with cystic fibrosis were surveyed during routine clinic visits for sinus specific and overall quality of life outcomes using both the sino-nasal outcome test (snot-20) and the cystic fibrosis questionaire (cfqr). analysis of snot-20 and cfqr scores were compared to %fev1 and sputum culture growth of pseudomonas for all patients. results: snot-20 scores ranged from 0.1 to 3.1 with a mean of 1.17 on a 0-5 point scale. for the purpose of data analysis, the patients were divided into two 13 patient cohorts: group 1 with minimal sinus symptoms (snot-20 < 1.0) and group 2 with mild to moderate sinus symptoms (snot-20 > 1.0). the mean %fev1 for group 1 was significantly higher than the mean %fev1 for group 2 (71.5% vs. 49.9% respectively, p<0.007). patients in group 2 were more likely to have sputum culture growth of pseudomonas although this trend did not reach statistical significance (92% vs 62%, p=0.16). cfqr scores differed significantly among the two groups with group 2 patients reporting more disability in the physical, role, vitality, body, eating, health, respiratory, weight, and digestion domains but not in the emotion, social, and treatment domains. conclusions: this cohort of adult cf patients show minimal to moderate symptoms of sinonasal disability. those patients with mild to moderate sinonasal symptoms have decreased pulmonary function and decreased overall quality of life compared to cf patients with minimal sinus symptoms. 1, 2 1. internal medicine, university of iowa hospital and clinics, iowa city, ia, usa; 2. translational lung imaging program, university of iowa hospitals and clinics, iowa city, ia, usa; 3. pulmonary and critical care medicine, mount sinai medical center, miami beach, fl, usa mucociliary transport is an important component of airway host defense against inhaled particles and microbial pathogens. mucociliary clearance is hypothesized to be defective in cystic fibrosis allowing for airway colonization and infection. current methods for measuring mucociliary transport and clearance lack the ability to measure mucociliary transport in specific proximal and distal airway segments. using a 64-slice high resolution ct scanner (somatom 64, siemens), we are developing a method to track radiopaque particle movement in the swine airway. swine were induced with ketamine/acepromazine, intubated, and then anesthesia maintained with propofol infusions. animals spontaneously breathed humidified air with the endotracheal tube cuff deflated. radiopaque teflon/bismuth trioxide disks (1 mm diameter, 0.8 mm thick) were instilled via a catheter into the airways. serial ct images (0.6 mm slice thickness and slice increment of 0.5 mm) were obtained every 2 minutes for a total of 20 minutes. airway tree segmentation was performed using pulmonary workstation 1.1 (vida diagnostics, iowa city, ia). this program allows for fully automated airway tree segmentation. baseline mucociliary transport rates (2-dimensional) were 15.6 ± 2.6 mm/min. following aerosol delivery of human sputum leukocyte elastase (150 mu), mucociliary transport was markedly diminished (5.0 ± 1.1 mm/min) at 45 minutes. thus far, we are able to measure mucociliary transport down to the 3rd generation airways. future studies include more distal deposition and measurement of particle movement. in summary, this novel method measures mucociliary transport in defined airways and will allow us to examine the effect of altered airway epithelial function on mucociliary transport. lung transplant. purpose: to design a ct scoring system to quantify structural abnormalities on ct scans from cf patients with severe advanced lung disease (sald) and to investigate the correlation between ct scores and survival. materials and methods: ct scans of 57 cf patients screened for lung transplant between 1990 and 2005 were collected from 3 transplant centres. all scans were scored with the brody ii scoring system. to design a new scoring system sensitive for the specific changes in sald, a panel of 3 experts reviewed a random set of 10 ct scans on eligible items to be used in a pilot analysis. the resulting sald scoring system consisted of four items: bulla/cysts; areas with consolidation/mucous; areas with hypo perfusion/air trapping and hyper/normal perfusion. in each ct slice (10 mm spacing) and for each of the items the surface area of corresponding lung tissue involved was estimated on a 0-100% scale. total surface area for the 5 items and each slice added up to 100%. the final sald score was a mean volume estimate of abnormal and normal lung tissue. results: pilot analysis of a set of 25 ct scans showed a a spectrum of abnormalities, ranging from predominantly bronchiectasis to predominantly mosaic perfusion. a moderate correlation was found between the sald components inflammation and hypo perfusion/ air trapping and brody score (p=0.004, r=0.55 and p=0.006, r=0.536). the inter-observer variability of both scoring systems was comparable. (ri ranging from 0.70 to 0.83) analysis of the complete cohort with the brody ii score showed a significant correlation with survival with a hazard ratio of 2.4 (95% ci of 1.07 to 5.33) for dying on the waiting list for every ten point increase in brody ii score. (p=0.02). the brody component scores for bronchiectasis, airway wall thickening and parenchyma showed a similar correlation with hazard ratios (95% ci's) of respectively 1.9 (0.98-3.73), 1.9 (1.03-3.35) and 5.3 (1.44-19.2) for every ten point increase in score. (p=0.044, 0.028 and 0.01). the preliminary results with the brody score suggest that it is useful to include ct information in prediction models. it is likely that the predictive value of the ct can for survival can be further improved using the dedicated sald-ct score. scoring of the remaining scans according to the sald system is ongoing and will be completed shortly. snell, g. 2 1. physiotherapy, the alfred, melbourne, vic, australia; 2. allergy, immunology and respiratory medicine, the alfred, melbourne, vic, australia; 3. epidemiology and preventative medicine, monash university, melbourne, vic, australia; 4. radiology, the alfred, melbourne, vic, australia background despite the widespread use of airway clearance (ac) techniques to clear excessive secretions and improve lung function, little is known about their efficacy following lung transplant (lt). this study aimed to compare the effects of two ac strategies (proactive vs reactive) on a range of clinical outcomes following lt. methods a prospective randomized trial design with stratification for suppurative pre-lt disease was used. consecutive patients at 1 month post lt were eligible for inclusion. subjects were excluded if medically unstable, ventilator dependent or had a contraindication to performing positive expiratory pressure (pep) therapy. patients performed ac using pep either twice daily (proactive strategy) or only in the presence chest infection (reactive strategy). lung function (fev 1 and fvc), chest radiography (brasfield score), exercise capacity (6 minute walk) and bronchoscopic airway characteristics (anastomotic healing, patency and secretions) were assessed at 1, 2 and 3 months post lt. patient adherence and satisfaction were measured. results of 60 consecutive patients, 36 (18 in each group) were recruited and completed the study. both groups improved lung function (fev 1 72 ± 4% to 81 ± 4 % p<0.0001; fvc 69 ± 3% to 81 ± 3% p<0.0001), brasfield scores (17.8 ± 0.5 to 19.8 ± 0.5 p<0.002) and 6 minute walk (451± 16m to 545 ± 16m p<0.0001) over the study period. no significant differences between groups for any outcome were found. the vast majority had fully healed, 100% patent anastomoses without secretions at 3 months. there were no significant differences between airway characteristics and incidence of chest infection. adherence to both strategies was high (84% proactive, 100% reactive). conclusion in the absence of significant differences in outcomes, it is recommended that ac only be performed in the presence of chest infection based on greater treatment cost and treatment time required by a proactive strategy. further studies in those with reduced anastomotic patency and in those with recurrent chest infections later post-transplant are warranted. supported by an alfred trusts grant. munro, p.e. 1 ; button, b. 1, 2 ; bailey, m. 3 ; holland, a. 1, 4 ; snell, g. 2 1. physiotherapy, the alfred, prahran, vic, australia; 2. allergy, immunology and respiratory medicine, the alfred, melbourne, vic, australia; 3. epidemiology and preventative medicine, monash university, melbourne, vic, australia; 4. school of physiotherapy, la trobe university, melbourne, vic, australia background the optimal duration and structure of pulmonary rehabilitation (pr) for lung transplant (lt) recipients is not known. this study aimed to describe changes in functional outcomes in lt recipients who participated in a post lt pr program at the alfred, melbourne, australia. methods prospective, repeated measures design. functional exercise capacity (six minute walk test-6mwt), lung function (fev 1 , fvc) and quality of life (sf 36) were assessed at 1, 2 and 3 months following lt. following discharge, all subjects attended a 1 hour supervised outpatient exercise training class 3 days per week until 12 weeks post lt and 6 education sessions. patients with post-operative complications were excluded. data were analysed using descriptive statistics and anova with repeated measures. results 60 consecutive lt recipients from sept 2003 to mar 2005 were assessed for inclusion. 36 (50% male) subjects, mean age 46 ± 14 yrs were recruited and completed the study. 81% had undergone bilateral lt. 33% had cystic fibrosis, 31% chronic obstructive pulmonary disease. significant improvements were demonstrated in 6mwt (451 ± 16m to 545 ± 16m, p<0.001), fev 1 (72 ± 4 % to 81 ± 4%, p<0.0001), fvc (74 ± 4% to 78 ± 4%, p<0.0001) and all quality of life domains, p<0.05. the greatest changes in 6mwt and lung function were seen between 1 and 2 months. conclusion functional exercise capacity, lung function and quality of life improved significantly over the first 3 months in lt recipients who participated in pr at our institution. these data will allow benchmarking with other centres and program structures. supported by an alfred trusts grant. does gender play a role in perception of cf quality of life domains before and after lung transplantation? the cf quality of life (cfq-r) for patients м14 years of age is a disease-specific instrument. previous studies using this instrument have reported that cf females have higher scores in weight and body perception than cf males. to determine the impact of lung transplantation on gender-based perception of quality of life, we reviewed the cfq-r on all cf patients м14 years of age and who underwent successful lung transplantation at our center. cfq-r was administered 1-3 months prior to transplant surgery and then 3 months after lung transplantation while the patients were in the outpatient clinic setting. domains measured: physical, role, vitality, emotion, social, body image, eating, treatment burden, health status, weight, respiratory symptoms, and digestion. the cfq-r responses were computer-scored and domain scores were then generated (score 0-100, 100=better). during the 2 1 ⁄2 year study period, 14 cf patients underwent successful lung transplantation (5 males:9 females; mean age 16.1 ± 2.5 years). prior to lung transplantation, female cf patients had lower physical domain scores but higher scores in health status perceptions than the male cf lung transplant candidates (see graph). all patients had marked improvement in all domains 3 months after lung transplantation (see graph). after transplant surgery, cf males had higher scores than cf females in emotion and in eating domains. scores for body image domain were similar for both genders before and 3-months after lung transplantation. we conclude that although there were differences in physical and health domain perceptions prior to lung transplantation, both male and female pediatric cf patients had marked improvement in all domains after lung transplantation. however, female cf lung transplant recipients had lower emotion and eating domain scores after transplantation. we speculate that lower scores in these domains may reflect increased post-traumatic stress and depression in female lung transplant recipients. further study on cf quality of life in pediatric lung transplant recipients is on-going. background cf liver disease (cfld) with severe cirrhosis develops in 5-10% of patients, usually within the first 2 decades of life. most patients with cfld suffer from complications of portal hypertension but hepatocellular function usually remains intact for many years, even decades. patients with cfld referred for lung transplantation (tx) may be offered lung tx, lung-liver tx or be excluded from lung tx. however, variceal hemorrhage due to portal hypertension can be managed by banding, sclerotherapy or shunt procedures without liver tx. the aim of this study was to examine outcome of cfld patients who underwent lung tx and the subsequent progression of cfld, particularly with the use of potentially hepatotoxic immunosuppressive drugs. we conducted a retrospective cohort study to compare cf patients undergoing lung tx with and without cfld. b. cepacia-negative cf patients undergoing lung tx at toronto general hospital from 1987-2007 were eligible for inclusion. liver cirrhosis was defined by histology, esophageal varices on endoscopy or imaging evidence of splenomegaly. each patient with cfld was matched for age and year of tx with 3 cf patients without cfld. demographic data, survival and liver function tests (lfts)(ast, alt, alp, inr, albumin, protein at week 1, month 1, 2, 3, 6, 9, 12, 24, 36, 48 and 60 post tx) were obtained by chart review. results of the 2 groups were compared using unpaired t-test (parametric data) or mann-whitney u test (non-parametric data). results 110 b. cepacia-negative cf patients underwent lung tx over this period, 6 (5.5%) having cfld with cirrhosis. data from the 6 cfld patients vs. 18 matched cf patients without cfld were analyzed. no significant difference was found between the groups in pre-tx age, gender, bmi, pi status, cfrd status, fev1% predicted, 6-minute walk distance or lfts. in the cfld group albumin was lower 1 week post-tx (18.2+1.7 g/l vs. 22.2+3.9 g/l, p<0.026), alp was higher 1 month post lung tx (389.5 vs. 137.9, p<0 .033) and alt was higher 60 months post-tx (26.7+8.3 u/l vs. 16.0+4.5 u/l, p<0.026). there was no significant difference in the other blood tests. azathioprine was changed to mycophenolate mofetil in 2 cfld patients due to liver test abnormalities. 1 cfld patient developed hepatic encephalopathy and ascites 4 years post lung tx and is being assessed for liver tx. no difference was seen in the number of episodes of acute rejection or post-tx survival (66.7% vs. 61.1% alive at 5 years). discussion cfld patients undergoing lung tx did not have a worse prognosis than patients without cfld. transient elevation of lfts were seen in the immediate post-tx period but settled either spontaneously or after discontinuation of azathioprine. we conclude that selected patients with cirrhosis due to cfld may be safely offered lung tx without concomitant liver tx. infectious mononucleosis, commonly called mononucleosis, or "mono," is an illness caused by the epstein-barr virus. mononucleosis has been nicknamed the "kissing disease" because the epstein-barr virus commonly is transmitted in saliva during kissing. however, sneezes and coughs also can transmit the virus occasionally. the epstein-barr virus (ebv) permanently infects more than 90% of the people on earth, but it causes symptomatic mononucleosis only in a small number. in developed nations, such as the united states, mononucleosis most often occurs between the ages of 15 and 25. unfortunately ebv can not only infect, but can also transform, b cells after transplant and may lead to lymphoma with a resulting ~50% mortality rate. the source of ebv is presumed to be passenger lymphocytes in the donor tissue and those at greatest risk are patients who are ebv naive before transplant. since most cf patients with endstage lung disease in the developed world are now being referred for transplant given the success of this intervention, we reviewed our entire lung transplant database to determine the nature of ebv infection and the likelihood of developing lymphoma following lung transplantation. of 272 first-time lung transplants, 178 (65%) had cf. ebv serology was not available in 27 (15 cf, 12 others) before transplant. 34 of 163 (21%) cf patients were ebv seronegative before transplant as compared to 4 of 82 (5%) patients with other end stage lung diseases (composed mostly of copd and ipf) in the control population (chi square p < 0.001). the cf ebv seronegative cohort was 24 (sd 9) years old on average, an age when most seroconversion has already occurred in our society. of the cf ebv seronegatives, 27 survived beyond 3 months and were thus at risk for post transplant lymphoma. of these 27, 8 developed lymphoma (incidence = 30%) in comparison to 1 of the 4 ebv seronegative controls (incidence = 25%, p = ns). all lymphomas were stage iv at presentation and the majority (9 of 11) arose in the first post transplant year. the cf ebv seronegatives who developed lymphoma did not differ from those who did not with regard to levels of immunosuppression (cyclosporine, methylprednisolone or prednisone) or doses of antiviral therapy (i.e., ganciclovir which is active against both cmv and ebv). in addition, the cessation of anti-lymphocyte antibody induction therapy in the summer of 1995, somewhat surprisingly, had no impact on the development of lymphoma. two cases of lymphoma developed in cf ebv seropositives (incidence = 1.2%) and no cases of lymphoma developed in the control ebv seropositives (p = ns). thus ebv seronegativity raises the lymphoma risk 25 fold in cf patients (p < 0.001). two of the 11 (18%) cases of lymphoma resulted in death (one cf and one other) and one cf patient probably died of complications related to chemotherapy even though he was tumor free at that time. lymphoma outcome was not stage or clonality dependent. in conclusion, cf lung transplant recipients are at an increased risk for lymphoma largely due to their lack of exposure to ebv before transplant. kissing more before transplant may lower the risk of lymphoma afterward. university hospital uzb, brussels, belgium; 2. chest medicine, erasme university hospital, brussels, belgium; 3. dermatology, erasme university hospital, brussels, belgium; 4. gynaecology, erasme university hospital, brussels, belgium; 5. gastroenterology, erasme university hospital, brussels, belgium; 6. pathology, erasme university hospital, brussels, belgium hpv infection is an underestimated phenomenon in organ transplantation (tx) recipients, being unable during immunosuppression (is) to mount an adequate anti-viral immune response and risking genital/anal warts (condylomata acuminata) as well as pre-cancerous/cancerous lesions. we retrospectively assessed the incidence/treatment of genital/anal hpv-associated lesions in cystic fibrosis (cf) lung tx recipients. the files of all 74 tx patients transplanted in the ulb center between 1988 and 01/2007 (38 men/36 women, median age 24±7.5, range 8-45 yrs) were reviewed. maintenance is consisted in chronic triple therapy combining a calcineurin inhibitor, a cell cycle inhibitor and corticosteroids. median survival was 46.5 ± 44.4 months (range 0-186). ten of 60 sexually active patients (17%) who survived ≥12 months post-tx developed hpv-associated genital/anal lesions: 3 men (35-41 yrs) and 7 women (25-41 yrs). genital/anal hpv-pcr proven condylomata were diagnosed between 62-97 months in the men and between 14-90 months post-tx in the women. all 10 underwent local treatment (cryotherapy ± laser ± topical imiquimod), 3/10 patients underwent multiple treatments under general anesthesia. recurrence of the condylomata was high. one male patient presented high-grade anal dysplasia and 3 women moderate to high-grade cervical dysplasia; 2 underwent conization and one complete hysterectomy after 2 conizations. one of the females presented both condylomata and a high grade cervical dysplasia. these retrospective data in a cf population indicate that 1) hpv infection may cause significant morbidity in young subjects with chronic disease needing organ tx. it may even compromise life expectancy after tx. 2) data on the effectiveness of treatment strategies -including topical immunomodulators -have to be collected 3) the potentially protective effects of the now available hpv vaccination in females and males? with chronic disease at risk for future organ tx, when administrated before sexual activity and before tx, should be rapidly evaluated in a multi-center effort. ballmann, m. 1 ; pfister, e. 2 ; schlueter, k. 1 ; becker, t. 3 ; melter, m. 2 ; junge, s. 1 1. pediatric pulmonology and neonatology, medical school, hannover, germany; 2. pediatric nephrology and hepatology, medical school, hannover, germany; 3. visceral and transplant surgury, medical school, hannover, germany liver disease is an important comortality and comorbidity in cf patients even in the pediatric age group. liver transplantation (ltx) is an accepted option in end stage liver disease. nevertheless the clinical outcome in cf patients is still under discussion. here we reported the clinical outcome 12 months after isolated liver tx in pediatric cf-patients.since 2000 n=9 ltx were performed in children (male n=7, female n=2; age (mean±sd) 14,1±3 years) . immunosuppressive therapy was done with ciclosporin ± basilixumab and steroid. we followed pulmonary function, cn-score, nutritional status (bmi z-score) and body com-position, airway cultures and igg.results:all were still alive 12 months after ltx. before ltx all patients had a mild to moderate pulmonary disease: fvc(%pred) 92,2±16 (71,4%-119), fev1(%pred) 88,7±16,1 (68-103), mef25(%pred) 61, 7± 28,5 (35-114) , cn-score 8±4,3 points. nutritional status: bmi(mean) 17,2±1,8% (range: 15,3%-20,3%, z-score: 0,88±0,86), upper arm circumference(cm)(mean±sd) 17,8±0,9, triceps skin fat fold(cm) 5,2±1,2. body composition: 13,5±7,2% of bodyweight were fat. one year after ltx all patients were still in a stable pulmonary situation: fvc(%pred) 93,9±14 (range:75,5%-114%), fev1(%pred) 88,6±12,9 (range:68%-108%), mef25(%pred) 61,6±30,4 (range:24,7%-106%), cn-score 7,5±4,8 points. there weren't any significant changes in the airway microbiology under immunosuppressive therapy (steroid, ciclosporin±basiliximab), the serum igg levels declined significant from 17,4±3,9g/l to 9,8±2,3/l(p<0.05). the growth over the year was 6,9±5 cm and the increase of weight come to 5,1±6,1kg, while the bmi didn't increase in this first year after transplantation. certainly we found an increase of the body fat mass (to 18,2±3,8% of body weight), of the upper arm circumference (to 20±4,2cm) und of the triceps skin fat fold (to 11,4± 8,1cm) .conclusion:liver transplantation is an effective therapy even in children with cf related liver disease and stabilise pulmonary function and improves nutritional status in patients with cf and mild or moderate pulmonary involvement before ltx. (neglia jp et al., n engl j med 1995; 332:494-499) , and colon cancer has been described in young adults with cf (chaun h et al., can j gastroenterol 1996; 10:440-442) . three of 62 lung transplant (ltx) recipients from our center developed colon cancer after successful bilateral lung transplant for end-stage lung disease. data from these 3 individuals are given in table below . an additional 8 recipients with cystic fibrosis were screened via colonoscopy. colonic polyps were detected in 2 and included lesions up to 3 cm in diameter. five years after a colonoscopy that had shown a 4 mm polyp, one patient underwent a 2nd colonoscopy that revealed multiple polyps, including a 3 cm diameter sessile polyp in the sigmoid colon. colonic malignancies appear to arise from mucosal polyps, and screening via colonoscopy can detect and remove premalignant lesions. lung transplant recipients have an increased risk of gi malignancies and tumor surveillance is impaired by post-transplant immunosuppression, colonoscopic screening should be considered for transplant recipients with cystic fibrosis. additionally, colonic malignancy should be considered as a potential cause of unexplained gastrointestinal symptoms such as constipation. nossent, g. 1 ; kastelijn, e. 1 ; teding van berkhout, f. 1 ; zanen, p. 1 ; van den bosch, j. 2 ; van de graaf, e. 1 1. respiratory dis, university medical centre, utrecht, netherlands; 2. respiratory diseases, st. antonius hospital, nieuwegein, netherlands objective: in the netherlands lung allocation is based on waiting time. to prioritize patients with a poor prognosis and to reduce waiting list mortality the high urgency (hu) status was introduced in 2001. to be considered for hu status there has to be a limited life expectancy on clinical judgement and all three transplantation centers have to audit this decision. the aim of this study is to analyze the potential effect of las on patients with cystic fibrosis (cf) who became hu or died on the waiting list. it would be expected that patients who became hu had a higher las. methods: from 2001 till 2007 in all cf patients placed on the hu waiting list, the median las (25th, 75th) was calculated at the moment of listing and at the moment the patients became hu. also for the cf patients who died on the waiting list the mean las (sd) was calculated at the moment of listing. results: from the 187 patients on the waiting list 52 patients had cf. of these patient 19 were placed on hu list and 4 died on the waiting list. in the cf patients that became hu the median las on the moment of listing was 35. 1 (33.9;35.9) . at the moment they were placed on the hu waiting list the median las was 35. 5 (34.8;37.4)(p= 0.2) . the median las of the 4 patients who died on the waitinglist was 35.3 (2.77) . this las did not differ significantly from the las of listing of the patients that became hu (p= 0.2). conclusion: the las does not detect the deterioration in patients with cf as diagnosed by clinical judgement. this may be due to the exclusion of specific prognostic factors of cf in the calculation of the las. besides that, the las does not differentiate cf patients on the waiting list with a high chance of mortality. introduction: lutx recipients have one of the highest rates of ia in solid organ transplantation causing high morbidity and mortality rates. nowadays antifungal prophylaxis is extensively used but clinical trials are rare. methods: all patients who received lutx in our hospital were included in this retrospective study. aspergillus airway colonization was defined as aspergillus cultured twice from airway specimens in the absence of ia or tracheobronchitis (tb). definition of ia and tb was according to literature. after the death of 2 cf-patients due to ia, a prophylaxis protocol was introduced containing avoidance of use of the cell saver during the operation in pretx colonized patients to avoid hematological fungal spreading and targeted prophylaxis with itraconazole or voriconazole in all pre-and posttx colonized patients during 3 months and inhaled amphotericin during hospital admission. results: a total of 93 lutx were performed (in 92 patients) (35% cf patients) from 15-7-2001 until 31-12-2006. 23 patients (24.5%) were colonized with aspergillus pretx and 16 patients posttx. only 4 patients were colonized before and after tx. before introduction of the protocol 4 patients (4/69=5.7%) (50% cf patients) developed an aspergillus infection: 3 (4.3%) patients developed a. fumigatus tb and 1 (1.4%) patient suffered from ia: cerebral a. fumigatus infection (proven ia). two patients, both cf patients, (2/4=50%) died due toaspergillus infection (cerebral and tb).after introduction of the protocol only one a. fumigatus tb occurred (1/24= 4.1%) but no invasive fungal infection. conclusions: the rate of fungal infections in our lutx patients was comparable with data from literature. since introduction of azole prophylaxis protocol and avoidance of use of cellsaver no ia occurred. fischer, a. 1 ; müllinger, b. 2 ; arendt, t. 1 ; bernhardt, t. 3 ; hannah, k. 4 ; scheuch, g. 1 1. activaero gmbh, gemuenden, germany; 2. biological products, bayer healthcare, leverkusen, germany; 3. activaero gmbh, gauting, germany; 4. talecris biotherapeutics, research triangle park, nc, usa background patient compliance during a study is an important factor in view of assessing the clinical effect of a treatment. this is especially true when patients administer the drug at home. usually, patient records, counting of returned doses or mechanical counters are used to track compliance, which may be biased by the study subjects. this report is about an aerosol study in cf which used a device for controlled breathing (akita® inhalation system, activaero, germany). the device works with a patient-individual smart card that records every single breath during a treatment including a date/time stamp in an encrypted manner. each patient's inhalation protocol can be displayed by loading the smart card into the proprietary "compliance manager" software. methods for this report, the data set of a recent controlled study (griese et al., 2007) was analyzed. 72 cf-patients were instructed to inhale with the akita inhalation system at home for 42 days. after the study, the smart cards were returned to activaero, and the inhalation records on the chip were analysed. we analysed the compliance of patients who participated at least 21 days (59 out of 72 patients, others deemed to be drop-outs . most of the patients showed a dsc which is lower than the tdc, indicating that they had missing treatment days, which were compensated by additional inhalations on other days. we found 9 patients with a dsc more than 10% lower than their tdc (max difference: 23.81%). conclusion this aerosol study with home treatment demonstrated a high compliance rate for tdc and dsc. this information is more reliable for compliance than study participation in days alone. for future studies it is recommended to define in the protocol not only a minimum of study participation in days but also a minimum in tdc and dsc. in addition the compliance thresholds may be defined with regard to the drug's pharmacokinetic profile. in general, the inhalation record of the akita smart card provides an unbiased view of the inhalations treatments during a study especially when the subjects perform the inhalations at home. compliance calculations as shown above may be performed and linked to other outcomes of a study for validation. compliance data like these may also be used routinely by the treating physician in order to guide and supervise his patients. introduction: adaptive aerosol delivery (the i-neb ® aad ® system) has been developed to deliver precise, reproducible doses of aerosol into inhalation. i-neb utilizes a medication chamber, which incorporates a metering chamber to deliver a preset (metered) dose of aerosol. inhalation of hypertonic (7%) saline has been shown to aid mucociliary clearance in patients with cystic fibrosis. [1] we performed an in vitro test to determine the emitted, delivered and residual doses of hypertonic saline from the i-neb aad system with 0.5 ml metering chamber, and a conventional jet nebulizer (lc plus ® ) with a 4 ml fill. the results were then entered into a model to predict the likely lung dose from the in vitro tests, in order to determine the equivalent i-neb dose to the 4 ml conventional jet nebulizer fill. methods:an i-neb device configured to operate at power level 10 (of 15) was fitted with a metering chamber designed to deliver a preset dose of 0.5 ml. the i-neb was weighed, loaded with 1 ml of 7% saline and run on the cen simulated breathing pattern. this process was repeated using an lc plus nebulizer loaded with 4 ml of 7% saline. aerosol was collected on a filter placed between a harvard pump and the device. emitted dose and residual dose were determined by gravimetric output, and delivered dose was determined by ion analysis. all tests were conducted in triplicate. results: as shown in table 1, the emitted dose approximated the delivered dose for i-neb, whereas for lc plus the emitted dose was approximately twice the delivered dose, due to the wastage of aerosol upon simulated exhalation. as i-neb has a delivered dose of 513 mg, and i-neb has been shown to deposit 62.8% of the dose in the lung, [2] this would result in a lung dose of 322 mg. the pari lc plus has a higher delivered dose, but the overall lung deposition is only 47% of the fill volume, i.e. 613 mg. [3] conclusion: the results of this test suggest that two treatments would be required in order to deliver an equivalent lung dose of hypertonic saline from an i-neb device fitted with a 0.5 ml metering chamber, compared with a 4 ml fill in an lc plus jet nebulizer. references introduction: inhaled colistimethate sodium can be used for the eradication of pseudomonas aeruginosa in patients with cystic fibrosis. the tonicity of colistimethate sodium inhalation solution has been linked to the occurrence of bronchospasm in some patients. it has been suggested that this bronchospasm can be avoided by using an isotonic solution of colistimethate sodium. [1] this can be complicated when using conventional jet nebulizers, since evaporation during nebulization tends to change the tonicity of the solution remaining in the nebulizer. however, because very little evaporation occurs within the i-neb medication chamber during nebulization, the tonicity of the loaded solution at the beginning and end of nebulization remains almost constant. aim: we investigated the effects of various saline concentrations upon the tonicity of two concentrations of colistimethate sodium solution (0.5 miu/ml and 1 miu/ml). methods: diluent containing sodium chloride concentrations of: 0% (water for injection), 0.225%, 0.45%, 0.675%, and 0.9% (normal saline) were used to reconstitute vials of colistimethate sodium (promixin ® , profile pharma, uk). each vial was reconstituted with either 1 or 2 ml of diluent to make up 1miu/ml or 0.5miu/ml colistimethate sodium solution, respectively. the tonicity of each diluent concentration/colistimethate sodium concentration combination was made up and tested in triplicate using an advanced micro osmometer (advanced instruments, norwood, usa). the results were plotted on a graph along with a line of best fit, to determine the diluent concentration that gave a mean tonicity closest to that of an isotonic solution. results: tonicity increased linearly with increasing concentrations of saline solution for both 1miu/ml and 0.5miu/ml concentrations. the tonicity of the 1miu/ml solution was approximately 116 mosm higher than the 0.5miu/ml solution across the range of saline concentrations tested. the best fit line passed through the isotonic solution point (290 mosm) closest to the 0.45% saline concentration point. conclusions: the diluent that produces the closest approximation of isotonicity for the mean of 0.5miu/ml and 1miu/ml colistimethate sodium concentrations was 0.45% saline. references 1. transave, inc., monmouth junction, nj, usa; 2. activaero, gmbh, gauting, germany arikace™ is an inhalation formulation of a liposomal amikacin suspension that is designed to treat chronic pseudomonas aeruginosa infections in cystic fibrosis patients. liposome encapsulation of amikacin reduces nonspecific binding of this cationic aminoglycoside drug to the negatively charged mucus and biofilm surfaces and allows penetration and delivery of packets of highly concentrated drug to the otherwise protected bacteria. as nebulized and delivered to the lungs, arikace™ comprises 65% liposomal amikacin and 35% 'free' amikacin that is not entrapped by liposomes; free drug is produced by liposome leakage during nebulization. this profile provides an initial high peak concentration of amikacin followed by a sustained level as drug leaks from the liposomes. nebulized arikace™ with this profile was evaluated previously in human clinical studies using the lc star® nebulizer. although the 0.3 µm liposomes of arikace™ are efficiently loaded with drug, the expected effective dose in humans will require relatively large volumes of drug solution to be administered. to minimize treatment time and improve patient convenience we sought to find a nebulizer that would efficiently deposit drug and produce aerosol at a high output rate, while still producing the same level of free drug during nebulization. methods: nebulization of liposomal amikacin was compared using several nebulizers, including 2 jet nebulizers: lc star® and lc sprint® (both from pari), and 3 electronic mesh nebulizers: microair® ne u22v (omron), aeronebgo® (nektar), and eflow® (pari). additionally, meshes of different pore sizes were examined for the microair® and eflow® devices. output rates were measured by gravimetric differences. droplet size distribution was assessed by a laser light scattering method and by cascade impaction; mass median aerodynamic diameters (mmad), geometric standard deviation (gsd), and fine particle fraction (fpf) (i.e., % droplet mass <5 µm). amikacin association with the liposomes was measured using a centrifugation-filtration assay. results: in terms of output rate, the order of performance was eflow® (~0.5 g/min) > lc sprint® > lc star® > aeronebgo® > microair®. while the aeronebgo® and microair® mesh nebulizers performed well with saline, their performance declined dramatically when nebulizing the liposomal amikacin solution, and, in fact, the microair device clogged. in additional studies with the eflow® where different meshes were compared, the 40l mesh was selected as it provided ideal aerosol properties (mmad = 3.7 µm, gsd = 1.7, fpf >70%) with a ratio of entrapped to free amikacin of approximately 65%/35%. importantly, from in vitro breath simulation studies it is estimated that about twice of the nominal drug dose is expected to deposit in the lungs compared to the lc star®. conclusions: for nebulization of liposomal amikacin, the eflow® configured with a 40l mesh provided optimum aerosol characteristics with a high output rate and ideal size for distribution throughout the lung. the eflow® reduces dose administration time not only because of its faster output rate but also because its higher deposition factor greatly reduces the amount of drug needed. zeman, k.l.; bennett, w.d. cemalb, university of north carolina, chapel hill, nc, usa many inhaled, therapeutic drugs have their site of action in the conducting airways, but may deposit elsewhere in the respiratory tract, resulting in unwanted side effects and waste. the quantity and location of particle deposition in the respiratory tract depends on both the particles' aerodynamic size and breathing pattern. in 8 subjects with normal lung function, we evaluated three methodologies for their ability to deliver radiolabeled particles (99mtc-labeled sulfur colloid) to the conducting airways: 1. inhalation of 5 µm mmad particles (devilbiss 646 jet nebulizer) using a resting tidal volume and flow (mean 0.43 l and 0.39 lps); 2. inhalation from the same nebulizer using a very large inspiratory capacity volume and higher flow rate (mean 2.6 l and 0.52 lps); and 3. inhalation of larger 9.5 µm mmad (heart jet nebulizer) particles with a large tidal volume and very low flow rate (mean 0.89 l and 0.08 lps). gamma scintigraphy gave an estimate of % conducting airway deposition (%cad) by multiplying the percent of activity in the lungs immediately post-deposition relative to the total deposition (i.e. lung+mouth +esophagus+stomach) times the percent of activity cleared from the lungs over 24 hours. %cad was 17%, 14%, and 31% for the three methods, respectively, significantly greater for the large particle, very slow inhalation technique when compared to the other two methods. the amount deposited in the mouth and larynx was 52%, 34 and 33%. in addition, we evaluated protocols 1 and 3 above in a preliminary group of 3 cf patients. for protocol 1, mean inhaled volume and rate was 0.48 l and 0.44 lps. mean inhaled volume and flow were 0.73 l and 0.078 lps for protocol 3. the %cad for the two protocols 1 and 3 were 16% and 25%, respectively. the amount deposited in the mouth and larynx was 41% and 33%. higher therapeutic value of a medication delivered to the airways where the primary defect is associated with cf disease, and with fewer losses to the extrathoracic airways, can be obtained by using a "very large particle/slow inhaled flow" routine when compared to normal or higher flow breathing associated with typical nebulizers. supported by cf foundation. positive effects of physical conditioning in cystic fibrosis (cf) have been reported for quality of life (qol) in addition to effectis on fitness and lung functions. the objective of this study was to identify the effects of different modes of training on qol in cf. 58 cf-patients (age 12-40 yr., fev1 >35%pred.) were randomized into 5 groups: germany: unsupervised homebased training, 3 hours per week, free choice of training mode and means (ut, n=18) and controls (con1, n=10); switzerland: aerobic training (at, n=11) or weight training (wt, n=11) in a fitness center, 3 x 30 min per week, or controls (con2, n=8). subjects in the training groups were asked to train for 6 months. at study entry and after 3 and 6 months, qol was assessed by questionnaire and maximal physical working capacity (pwc, %pred) was determined during cycle ergometry (godfrey protocol). changes from baseline values were calculated and anova for repeated measures was used to test for differences among groups. weight training resulted in a significant decrease in qol (generic dimension) at 3 and 6 months compared with all other training modes including controls. there was no significant difference between at or ut and controls in qol during the program. however, there was a positive association between the change in pwc and the change in qol (r=0.32, p<0.05) in the entire sample. in conclusion, physical training may not always result in an increase in ql in cf. possibly, the decrease in qol in the wt group may have resulted from tiredness induced by the training. thus, a different outcome might have been found with less intense training. positive effects may be achieved by improving aerobic fitness. supported introduction: exercise and exercise training programs are felt to be important to improve aerobic exercise capacity, muscle strength, lung function, and quality of life in cf-patients. recent studies in children and adults with cf have demonstrated an increase of motor performance after exercise training. less is known about trainability of motor performance in younger children with cf. the aim of this study was to assess the effects of a training program on motor performance in preschool children with cf. methods: 19 preschool children with cf (10 female and 9 male patients, mean age 5.2 yrs; range 4-6 yrs, fev1%pred. 100,1±15.6 rage from 65.8-123.1 ) participated. at baseline (t1) and at the end of the training program (t2) the "motorik test (mot)" was used to assess motor performance and to evaluate training effects. components of motor performance tested with the mot are balance, agility and flexibility, strength, coordination, fine motor skills, reactivity, accuracy of movement). lung function in patients with cf was measured by spirometric techniques. the duration of training was 4-6 weeks (3 times/week, 45 minutes per session) with different kinds of sport activities. results: at t2, strength and balance had increased significantly (p<0.05). no other components of the mot increased, but the increases in strength and balance were enough to cause a significant (p<0.05) increase overall motor performance, as indicated by an increase of 10.7% in mq. the mq is the sum of all 18 test-items of the mot. no correlation was found between lung function and motor performance. discussion: to our knowledge this was the first study that examined the effects of an exercise training program on motor performance in preschool children with cf. main findings of this study were that a training program of 4-6 week of duration improved some aspects of motor performance. the improvements may be explained by different kinds of sports activities during training when compared with activities before training. classification of motor performance of the tested children was normal (mq =3.1±0.8). this could be the reason that only some aspects of motor performance increased. parents of the children were asked about habitual physical activity at home. all children were physically active and some of the children participated in organized sports. a recent study showed that with an increase in age lower motor performance in children and adolescent with cf was seen compared to healthy children. we speculate that time spent in physical activity in preschool children with cf is comparable to healthy children. conclusion: an exercise program offered to preschool-children with cf may lead to an improvement in motor performance. pre-school children with cf have normal motor performance. to prevent a decline of motor performance, exercise programs should be available for preschool children. gruber, w. 1 ; braumann, k.m. 2 ; orenstein, d.m. 3 ; huels, g. 1 1. dept. for sports and exercise, clinic sattelduene for children and adolescent, nebel/amrum, germany; 2. institute for sport and exercise medicine, university of hamburg, hamburg, germany; 3. school of medicine, university of pittsburgh, pittsburgh, pa, usa; 4. dept. of medicine, clinic sattelduene for children and adolescent, nebel/amrum, germany introduction: a decline in physical activity with age is one of the main problems in patients with cf. as a consequence, lower exercise capacity and a more rapid decline in lung function may be seen in those patients who are physical inactive. furthermore, motor performance is lower in children and adolescents with cf compared to healthy children of the same age. little attention has been paid to younger children with cf and motor performance. the aim of the study was to compare motor performance in preschool-children with cf with healthy children of the same age. methods: 19 children with cf (mean age 5.2 yrs; range 4-6 yrs) and 21 healthy children (mean age 5.5 yrs; range 4-6 yrs) participated. the "motorik test" (mot) was used to assess motor performance. the mot consisted of 18 test-items which evaluated seven different components of motor performance (balance, agility and flexibility, strength, coordination, fine motor skills, reactivity, accuracy of movement). lung function in patients with cf was measured by spirometric techniques results: results of the mot showed no difference either for single testitems or for parameter "motorische quotient (mq)" (p>0.05). this parameter classified motor performance of all aspects tested with the mot. classification of children with cf and of healthy children was normal. mean value for parameter mq (cf: 3.1±0.8 vs. healthy 3.29±0.7) was slight higher in patients with cf than in healthy children (p>0.05). differences between healthy and cf for different components of motor performance were not significant (p<0.05) discussion: the mot is a reliable and valid test method in germany to determine motor performance in younger children. to our knowledge this was the first study to examine motor performance in preschool-children with cf. furthermore, we compared patients with cf and healthy children. we observed no differences between groups for motor performance. in older children with cf, lower motor performance has previously been found compared to healthy children of the same age. we do not have any information about the level of habitual activity in tested healthy children. we therefore speculate that time spent in physical activity in cf is similar to that of healthy children at this age. a recent study showed a decline in physical activity in cf when age and problems during physical activity increased. we think that motor performance is normal in children with cf up to an age of 6 yrs. when age increased, time spent in physical activity decreases (treatment, school) and therefore motor performance decreases. conclusion: motor performance is normal in preschool-children with cf compared to healthy children. to prevent a decline or to maintain motor performance it is recommended to motivate cf-children from early infancy to participate in sports activities or other physical activities to maintain or to increase their physical fitness. 1 1. respiratory medicine, the hospital for sick children, toronto, on, canada; 2. respirology, st. michael's hospital, toronto, on, canada; 3. respiratory medicine, montreal children's hospital, montreal, qc, canada purpose: the habitual activity estimation scale (haes) questionnaire has been shown to be a feasible tool to measure physical activity however the reliability has yet to be determined in cf populations. we therefore assessed the reliability and validity of the haes questionnaire in paediatric and adult cystic fibrosis (cf) populations. methods: fourteen (7 male, 7 female) patients aged 16.2 + 4.2 yrs with cystic fibrosis from the cf clinics at the hospital for sick children and st. michael's hospital participated in this study. participants were clinically stable at the time of the study (fev1 > 70% predicted) and participating in their 'habitual' physical activity. to assess test-retest reliability, patients completed each of the haes (schneiderman-walker et al., j pediatr. 2005; 147 (3):321-6.), and a validated 3-day activity diary (bratteby et al., eur j clin nutr. 1997; 51(9) :585-91), and wore an actigraphtm tri-axial accelerometer for two consecutive weeks. validity was assessed by comparing the activity results of each of the three instruments over a single week time period. results: intra-class correlation coefficient estimates of reliability for the haes, diary and accelerometer were 0.72 (p<0.0001), 0.76 (p<0.0001), 0.63 (p<0.0001), respectively. validity was estimated by comparing the results from each instrument using a one-way anova with block design. there were no overall differences among the participants' activity results as estimated by the haes, diary and accelerometer. furthermore, there were no significant differences between activity measures among the 3 instruments when broken into morning, afternoon, or evening periods, or between measures from weekday or weekend days. while there were no significant differences among the 3 instruments when recording 'very active' intensity levels, they did detect differences during the inactive / somewhat inactive (p = 0.02) and somewhat active (p = 0.001) intensity levels. conclusion: the findings of this study suggest that the haes questionnaire is a reliable and valid instrument that can be used to assess activity levels in patients with cystic fibrosis. in agreement with others, our patients found it easiest to recall and record accurately those periods in which they are participating in vigorous activity. acknowledgement: this research was supported by the canadian cystic fibrosis foundation. 1 1. respiratory medicine, hospital for sick children, toronto, on, canada; 2. respiratory medicine, montreal children's hospital, montreal, qc, canada; 3. respirology, st. michael's hospital, toronto, on, canada objective: previous work showed a relationship between habitual physical activity (hpa) and decline of lung function (fev1) in cf females (sneiderman-walker, j peds 147:321,2005) . this study only included a small number of patients and the follow-up period was limited to 18 months. the purpose of this study was to evaluate daily activity levels and decline in fev1 in a larger cohort over a longer period of time. methods: a total of 187 (n= 99 female) patients with cf (age 7-25 yrs) were studied over a six year period at the hospital for sick children, st. michael's hospital and montreal children's hospital. at each quarterly cf clinic visit, patients performed pulmonary function tests to determine fev1 and completed the habitual activity estimation scale (haes). weekday total activity (wdta), expressed as hours/day (h/day), was calculated from the sum of 'very active' (wdva) and 'somewhat active' (wdsa) categories as previously described. a repeated measures linear regression was performed to evaluate the relationship of fev1 and hpa over time. results: at baseline, mean fev1 was 85% predicted (range 29-119% pred). mean baseline wdta was 5.3 h/d (wdva = 2.3 h/d and wdsa = 3.0 h/d) similar to that previously reported. subjects were divided into high and low activity groups based on the group median wdta. those in the low activity group had a significantly (p=0.004) steeper rate of decline (-1.47 % predicted per year) compared to those in the high activity group (-1.06 % predicted per year) . unlike in the in the previous study this difference was observed in males as well as females. conclusions: six year follow-up of this patient cohort has shown that higher activity levels are clearly associated with a slower rate of decline of fev1. we are currently evaluating whether strategies aimed at increasing physical activity will have an impact on lung function decline in cf patients. background: knowledge of exercise principles, benefits, and techniques has not been assessed in children with cystic fibrosis (cf) or their parents. regular exercise is a recommended cf treatment regimen that is often not followed, but one which might improve cardiovascular fitness, lung function, sputum clearance, and health-related quality of life. further, aerobic fitness, measured as peak oxygen uptake (vo 2 ), is positively related to survival in cf. construction of a reliable and valid measure of exercise knowledge is necessary to assess level of knowledge and study its relationship to exercise behavior. the aim of this study was to develop and test a measure of exercise knowledge for pediatric patients with cf and their parents. methods: we constructed a 22-item exercise knowledge test (ekt) and then examined its psychometric properties. a comprehensive review of the exercise literature guided the formulation of objectives and development of items. to establish content validity, a panel of experts (cf physician, two exercise physiologists, and a clinical cf nurse) evaluated the objectives and test items for relevance, clarity, and accuracy. after feedback from the panel, we revised the ekt through an iterative process. in consultation with a reading level expert, we wrote the ekt items so that the test could be administered to children and adults. a measurement and evaluation expert also reviewed the test, and additional revisions were made. during routine clinic visits, the ekt was administered to 50 subjects with cf and 50 parents. the cf subjects included 31 boys and 19 girls, age 8 to 18 years, fev 1 range: 26-123 % predicted. the parents had an average age of 41 years and most (n=44) were female. results: the ekt includes 22 items, 12 true-false and 10 multiple choice. it is self-administered, yields a single summed score, and has a flesch-kincaid 3rd grade reading level. the means were 17.96 + 2.7 (82% correct) and 20.24 + 1.4 (92% correct) for the cf subjects and parents, respectively; 74% of the parents scored between 20 and 22. internal consistency reliability estimates were .64 for the children and .26 for the parents, reflecting both the multidimensionality of the material and the lack of variability in the parent scores. item difficulty ranged from 0.50 to 0.98 for the cf subjects and from 0.64 to 1.00 for the parents. in both groups, the discrimination values were positive for all items with difficulty values less than 1.00. conclusions: we were able to develop a measure of exercise knowledge with an acceptable reading level for children with cf and their parents. this sample of cf subjects and parents showed substantial exercise knowledge. the ekt can be a useful tool to identify gaps in exercise knowledge in cf patients and their parents, develop educational programs to increase knowledge, and examine how knowledge relates to exercise behavior in children with cf. supported by cystic fibrosis foundation therapeutics, inc. purpose: although physical activity is routinely recommended for adolescents with cystic fibrosis, past research suggests that many patients do not regularly participate in physical activity. there may be a variety of reasons, both related to disease and otherwise, for this inconsistent participation. in this study, qualitative methods were used to describe factors that facilitated or served as barriers to physical activity in adolescents with cf and compared these factors to those in a healthy peer group. methods: ten subjects with cf and ten subjects without cf aged 13-17 volunteered for this study. each subject participated in two, open-ended semi-structured interviews with 3-4 weeks between each interview. subjects were questioned about their current and past physical activity as well as the benefits and barriers to participation. the adolescents with cf were also asked about their perceptions of physical activity on their own health and other adolescents with cf. interviews were transcribed and coded by three investigator groups. analysis was conducted using the grounded theory approach at two points in the study, following each interview and again at the completion of all interviews. at the first point, individual interviews were coded using line-by-line coding, and similar information was grouped into categories. in the second interview, categories were confirmed and subjects were also asked to elaborate or clarify points from the first interview. interviews were continued until themes reached a saturation point. upon completion of all interviews, data for each group were organized by question. data categories appearing throughout the interviews became major themes describing facilitators and barriers to exercise. the major themes were identified by the three researcher groups and then through collaborative group analysis. results: the central theme for both groups was perceived importance of physical activity for health benefits. for the cf group, facilitators and barriers were both psychological and physical. physical benefits were categorized as either relating to their general health (such as feeling energized) or as benefiting their disease (such as improving breathing). in the non-cf group, facilitators included social factors in addition to general health-related and psychological benefits. in the non-cf group, barriers were categorized as either internal or external, with the internal barriers being similar to the cf group (physical and psychological), however, the non-cf participants also indicated external barriers (such as time) not articulated by the cf group. conclusions: in general, we found very similar facilitators and barriers to physical activity in adolescents with cf and those without. however, the adolescents with cf strongly indicated that physical activity would have positive effects on their physical health. further work is needed to determine ways to accentuate facilitators and remove barriers to improve physical activity levels in adolescents with cf. this project was supported by a research grant from the cardiovascular & pulmonary section, apta. lloyd, e. 1 ; dodd, m. 2 1. paediatric cystic fibrosis service, central manchester and manchester children's university hospital, manchester, united kingdom; 2. adult cystic fibrosis centre, wythenshawe hospital, university hospital of south manchester nhs foundation trust, manchester, united kingdom background: training in the skills of communication and self care are considered important components of transitional care1. physiotherapists work to involve families and young people to encourage this independence. the cf manager training programme2 (cfmtp) is a recognised tool for measuring age appropriate tasks in cf care. aim: the aim of the study was to survey the current levels of autonomy in all patients with cystic fibrosis in the north west of england from birth to 18 years to provide a baseline measure for encouraging autonomy in preparation for transition. method: three areas of the cfmtp were selected for the survey: physiotherapy (p), inhalation therapy ( it) and communication (c) for the 10 age groups: 0-1. 5, 1.5-3, 3-5, 6-7, 7-8, 8-9, 9-10, 11-12, 13-15,and 16+ years. physiotherapists from all the north west cf clinics were invited to assess all the patients under their care. data collected were age, gender and whether the patients skills fell "below", "same" or "above" the age appropriate self management tasks as described in the cfmtp. 'not applicable' was indicated where the skills did not apply. results: 14/16 clinics responded. 401out of a total of 474 patients in the nw were assessed, three patients were excluded. population by clinic ranged from 6-133. population by age category: 15 (0-1.5 years) 24 (1.5-3years) 45 (3-5 years) 25 (6-7 years) 36 (7-8 years) 16 (8-9 years) 45 (9-10 years) 50 (11-12 years) 78 (13-15 years and 64 (16+ years). 213/398 (54%) were male. for p (n=390), it (n=261), c (n=367), 32%, 40%, 22% = below, 59%, 51%, 65% = same, 9%, 9%, 13% = above respectively. there were no gender differences. conclusions: overall scores revealed that 2/3rd of patients were gaining age appropriate independence in all areas. there was the least autonomy with it. it was rare for a child to be setting up and cleaning equipment regularly at 7-8years, but this continued throughout the adolescent years. overall 35% were 'below' for p and it; this was highest after the age of 11years. autonomy with physiotherapy appeared slow to develop. some categories in cfmtp were different from our practice for e.g. patients are not invited to be seen alone in our clinics until 13 years v 9-10 years and this may account for the lower scores in communication below this age. this study has highlighted that some of our patients are not fully independent in the time up to transfer. we need to encourage independence from an early age with all aspects of care and integrate the process into our routine clinical practice. refs 1 mcdonagh and viner bmj 2006; 332:435-6, 2. parents' guide to the cf manager training programme. children's hospital of eastern ontario planner, s. 1 ; morrison, l. 2 1. physiotherapy, gartnavel general hospital, glasgow, united kingdom; 2 cystic fibrosis (cf) causes a deterioration in lung function and exercise tolerance. assessing exercise capacity is therefore essential in order to monitor changes in disease severity. the 6-minute walk test (6mwt) and 3 minute step test (3mst) are validated and widely used outcome measures for identifying exercise capacity in patients with cf. exercise testing is part of annual review of all patients in the west of scotland. previous studies identified that the 3mst was a sensitive measure of exercise tolerance, but did not successfully challenge patients with mild or moderate lung disease as defined by fev(sub)1(/sub)>40% (morrison 2005) . further analysis of the 3mst, where step height was increased to 8 inches did not show any difference in sao(sub)2(/sub). decline or rate of perceived exertion (rpe) in patients with mild disease. the chester step test (cst) was developed to assess aerobic fitness which is used in the uk cardiac population for exercise prescription (sykes 1999) . it is a 10 minute progressive, sub-maximal test with a variable step height (6-12inches) , making it suitable for those with a wide range of exercise capacities. in addition, cst results extrapolate to a mets value (oxygen utilisation: 1met = rate of oxygen utilisation at rest) as an outcome measure which can facilitate exercise prescription based on established values for physical activities. we aim to determine whether the cst is a more sensitive measure of exercise capacity than the 6mwt or 3mst when considering decrease in sao(sub)2(/sub) and rpe. 13 patients ( aged 16-49 mean fev(sub)1(/sub)2.39l) with mild-moderate cf performed the cst and 6mwt and results were correlated with fev(sub)1(/sub). decrease in sao(sub)2(/sub), increase in hr and rpe scores were also compared between the cst, 6mwt and recent 3mst data. patients were exercised to a symptom limited maximum which differed from the cardiac cst where patients are exercised to 80% of maximal hr. the cst appears to be more challenging as only 4 patients completed it, all with an fev(sub)1(/sub)of >50% predicted, whereas in the 3mst and the 6mwt all patients completed the test. mets levels for the cst were weakly correlated with fev(sub)1(/sub)(r=0.456) and the 6mwt (r=0.29) . this could be explained by the narrow range of mets data available or the size of the sample population. there were significantly greater rises in hr (p= 0.01, p= 0.01), decrease in sao(sub)2(/sub) (p=0. 03, p=0.0001) and rpe (p=0.04, p=0.013) , between the cst, 3mst and 6mwt respectively, suggesting that the cst is a more sensitive measure of cardiorespiratory effort. conclusions the cst appears to be a more challenging exercise test for those with mild to moderate disease and therefore may be a better reflection of exercise capabilities. cst results are useful for prescribing exercise and monitoring programmes appropriately for this patient group. further study could determine whether the cst is also suitable for those with severe disease. lee, a.l. 1 ; button, b. 1 ; holdsworth, m. 1 ; holland, a. 2 1. physiotherapy, the alfred, melbourne, vic, australia; 2. latrobe university, melbourne, vic, australia musculoskeletal pain is prevalent in cystic fibrosis (cf). while the chest and back are frequently reported regions of acute or chronic pain, it is unclear if pain in these areas is related to disease severity or respiratory symptoms. manual therapy and postural advice has been shown to alleviate pain, but the effects of a combined approach of musculoskeletal physiotherapy and massage has not been studied. the aim of this study was to identify the primary regions of musculoskeletal pain, the relationship between pain severity and respiratory symptoms and to examine the effect of musculoskeletal physiotherapy techniques and soft tissue therapy, including remedial massage on pain and ease of breathing (eob) in adults with cf. one hundred and twenty-nine adults with cf (70 with acute exacerbation, 24 post lung transplant) aged 31 ± 9 years (mean ±sd) with fev(sub)1(/sub) of 51± 21% participated in this study. following assessment of primary pain regions, each subject underwent a single treatment session including spinal joint / intercostal mobilisation and soft tissue therapy. pain and eob on a visual analogue scale were measured before and after treatment. changes were compared using paired-samples t-tests. pain was frequently reported in the thoracic spine region (38% of subjects), followed by the shoulder region (31%), cervical spine region (16%) and chest wall region (9%). equivalent ratings of pain and eob prior to treatment were reported for all regions of pain. eob rating prior to treatment were worse in those with low bmi (r=-0.21, p=0.02) and low fev(sub)1(/sub) (r=-0.24, p=0.01). a single treatment session was associated with reduction in pain (95%ci 1.6 to 2.1, p<0.001) and improvement in eob (95%ci 0.4 to 0.7, p<0.001), irrespective of clinical status. improvement in pain was equivalent for all primary pain regions. however, greater improvement in eob was evident in subjects with shoulder pain compared to other regions (95%ci -1.1 to -0.02, p=0.04). a combination of musculoskeletal physiotherapy techniques and soft tissue therapy reduces musculoskeletal pain and improves eob in adults with stable cf, during an acute exacerbation and post lung transplantation. service cystic fibrosis is characterized by an excessive production of airway secretions responsible for bronchial obstruction and recurrent infections of the respiratory tract. the transport by ciliary activity and cough may be dependent on the unsteady rheological properties of the respiratory mucus such as thixotropy and shear-thinning properties which correspond to a decrease of viscosity with time or flow rate, respectively. we have previously shown that respiratory mucus with high shear-thinning and thixotropic properties is better transported by the cough mechanism (zahm et al, eur. respir. j., 1991) . to promote mucus clearance and to finally expectorate the mucus by a cough manoeuvre, an innovative device, the airhelp, has been developed and aimed to improve chest physiotherapy effectiveness. during a prolonged expiratory manoeuvre, the airhelp delivers negative pressure variations (maximal amplitude: -4 kpa) at an oscillatory frequency of 13 hz. the aim of the present work was to test the effect of the airhelp device on the in vitro clearance of mucus by airflow. a plexiglass tube of 9 mm in diameter and 210 mm in length has been connected by one part on the airhelp and by the other part on a 30 l thank mimicking the lung compliance. a 100 µl drop of mucus simulant at different concentrations (viscogum at 0.25% or 0.5% with actigum at 0.5% to 1.75%) or of respiratory mucus, was deposited within the plastic tube and the distance travelled by the sample under a 5sec-airflow was measured. twelve respiratory mucus samples collected from cf patients were analyzed and the viscosity of the samples was measured before and after the airhelp experiment. we observed a significant (p < 0.03) and negative relationship (r = -0.77) between the viscosity of mucus simulants and oscillatory airflow transport: the lower the viscosity, the higher the mucus transport by oscillatory airflow. using cf respiratory mucus samples, we observed that without the oscillatory airflow, no transport occurred, whereas when the samples were submitted to the oscillatory airflow, a significant increase in mucus transportability was measured. in addition, the viscosity of the cf respiratory mucus samples was significantly decreased after oscillatory airflow treatment (485 ± 148 pa.s to 85 ± 55 pa.s, p <0.02). these results demonstrate that the mucus transport efficiency by an oscillatory airflow is dependent on mucus viscosity and that the improvement in mucus transport is related to the thixotropic and shear thinning properties of the airway mucus. we conclude that the airhelp device may improve chest physiotherapy effectiveness. potter, e. 2 ; nufer, j. 2 ; cullina, j. 2, 4 ; quinton, h. 3 ; jain, m. 1, 4 ; mccolley, s.a. 1, 2 1. northwestern university feinberg school of medicine, chicago, il, usa; 2. children's memorial hospital, chicago, il, usa; lebanon, nh, usa; 4. northwestern memorial hospital, chicago, il, usa socioeconomic status (ses) significantly impacts health outcomes in cystic fibrosis (cf). the cystic fibrosis foundation (cff) is currently using median income estimated by zip code from the 2000 us census of the population for ses risk adjustment of data reported publicly from the cff patient registry. median income estimated by zip code is an "ecologic" variable that may misclassify patient and family income. in order to more fully assess the impact of ses on cf health outcomes, the cff began to ask centers to collect data on household income directly from patients and families. these data were captured for 6.5% of patients and families in 2005 and 16.1% in 2006 in the national registry. we report a method of collecting ses data from children and adults implemented at the children's memorial hospital and northwestern university cystic fibrosis center, and compare directly collected income to income based on zip code. methods: a short survey with the questions on household composition, income, and education level from the cff patient registry was developed. a letter from the center director and adult program director was mailed to patients and families informing them of the survey's purpose and confidentiality measures, i.e. responses would be seen only by the individual entering the data and identified by cff id number. the survey was distributed to cf patients and families in clinic by clerical staff. completed surveys were placed in an envelope and sealed by the patient or family. both directly collected income and income from zip code was classified into 9 groups (<$10k, 10-20k, 20-30k, 40-50k, 50-60k, 60-70k, 70-80k, 80-90k, and >90k) . percent concordance and spearman's rank correlation were calculated. 2005 data were used for patients who provided data in 2005; otherwise, 2006 data were used. each patient's data was used only once in the analysis. results: 202 unique patients were identified, 127 pediatric and 75 adult patients. eighteen percent of pediatric and 39% of adult patients preferred not to answer income questions or did not complete the survey. the mean income by zip code was $60 k for patients who did not report income data and $61k for the entire group. for the 150 responses, reported income data was moderately correlated with income by zip code (rho=0.48, p<0.001). concordance between the 9 categories was 15%. the correlation was stronger for pediatric patients (rho=0.53, p<0.001) than for adults (rho=0.27, p=0.07). re-classifying income as "low", "medium" and "high" (<$40k, $40-70k, >$70) led to a concordance of 40%. conclusion: survey methods that protect patient and family confidentiality result in good return rates of full ses data from patients and families. zip code derived median income shows moderate concordance with reported income. comparing the associations of the 2 forms of income data with important cf outcomes needs to be done in a larger data set. we acknowledge the cystic fibrosis foundation, gerald o'connor, kathryn sabadosa, and participating patients and families. the patient registry provided a unique opportunity to assess changes in the epidemiology of respiratory pathogens in cf. while the incidence of p. aeruginosa remained stable, the prevalence decreased, suggestive of the impact of antibiotic eradication strategies. in contrast, the increasing incidence and prevalence of s. aureus and mrsa may reflect improved microbiological surveillance and laboratory techniques. the trends noted for b. cepacia complex may reflect successful implementation of infection control strategies. future studies are needed to better define the association between these observed trends and improving care for cf patients. vanderwyden, a. 1, 2 ; drumm, m.l. 1, 3 ; schluchter, m. 2 1. pediatrics, case western reserve university, cleveland, oh, usa; 2. epidemiology and biostatistics, case western reserve university, cleveland, oh, usa; 3. genetics, case western reserve university, cleveland, oh, usa in the genetic modifier study (gms) of lung disease of cystic fibrosis (cf), patients, homozygous for the ∆f508 mutation, were classified as having either severe or mild lung disease, as defined by the lowest or highest quartile of forced expiratory volume in one second (fev1) and a linear model was fit from these patients' longitudinal data (drumm 2005 , schluchter 2006 . while these models successfully dichotomized patients for the association study, we assessed whether a single slope model could be improved. here we have begun to compare models with one, two and three slopes, each representing different age ranges, for their use in this type of association study. as an example, interleukin-8 (il-8), a neutrophil chemoattractant, associates with the severe lung inflammation seen in cf patients. we have observed the tt genotype of the il-8 snp rs4073, correlated with severe pulmonary function in the gms sample of 808 patients, relative to the aa and at genotypes of this snp. in a replicate study of 268 patients, with cftr genotypes associated with exocrine insuffiency, longitudinal linear mixed models were used to further characterize the decline in lung function over time as associated with the il-8 snp, rs4073. three linear regression models of fev1% predicted of patients were fit over age and compared with likelihood ratio tests. the three models differed with respect to the number of nodes. the first model had a single slope while the second and the third were piecewise linear models, with nodes at age 15, and ages 15 and 25, respectively. age 15 was chosen as the first node, as by this age most cf patients have reached puberty, an event believed to coincide with changes in pulmonary function. the second node, age 25, was arbitrarily chosen as a point in the third decade, as survivor effect becomes a significant issue during this period. when compared to the single slope and two slope models, the three slope model with nodes at age 15 and age 25 better explained the pulmonary function of this study (p<0.001, -2loglikelihood chi-squared tests). when the explanatory variable, il-8 rs4073 genotype, was added, the single slope model and the three slope model were again compared. the three slope with nodes at age 15 and age 25 again resulted in a better fit of fev1% predicted over age as correlated with rs4073 genotype (p<0.001, -2loglikelihood chi-squared test). estimates of fev1% predicted at ages 6, 15, 25, and 30 for each rs4073 genotype (aa, at/ta, or tt) were determined using the three slope model (p<0.001 for all estimates). other explanatory variables, like gender and survival (p<0.001), were added to further elucidate the relation between il-8 genotype and lung disease phenotype. we propose to use this piecewise three slope model with nodes at age 15 and age 25 to look at the pulmonary function of patients in the cf foundation registry data to better explain the decline in fev1% predicted over time seen in cf patients. (supported by hl-68890 and grants from the cf foundation) stephenson, a.l. 1 purpose: the purpose of this study is to determine the annual incidence of hospitalizations for individuals with cf living in ontario and to examine predictors of hospitalization, specifically gender. methods: this is a retrospective cohort study from 1993 to 2002, using newly linked clinical and administrative databases. the canadian cf foundation patient data registry (cpdr), contains detailed clinical information on all cf patients receiving care at one of 38 accredited cf centres across canada. the institute for clinical evaluative sciences (ices), holds linked administrative databases containing information on all publicly reimbursed health care services delivered in ontario. canadian institute for health information (cihi) discharge abstracts database (dad), ontario health insurance plan (ohip), and homecare databases were used for this study. the cpdr was probabilistically linked (automatch software) to the ices administrative database using name, gender and date of birth. all individuals were then linked to the cihi dad, ohip, and homecare databases using unique numeric identifiers. with these databases, longitudinal records for each individual were created. cihi-dad, ohip, and homecare claims data were used to calculate the number of hospitalizations per year for pulmonary infections. males and females were analyzed separately to elucidate any gender disparities. other predictor variables include age, geographical location, number of clinic visits, socioeconomic status (income quintile) as well as clinical variables such as pulmonary function tests, nutritional status, diabetes mellitus, sputum bacteriology and pancreatic status. results: the cpdr contained data on 4244 individuals with cf followed within canada between 1993-2002. of those individuals, 1452 could be found within the ices administrative databases using probabilistic linkage. of those 1452 individuals matched to the administrative databases, 1170 (45.6% female) had 7244 hospitalizations. a total of 312,015 ohip claims were made during this 10-year period which represents any encounter between an individual with cf and a physician in ontario. multivariable analysis of hospitalization predictors is in progress. zhang, z.; lai, h. uw-madison, madison, wi, usa the 2002 cff practice guidelines recommend adjusting for genetic potential when evaluating height status in children with cf. however, calculation of adjusted height percentile is not recommended due to the complexity of method involved in such calculation. instead, a simple method to estimate a target height based on mid parental height was recommended. however, this target height includes a 10-cm confidence interval, which spans most of the channels on the growth chart. therefore, a child's unadjusted height may be considerably below the target height, yet remains above the lower bound of the target height range. in addition, there is a paucity of data on the associations between adjusted height to lung disease outcomes to justify whether adjustment for genetic potential is necessary. the objective of this study is to compare two methods of incorporating genetic potential in identifying short stature, namely, the cff's target method and the method developed by himes et al (pediatr 1985; 75:304-313) , which applies positive adjustments to children with short parents and negative adjustments to those with tall parents. data from 1986-2005 cff registry were utilized. a total of 558 children born between 1984-1988 who had complete height data from age 2-18 years and their parental height data were analyzed. short stature was defined by unadjusted height < 10th percentile, unadjusted height below the lower bound of cff's target height, or himes' adjusted height <10th percentile. our results showed that adjusted height percentiles are consistently lower than unadjusted height percentiles, with an average difference of 0.2 z-score unit between ages 2-14 years and 0.3 z-score unit between ages 15-18. proportionately more children were classified as short stature based on himes method compared to unadjusted height (31% vs. 26%, p = 0.0003). in contrast, proportionately fewer children were below the lower bound of cff's target height (23%) compared to unadjusted height < 10th percentile (26%), p < 0.001. among children with average parents, unadjusted and adjusted height percentiles were similar. however, among children with short parents (< 25th percentile), the percentage children with of short stature decreased from 68% with unadjusted height to 35% with himes' adjusted height and 14% with cff's target height method. among children with tall parents (> 75th percentile), the percentage of short stature increased from 7% with unadjusted height to 32% with himes method and 35% with cff's target height method. taken together, these results demonstrate that, without incorporating genetic height potential, 2-5 times more cf children who have short parents would be classified as short stature, while 5 times fewer cf children who have tall parents would be classified as short stature. further analyses show that, compared to unadjusted height percentile, himes adjusted height percentile is more sensitive to, and has stronger association with, percent predicted fev-1. in conclusion, our findings provide evidence that genetic potential should be incorporated in evaluating short statue in cf children, particularly for children with short or tall parents. the cff's target height method and himes method differ significantly in identifying short stature in cf children with short parents. cohort study of individuals in the cff patient registry from 1995-2005. all individuals 6 years of age or older and diagnosed before age 45 were included. the new, persistent mrsa infection cohort was defined by having at least two cultures negative for mrsa over a two-year lead-in period followed by at least two mrsa positive cultures. individuals with only one mrsa culture (transient infection) were excluded. we developed multiple linear regression models using generalized estimating equations to assess the effect of mrsa on fev 1 %predicted (%fev 1 ) and rate of change of %fev 1 . results: during the study period 5,955 individuals cultured mrsa. of these, 2,025(34%) cultured mrsa only once (transient infection) and were excluded from subsequent analysis. 3,116 individuals met the criteria for new and persistent mrsa infection. 16,088 met the criteria for never having mrsa resulting in a total study cohort of 19,204. participants were followed for a median of 10 years. the median time to first isolation of mrsa in the persistent mrsa cohort was 5.5 years and the mean number of positive mrsa cultures per patient was 7.5. a comparison of baseline characteristics between the persistent mrsa and never mrsa cohorts revealed the mrsa cohort to be younger (13.1 vs 15.0 years p<0.001), equivalent in lung function (%fev 1 79.1% vs 78.3% p=ns), and with higher likelihood of methicillin sensitive staph aureus colonization (45% vs. 39.3% p<0.001). 5.5 years into the study period (the median time of acquisition for new mrsa), the mrsa cohort had a lower %fev 1 (68.1% vs 71.1% p<0.001), and averaged twice as many hospital admissions per year (0.88 vs 0.44 p<0.001) and home iv courses per year (0.40 vs 0.21 p<0.001). in an unadjusted analysis, the presence of mrsa was associated with a mean %fev 1 that was 9.8% lower than the mrsa cohort (95%ci 9.6%-10.0%). however, after adjusting for sex, age, pancreatic status, p. aeruginosa, and b. cenocepacia, the presence of mrsa was associated with a mean %fev 1 3.3% lower (95%ci 3.1%-3.5%). most importantly, an analysis investigating the interaction between mrsa and time, after adjusting for confounders, indicated no statistically nor clinically meaningful difference in the mean rate of lung function decline between the two groups. conclusions: approximately one-third of patients who culture mrsa do so only once and do not subsequently develop persistent mrsa infection. mrsa infection occurs in a group with more severe structural lung disease as measured by fev 1 , and frequent hospitalizations and iv antibiotics are strongly associated with mrsa acquisition. after adjusting for confounders, mrsa infection is not statistically significantly associated with a more rapid decline in %fev 1 over time. simmonds, n.j. 1, 2 ; macneill, s. 2 ; cullinan, p. 2, 1 ; hodson, m.e. 1, 2 1. department of cystic fibrosis, royal brompton hospital, london, united kingdom; 2. national heart and lung institute, imperial college, london, united kingdom introduction: disentangling the influence different factors have on long-term survival in cf is complex. there are a myriad of influences (including environmental, healthcare-related, psychological and socioeconomic) which may be important, as there is generally a poor genotype-phenotype correlation. the aim of this case-control study was to identify these factors. the cf database at the royal brompton hospital was used to identify long-term survivors. they were classified as cases and were patients who had reached 40 years of age (without transplantation). each case was agematched with at least one control who died of cf (or a cf-related condition) or was transplanted before reaching 30 years of age. late diagnosis patients were excluded. conditional logistic regression models were used to identify potential influences on survival. results: 78 cases and 152 controls were analysed, producing 1811 matched pairs. 97% of the 230 patients were pancreatic insufficient. of the many factors investigated, those resulting in increased probabilities of survival included: increased body mass index (or=1.52, 95% ci 1.35-1.72), fev 1 (or per 5% increase=1.62, ) at transfer to the adult clinic (after adjusting for age and sex) and the use of oral antibiotics before attending the adult clinic (or=3.01, 1.38-6.56) after adjusting for age at first attendance. factors associated with a reduced probability of survival included: cf diagnosis <5 years old (or=0.46, 0.28-0.76); initial presentation of chest symptoms or malabsorption (or=0.44, 0.27-0.71 and or=0.38, 0.24-0.61, respectively); referral from a paediatric clinic in a deprived area (or=0.12, 0.04-0.34); pneumothorax before transfer to the adult clinic (or=0.03, 0.005-0.24) after adjusting for age at first attendance; and s. aureus or p. aeruginosa colonisation before 16 years of age (or=0.36, 0.14-0.89 and 0.18, 0.06-0.58, respectively). factors not influencing survival included: sex, h. influenzae colonisation before 16 years of age, development of diabetes before 16 years of age, major haemoptysis before transfer to the adult clinic, parents' occupation, number of siblings (cf and non-cf) and school achievements. in a very carefully matched study we failed to identify major non-clinical determinants of long-term survival in early diagnosis cf. findings suggest that the majority of significant factors were directly related to optimal physical parameters, the use of oral antibiotics, avoiding bacterial colonisation and a low pneumothorax rate. adler, a. 1, 2 ; bilton, d. 2 ; haworth, c. 2 ; gunn, e. 2 ; shine, b. 3 1. addenbrooke's cambridge university hospitals, cambridge, united kingdom; 2. papworth hospital, papworth everard, united kingdom; 3. oxford centre for diabetes and endocrinology, oxford, united kingdom background and aims: although the prevalence of diabetes in patients with cf (cfrd) is high, few prospective studies exist, and even for genet-ic factors, cross-sectional studies may bias the magnitude of associations. this study sought to identify risk factors for cfrd from a predominantly white cohort of individuals with cf in the united kingdom. methods: 8,029 individuals aged 0 -64 were identified from the uk cf database, a registry of patients coordinated by the uk cystic fibrosis trust with data from over 44 specialists centres from england, scotland and wales, countries which provide medical care free of charge. of the patients, 5,196 had no diabetes at baseline (1996-2004) , and also had at least one follow-up annual visit. diabetes was defined as either a physician diagnosis of diabetes, a blood glucose of >11.1 mmol/l two hours following an oral glucose tolerance test, or treatment with insulin or oral hypoglycemic drugs. follow-up was calculated as time from baseline to the first detection of new diabetes or censoring. risk factors included clinical, genetic (functional classes i,ii,iii vs iv,v), and anthropometric characteristics measured at baseline. survival analyses were limited to patients with complete data. cox proportional hazards modeling was used to estimate the magnitude of the association between potential risk factors and incident diabetes. one-way interactions with sex were tested. results: the median age of entry into the cohort was 12 years and bmi was 17.9 kg/m2. 54% were male, and 96% were white. 526 patients developed diabetes during 15,718 person-years of follow-up (mean 3.0 years); the incidence was 3.4% per year. patients who developed diabetes were more likely in univariate analyses to be older, have a high body mass index (bmi), bacterial pulmonary infections, liver and pulmonary function abnormalities, poor oxygen saturation, a history of organ transplantation, supplemental feeds, and take pancreatic enzymes or bile acids. in addition, patients with class iv,v genotypes were less likely to develop diabetes relative to patients with other genotypes, as were patients screened for cf at birth. not associated with incident diabetes was age at diagnosis of cf and ethnicity. in multivariate analyses (n= 4,004) age (hazard ratio 1.02 per year, 95% ci 1.01 -1.03), female sex (1.67 (95% ci 1.39-2.00), % predicted forced expiratory volume in 1 second (1.019, 1.014 -1.024 per 1% decrement), dose of pancreatic enzyme replacement (1.33, 1.10 -1.62), above vs below median dosage, use of bile acid supplements (1.51, 1.20 -1.91) , and genotype class i,ii, iii (5.60, 2.08 -15.1) relative to class iv/v. these associations were not confounded by bmi or history of transplantation. there were no significant interactions between sex and other risk factors. conclusions: the study confirms the high incidence of cfrd, and shows that patients with class iv/v genotypes are less likely to develop diabetes independent of other cf-related complications. females are disproportionately at risk for diabetes which is not explained by a higher prevalence of known risk factors nor by effect modification. cystic fibrosis children are at increased risk for hearing disorders due aminoglycoside exposure, mucopurulent upper respiratory tract infections, and increased inflammatory responses. pure tone audiometry is the standard instrument used to determine hearing loss. however, it can be expensive and time-consuming to use audiometry for routine hearing screening. hearing questionnaires have been used as a screening tool to assess possible hearing loss in otherwise healthy populations. what are the results of hearing questionnaires administered to pediatric cf patients? does cf patient age or ethnicity increase the likelihood of hearing loss as determined by a hearing questionnaire? a hearing questionnaire (english and spanish versions) was administered to pediatric cf patients over a 2 week period at our accredited cf center. data collected included demographic information. inclusion criteria: age м10 years and in baseline health status. possible hearing loss was defined as at least 3 "yes" responses (>3) to the 14 quesions administered to the patients. fisher's exact test was used to perform the analyses. a total of 20 cf patients were seen in the cf clinic during the study period. 14 cf patients completed the hearing questionnaire. mean age was 14.3 ± 3.6 years (median 13.6 years). there were 9 males and 5 females; 10 were classified as hispanic and 4 were classified as caucasian. 7 patients were older than 13.6 years (the median age) and 7 patients were less than 13.6 years. 3 patients who were older than age 13.6 years had a score of >3 and 4 patients < age 13.6 years had scores >3 (p=1.0, ns). thus, 7 of 14 cf patients (50%) were classified as having possible hearing loss as per their questionnaire scores. 6 of 10 hispanic cf patients had hearing scores >3 and 1 of 4 caucasian patients had hearing score >3 (p=0.56, ns) . we found that a significant proportion (50%) of our cf children may require formal hearing evaluation. we speculate that hearing questionnaires may be useful to screen for hearing loss in the cf patient population. hispanic cf patients have increased morbidity and mortality risks compared to caucasian cf patients who attend our accredited cystic fibrosis center in southern california. is the increased morbidity/mortality risk due to decreased access to outpatient cf care? we performed a retrospective review of all patient visits to our cf clinic between january 1, 2005 through december 31, 2006 . data collected included demographic information (age, ethnicity, gender) , and the number of clinic visits during the study period. data was analyzed by unpaired student's t-test as well as by chi-square. during the study period, a total of 197 cf patients (106 males:91 females; mean age 10.8 ± 5.7 years; range 3 months to 21 years) were routinely followed in our cf clinic. these patients had a total of 1806 outpatient visits to the cf clinic (9.2 clinic visits/patient over the 2 year study period). there were 82 hispanic cf patients (46 males:36 females; mean age 11.0 ± 5.3 years) and 99 caucasian cf patients (49 males:50 females; mean age 10.8 ± 6.2 years; p=0.42, ns). there was no difference in gender distribution between the 2 group (p=0.52, ns). hispanic patients had a greater number of cf clinic visits (11.2 visits/patient) as compared to caucasian patients (7.9 vists/patient; p=0.03) during the same period. we conclude that the increased morbidity and mortality of hispanic cf patients in california cannot be attributed to barriers to outpatient care. in fact, hispanic cf patients were seen more frequently in cf clinic compared to caucasian cf patients who attend the same clinic. we speculate that hispanic cf patients are seen more frequently in cf clinic due to increased severity of cf disease manifestations. duguépéroux, i. 1 ; scotet, v. 1 ; audrézet, m. 1, 2 ; blayau, m. 3 ; parent, p. 4 ; journel, h. 5 ; boisseau, p. 6 ; férec, c. 1, 2 1. inserm u 613, brest, france; 2. dep. of genetics, brest, france; 3. dep. of genetics, rennes, france; 4. unit of medical genetics, brest, france; 5. unit of medical genetics, vannes, france; 6. dep. of genetics, nantes, france aim: the aim of this study was to describe 15-year experience of prenatal diagnosis (pd) for cf in brittany (western france) and to assess its impact on incidence. method: we registered, by the genetic laboratories of our region, all the pds performed in women living in brittany over the period 1991-2005. we described the number of pds made for each reason (way by which the one-in-four risk was identified: previous affected child, family testing, echogenic bowel, etc). we reported the proportion of cf-affected fetuses and of consecutive terminations, and assessed the incidence modification due to pd. results: over the 15-year period, a total of 253 pds were performed in couples living in brittany. most of them were done in couples already having cf child(ren) (n=167, 66.0%). extended testing in families -a practice largely proposed in brittany -led to the identification of 18 new onein-four risk couples among the relatives of cf patients who opted for pd 38 times over the study period (15.0%). the 48 other pds were made in couples without previous history of cf. the one-in-four risk was mainly identified following the detection of an echogenic bowel during pregnancy ultrasound examination (21 pds were done directly following the positive ultrasound (8.3%), whereas 19 others were done for subsequent pregnancies (7.5%)). the other pds were consecutive to the detection of an heterozygote through newborn screening (n=6, 2.4%) or for an other reasons (n=2, 0.8%). over the study period, the number of pds per couple varied between one and five, the mean being 1.6. overall, a total of 88 cf fetuses were identified, among whom 77 were terminated (87.5%). the inclusion in the incidence calculation of these 77 pregnancy terminations led that to an incidence modification of 28.8% over the study period. conclusion: this study shows that pd for cf is commonly used in brittany and highlights the impact of family testing and of routine ultrasound examination of pregnancies in that region. supported context: hispanics with cystic fibrosis (cf) in the united states experience an 85% increased annual risk of death from cf compared to non-hispanic patients. when adjusted for socioeconomic status this disparity persists. studies characterizing this at-risk population do not exist. objective: to characterize the center-reported us hispanic population with cystic fibrosis through a cystic fibrosis foundation (cff) patient registry analysis and to elucidate factors present within this hispanic population that may affect outcomes. design: retrospective cross sectional study of the 2004 cff patient registry. patients: all 22,714 patients in the 2004 cff patient registry were included in the analysis. there were no exclusion criteria. the hispanic population is defined as those who were entered by their respective care center as hispanic ethnicity regardless of race. main study measures: prior to analysis, study measures were selected to demographically and genetically characterize the hispanic cf population. genotype, state of residence, mean age and mean age of diagnosis were obtained in the entire population. maternal education (for patients <18 years of age) and insurance status data were captured as indicators of socioeconomic status. complication rates, fev1 percent predicted (under 18 years of age) and absolute fev1 (over 18 years old of age) were obtained as measures of clinical status. t-tests and logistic regression analyses were used to compare measures between hispanic and non-hispanic groups. odds ratios (or) and 95% confidence intervals are reported. results: 1511 center-reported hispanic patients with cf were identified. over 50% of hispanic patients resided in california, florida, texas or new york. the most common genotype was delta f508 homozygous, accounting for 32% of the population. mean age was 12.3 years +/-9.2 years for hispanic patients and 17.3 years +/-12.1 years for non-hispanic patients. hispanic patients were diagnosed at an earlier age (2.8 years vs. 3.3 years, p=0.005). they were more likely to have mothers with education at less than a high school level (or 5.25 (4.12, 6.71) ) and have government insurance (or 2.73 (2.45, 3.04) ). complication patterns differed between the two groups; non-hispanics were more likely to have complications reported that were related to depression (or 1.94 (1.48, 2.54) ), bone health (or 1.82 (1.41, 2.41) ) and cf related diabetes (or 1.9 (1.6, 2. 3)) compared to hispanics. hispanics however, were more likely to have complications reported from cf liver disease (or 1.31 (1.1, 1.56) ). pulmonary function, as measured by fev1 percent predicted in children (p<0.001) and absolute fev1 for adults (p=0.01) were lower for hispanic patients. conclusions: differences exist between hispanics and non-hispanics with cf with respect to maternal education, insurance status, complications rates and pulmonary function. the lower reported rates of depression, bone complications and cf related diabetes may represent ascertainment bias. further study is needed into the etiology of health outcome disparities in this population, and to design interventions for this high-risk population. mastella, g.; baldo, e.; forneris, m.; furnari, m.; lucidi, v.; manunza, d.; marinelli, i.; messore, b.; neri, a.; raia, v.; salvatore, d.; buzzetti, r.; cairo group, italian cf research foundation, verona, italy with our work we reviewed the international scientific literature coming from cf registries worldwide. our aims were: to verify what has been produced in scientific literature thanks to the material derived from fc registries and to see which clinical problems have been tackled and which clinical questions were answered correctly and exhaustively. a search in medline and embase produced 160 articles (our strategy was "cystic fibrosis"[mesh] and ("registry"[mesh]) or registr$) updated to the 04/30/07). out of these articles 88 have been selected by two independent assessors on the basis of their pertinence (primary studies that used data taken from a formally established registry, at least national, to test some hypothesis of research). each article has been examined with the help of a pre-defined form that in particular evaluated skills of different registry data, selected clinical characteristics of the study populations and statistical methods. more than one half of cases data came from usa-cff foundation patient registry, the remaining coming from canadian, uk, french, italian, german, and other european registries. the main focuses of the articles were included in the following topics: 1) incidence/prevalence of the disease and survival (30 studies); 2) neonatal screening, growth and nutrition (18); 3) genotype/phenotype correlation/ different ethnical groups and twin/brothers (12); 4) complications and outcomes (abpa, diabetes, nasal polyposis, pregnancy and paternity, etc) (19); 5) microbiology (9). our assessment scores were good/excellent for 62/88 studies for the relevance of the problem addressed; 54/88 for the usefulness of the outcomes and 46/88 for the usefulness of the implications for research from the italian registry. thirty-two articles gave a significant contribution to the analysis of the studied problem, but 47 of them left a partial uncertainty, while 9 left complete uncertainty. cf registries are a very important source of data to provide a powerful tool for clinical and research questions. however, further comparable studies have to be planned to assess registry data as a platform for improvement in clinical practice. vandenbranden, s.l. 1 ; mcmullen, a. 7 ; konstan, m. 4 ; morgan, w. 3 ; wagener, j. 6 ; schechter, m. 2 ; watts, k. 1, 5 ; mccolley, s. 1, 5 1. children's memorial medical center, chicago, il, usa; 2. emory university, atlanta, ga, usa; 3. university of arizona, tuscon, az, usa; 4. case western reserve university, cleveland, oh, usa; 5. northwestern university feinberg school of medicine, chicago, il, usa; 6. university of colorado, denver, co, usa; 7. university of rochester, rochester, ny, usa background: although life expectancy in cf has dramatically improved over the last 20 years, most of the improvement is due to improved survival during early childhood. adolescence and young adulthood continue to be a time of worsening pulmonary disease and mortality. study objective: data from a large longitudinal observational study, the epidemiologic study of cystic fibrosis (escf), was examined to characterize lung function decline in subjects with cf during the periods preceding and after the age of 18. design: escf data from 2136 individuals with cf collected during the period of 1994-2005 were analyzed. inclusion criteria required individuals to have a minimum of 5 encounters in each of the 3.5 year time periods before and after the age of 18. the cohort was strati-fied by disease severity based on the best fev 1 at the 18th year (+/-6 months). one hundred thirty seven subjects had severe lung disease (fev 1 <40% predicted), 668 had moderate-severe lung disease (fev 1 40-<70 % predicted), 1049 had mild lung disease (fev 1 70-<100% predicted), and 282 had very mild disease (fev 1 >100% predicted) the study compared the annualized rate of fev 1 decline during the adolescent period, defined as ages 14-17.5 years, and in the young adult period defined as 18.5-22 years. results: in the entire cohort, the annualized rate of decline was less in the young adult period than in the adolescent period (p<0.001). the most dramatic improvement in slope of decline was seen in the severe group, and significant improvements were also noted in the moderate group (p<0.001). the mild group demonstrated insignificant change; the very mild group was the only group that demonstrated a more negative annualized rate of the decline in young adulthood than in adolescence (p=0.027). conclusions: these data suggest that, overall, young adults have a slower rate of decline in fev 1 in the period after age 18. this "stabilizing" of lung function is most prominent in those individuals with moderate-to-severe lung disease. only those with very mild lung disease have a slightly more rapid rate of decline between the ages of 18 and 22. overall there is less variation in the rate of fev 1 decline across disease strata after the 18th birthday. further analysis is underway to better understand the factors leading to these findings. 1 1. department of paediatrics, university of florence, cystic fibrosis centre, florence, italy; 2. italian cystic fibrosis microbiology group, florence, italy; 3. institute of medical microbiology and hygiene, tübingen, germany mrsa is now a worldwide public health problem, due to the increasing rate of infection in several settings as well as in cf patients. mrsa was first recognized as being acquired from hospitalized patients (ha-mrsa), but the onset of mrsa infection outside the hospital setting, in communityassociated strains (ca-mrsa), has recently been described with increasing frequency. ha-mrsa are known to be responsible for infections in hospitalized patients, although highly virulent ca-mrsa are increasingly reported worldwide as the cause of severe outbreaks, replacing ha-mrsa strains in nosocomial infections. little evidence is available about the characterization (sccmec type) and epidemiology of community-acquired mrsa (ca-mrsa) or hospital-acquired mrsa (ha-mrsa), transmissibility, antibiotic susceptibility and virulence in the cf population. the present multicenter study investigated the susceptibility pattern, epidemiology, and sccmec type of mrsa strains isolated from nine italian cf centers. the susceptibility pattern was determined by the disk diffusion method and pulsed field gel electrophoresis (pfge) was performed for epidemiological purposes. the sccmec type in order to identify hospital-and community-acquired mrsa strains (ha-mrsa and ca-mrsa respectively) was determined with molecular methods, and the presence of gene encoding panton-valentine leukocidin (pvl) was tested. during the study period, 181 mrsa strains were isolated from 178 out of 2362 (7.5%) patients attending these cf centers. of the sccmec type representing ha-mrsa, our data showed an high prevalence of sccmec i (49.4%) while the prevalence of sccmec ii, (reported as the most represented among ha-mrsa) was only 1.1%. furthermore a high prevalence (31.4%) was found of sccmec iv (suggestive of ca-mrsa strains). epidemiological analysis showed that 31 (17.1%) out of 178 analyzed mrsa strains belong to the same pfge clone shared among six centers, belonging to the sccmec type iv, suggestive of ca-mrsa isolates. twenty-four (77.4 %) out of these 31 strains were sccmec type iv, indicative of ca-mrsa, as suggested by the good susceptibility rate to trimethoprim-sulfamethoxazole and rifampin. surprisingly all the isolates belonging to this epidemic clone were negative for the pvl genes. these results show that ca-mrsa is now spreading in the cf population, documenting the first epidemic of ca-mrsa in cf patients who are considered at risk for ha-mrsa acquisition due to frequent hospitalizations. these data show a peculiar picture of mrsa epidemiology indicating that further studies are required to clarify the role of ca-mrsa in global epidemiology, their pathogenicity and clinical impact on cf patients. we thank the fondazione per la ricerca sulla fibrosi cistica-onlus for its grant (ffc#12 2006) . there are concerns regarding an increased risk of cancer in patients with cystic fibrosis (cf). this study aimed to review the occurrence of malignant disease in a large population of adult patients with cf. the case notes of 372 patients attending the leeds adult cf unit were reviewed. demographics and data regarding a diagnosis of malignant disease were recorded. a total of eight patients (3 male, 5 female) were diagnosed with a malignant condition over the 10 year period. the median (range) age of diagnosis of a malignant condition was 30.5 years (23-55 years). five patients were diagnosed with malignancy post-transplantation. within this group of patients there were two cases of lymphoproliferative disease, one case of basal cell carcinoma, one case of liver cancer and one case of small cell lung cancer related to the donor lung. the median (range) time to diagnosis post-transplantation was two years (1-6 years) with a median (range) age of 26 years (23-55 years). three patients were diagnosed with a malignant condition without solid organ transplantation. this group consisted of one case of pancreatic carcinoma, one case of oesophageal carcinoma and one case of bowel carcinoma. the median age of diagnosis in this group of patients was 44 years (27-55 years) . prior to transplantation we found all malignant diagnoses were related to the gastrointestinal tract. post-transplantation we found a wider variety of malignant diagnoses, with lymphoproliferative disease being most common. awareness of the increased risk of malignant disease is important and appropriate investigations should be undertaken particularly in patients with atypical symptoms. background: patient outcomes can be improved by implementing evidence-based guidelines for initiating therapies. in our cf center, practitioners have traditionally prescribed medications without a centralized database to screen patients for evidence-based criteria. goals: the primary goal of this project was to increase cf practitioner, family and patient awareness of evidence-based medication guidelines to facilitate discussions regarding therapies. the secondary goal of this quality improvement (qi) project was to determine if cf practitioner prescribing patterns would be altered by increasing awareness. methods: the cf steering committee at cincinnati children's hospital medical center (cchmc) identified oral azithromycin, inhaled tobramycin, and dornase alpha as target medications for this qi project. an evidence-based medicine committee was formed, including a parent advocate, to direct the project. a consensus was reached among cf practitioners for when to initiate therapy discussions. dornase alpha was recommended for cf patients ages six and older. inhaled tobramycin was recommended for cf patients ages six and older with chronic pseudomonas (defined as 2 positive cultures in past 12 months). oral azithromycin was recommended for cf patients ages six and older, with chronic pseudomonas, and a weight of 25 kilograms or more. the cf database was screened to determine each patient's medication eligibilities. eligibilities were reviewed weekly at our cf chart conference. medication specific family and patient handouts were developed, reviewed by the parent advocate, and given to eligible families and patients on arrival to clinic. data was collected to document the number of discussions facilitated at clinic visits, the decisions regarding medications, and the number of new treatment prescriptions that were facilitated. after seven weeks of the initiative, statistical analysis was conducted using mcnemar's test for differences. a chi-square test with a two sided alpha of p <0.05 was used to determine significance. results: 148 of the 203 cf patients in our center were eligible for pulmonary medications using our evidence-based criteria. there were 253 individual prescribing opportunities in these 148 patients (taking each medication for each patient as a unique prescribing opportunity). at the start of the project, 64.3% (163/253) of the prescribing opportunities were fulfilled. 50% (74/148) of patients were prescribed all of their recommended medications. during the first seven weeks of the project, cf practitioners discussed initiating new pulmonary medications with 40 cf families and patients. the proportion of prescribing opportunities fulfilled increased from 64.3% (163/253) to 75.5% (191/253) (p value < .0001; 95% ci 65.3%-82.8%). the proportion of patients prescribed all of their recommended medications increased from 50% (74/148) to 65.5% (97/148) (p value < .0001; 95% ci 57.8%-80.0%). conclusions: increasing awareness of evidence-based pulmonary medication recommendations with cf practitioners, families and patients facilitates therapy discussions, increases the proportion of therapies prescribed and increases the proportion of patients on all recommended therapies. purpose: our purpose was to optimize lung function (fev1) by consistently and aggressively diagnosing and treating pulmonary exacerbations (pex) in all cf patients at our center. it was important to work on this because our families wanted standardized care and we felt our patients' fev1's were suboptimal. methods: our core llc team included two parents of cf patients and one adult patient. we met weekly, received coaching every other week, and learned and applied quality improvement techniques. physicians began monthly meetings, and monthly meetings of the entire cf team continued. the fab met every other month. we identified our specific aims, and approached them systematically. the providers, starting with criteria used by several authors, agreed upon the definition of pex and created a pulmonary exacerbation score sheet (pexss). we selected a score of 4 as the threshold for defining a pex. after using the pexss for three months, all providers reviewed the individual pexsss and validated our original choice of 4. run charts were produced and reviewed periodically to display each provider's percent of pexsss used. when the use of the pexss was not standardized after a year, it was reprinted on bright yellow paper to make it stand out. to educate the families and patients about the early symptoms of pex, a refrigerator magnet was designed and distributed that included all pex criteria. results: we standardized the use of our pexss by all providers on all patients seen in cf clinic with ongoing measurement of its use and regular feedback. many patients and families gained greater knowledge about what a pex was, and the importance of early recognition and treatment. the cf nurses note that 1) many patients call with milder pex symptoms than they did before, 2) providers treat pex earlier, 3) many patients call with their pex score as well as their symptoms. we hospitalized more patients for pex after this project began, with an average of 62 patients/year in the 3 years prior, and 84 in 2006. the median fev1 % predicted of our 6conclusions: participating in the llc changed the culture and functioning of our cf team. limitations to our study include: 1) fev1 change may not have been due solely to this project, 2) the groups who's fev1's were averaged over the various years are different. we will continue to optimize and standardize 1) prevention of pex, 2) inpatient care, 3) care of pex in cf providers' private offices, and 4) follow up of patients with pex. patient adherence to airway clearance techniques (act) in the treatment of cystic fibrosis is known to be sub-optimal resulting in deterioration of lung function. the global aim of our project was to improve median fev1. the specific aim was to improve adherence to act. we believed that these goals could be acheived by implementing a program which included re-education of airway clearance techniques (react) for our cystic fibrosis patients. methods: the initial phase of this project began with the administration of an anonymous survey to patients/families. this survey was designed to assess act practices, knowledge of the rationale for performing act and barriers that prevent patients/families from adhereing to act. the next step included an in-clinic questionnaire and patient/family demonstration of current aerosol therapy and act. based on individual results patients were identified as: adherent with correct technique, adherent with incorrect technique or non-adherent. we defined adherent as correct performance of act one to two times daily as prescribed. then all patients entered the react program which included a standardized tutorial on proper act and correction of techniques if necessary. in the non-adherent group barriers to performing act were discussed and problem solving techniques were used to overcome barriers. the patients in the non-adherent group were re-evaluated in clinic monthly while the adherent patients returned on an every two month schedule. results: analysis of our initial survey revealed that while the majority of patients/families reported performing act daily, the duration of treatments was less then medically prescribed. many patients/families reported barriers which decreased adherence to therapy. to date 85% (74/87) of our patients have completed the in-clinic questionnaire and entered the react program. the results of these evaluations and patient demonstrations revealed: 37% (27/74) were adherent with correct technique, 3% (2/74) were adherent with incorrect technique and 60% (44/74) were non-adherent. in the six months since the implementation of the react program, patients/families are self-reporting an increased adherence to act and the median fev1 for our center has increased form 84.7% (2005 cff registry data, ages 6-18 years) to 87.4% (cff registry, first quarter, 2007, ages 6-17 years). conclusion: while adherence to act in our center was poor, we were able to improve adherence and ultimately improve the median fev1 by implementing a program of re-education for act (react). we believe re-assessment of adherence to therapy and reviewing correct technique is necessary at every clinic visit. continuous quality improvement (cqi) techniques have recently been employed in medicine to improve quality of care. although there is evidence that cqi improves efficiency in hospitals, little is known about effects of cqi on improving patient outcomes. we have previously reported creation of a unique nutritional risk pathway (nrp) to care for cystic fibrosis (cf) patients who are at nutritional risk. the goal of this study is to assess perceived impact and objective clinical efficiency of our nrp. patient data was recorded from all children attending the cf clinic over an 18-month observation period (between sep 05 and mar 07). bmi percentiles were assessed and nutritional risk zones -green: acceptable (bmi > 25%ile), yellow: at risk (bmi 10-25%ile) or red: at urgent risk (bmi < 10%ile) -assigned at each visit. the visit intervals were every 4 to 12 weeks based on severity of nutritional risk. patients that remained in the red zone for an average of 3 to 6 months with no improvement were considered for gastrostomy tube (gt) placement. the cf dietitian updated the data weekly and e-mailed quarterly to cf physicians for review of their patients' status. patient/family perceptions and understanding of the nrp was assessed by questionnaire. of the 164 children in the study, 29 (18%) were identified to be at nutritional risk and were enrolled into the nrp: 19 in red and 10 in yellow zones; 13 (45 %) male, 16 (55%) female; 18 (62%) age < 12 yrs, 11 (38%) age ≥ 12 yrs. over the 18-month observation period, 21 (72%) showed improvement (7 moved from red to yellow, 7 from red to green and 7 from yellow to green zones). thirteen (81%) females improved, whereas 8 (62%) males improved. in the under 12 yrs age group, 17 (94%) improved compared to 4 (36%) in the ≥ 12 yrs age group. as a result of participation in the nrp, 9 (31%) children had gt placed: six of the 9 showed improvement with four moving from the red to green zone. from the survey responders, 80% stated they understood the meaning of the cf nrp. all responders were extremely to somewhat motivated to focus on their child's nutrition when asked to return for growth monitoring or follow-up visits. having specific goals for weight gain, calorie requirements and bmi were considered extremely helpful by 100% of families. when gt placement for their child was recommended, 40% felt they lacked all the necessary information to make a decision. our new cf nutrition education booklet was not received by 40% of families. some of the physicians who received the quarterly nutritional patient data provided positive feedback and found this information beneficial for patient care. nutritional cqi in our cf center utilizing a specific nrp and monitoring of nutritional risk status resulted in improved patient outcomes. overall perceptions of our nrp were positive from both patients and cf physicians. these results suggest that this cqi strategy improved clinical outcomes in our patient population. patients with cf require frequent healthcare visits to optimize growth, initiate early interventions and delay progression of lung disease. the cff recommends evaluation at a cf center at least quarterly. as part of a llciv quality improvement (qi) initiative, our cf team set a goal to increase the percent of cf patients attending clinic 4 or more times a year to 90% or greater by developing strategies to monitor and improve adherence rates. methods: we reviewed cf patients who attended clinic 3 times or less in 2005 to determine patterns of non-adherence in regards to age, gender, distance from center, season, physician or insurer. a patient survey was used to assess clinic attendance barriers from a patient/family perspective. a brainstorming session identified other barriers. a fishbone diagram was drafted into 4 barrier categories: external, internal, communication, and patient/family perceptions. after reviewing all barriers, we concluded the most effective improvements could be made to internal clinic barriers. clinic processes were then assessed including making of appointments, reminder letters/calls, a time cycle analysis, and number of cf appointments available per patient per physician. our family advisory board participated by focusing on the importance of 4 or more clinic visits in their newsletter and by assignment of a member to the llciv team. we discovered a lack of a standardized process to follow-up (fu) patients who "did not keep appointments" (dnka). a spreadsheet was created to monitor physician dnka rates. a primary fu strategy was devised to identify patients who dnka through daily auto-emails to the cf office assistant and utilization of a rescheduling decision tree for reappointment of these patients by our appointment center. two secondary fu strategies were developed. the first involves the clinical coordinator printing a monthly report off port cf entitled "patients due a visit" then emailing this list to the cf team to contact and reappoint. the second involves the cf social worker tracking patients seen per quarter on a spreadsheet and emailing the spreadsheet quarterly to the cf team as well as posting on our datawall. results: in 2004 , and 2006 , 35%, 56%, and 89% of our patients attended clinic 4 or more times, respectively. in 2004 in , and 2006 , 33%, 25%, and 23% of our patients were at nutritional risk, respectively. in 2004 and 2005, mean fev1% predicted was 88.3% and 90.4%, respectively. conclusion: the number of cf patients attending clinic quarterly at our center increased by implementing processes to track dnka patients and reappoint them to clinic promptly. however, some of the early improvement in clinic attendance was partially due to an increased awareness of our qi efforts by our clinic staff, cf team and patients/families. we speculate that improvements in nutritional status and lung function are at least partially related to more frequent clinic attendance by our patients. continued monitoring of the number of dnka patients and patients who attend clinic quarterly is required to sustain these gains in improved patient outcomes. data when we first measured patient/family satisfaction in 2003, 31 parents and 14 children were given questionnaires to measure their satisfaction with services provided at the clinic. at that time, responses in our survey were very positive. with regards to our new model we received very positive feedback, encouraging us to continue with the model. parents and children liked the elimination of duplication in questions and they also like having the whole team involved in the meeting and problem-solving. final results for our second satisfaction questionnaire will not be available until june of this year as the questionnaires are being given at clinics over a 3 month period this spring. the children complete a questionnaire with the help of their parents if needed and the parents also complete a survey. results to date are very positive for both the parents and the children. of the 14 children who have completed the survey to date, all stated they would like to continue to meet with the group as a whole as opposed to meeting individually with team members. of the 14 parents surveyed to date, all are either very satisfied or satisfied with the group format. conclusion: we have not yet completed the survey but results to date clearly show that both the patients and their families are happy with the round table model. results will be compiled early this summer and would be ready for the poster and/or presentation in the fall. the cff clinical care guidelines recommend routine clinic visits for assessments, interventions, monitoring, education and counseling every 3 months or more often as indicated. adherence to these guidelines is important in ensuring better clinical outcomes. in reviewing our cf center data from the 2003 cff registry report, 46.2% of pediatric patients (6-18 yrs.) and 47.8% of adults (18 yrs. and older) adhered to the guidelines. national goals were not provided then; instead data was given for the top ten cf centers (80.9% and 78.5% for pediatric and adult patients respectively) as well as the national rates (55.5% and 44.8% for pediatric and adult patients respectively). our cf care center acknowledged the need to improve adherence to these guidelines and at the beginning of the year 2005, embarked on a quality improvement project to address this issue. following the pdsa (plan-do-study-act) steps, we agreed on the objective of increasing the percentage of pediatric and adult patients adhering to the guidelines by at least 50% and at the same time, to exceed the national average in 1 year or by the end of the year 2005. the physicians and social worker obtained feedback from patients and/or families during a clinic visit regarding barriers in complying with scheduled clinic visits. suggestions were elicited to overcome these barriers. the team members then identified barriers in meeting the guidelines and implemented measures to improve compliance with clinical care guidelines. among these measures were sharing the information about clinical care guidelines and importance of adherence through our cf newsletter and at every clinic visit, reminder calls to patients or caregivers at least 2 days prior to the visit, appointments for the next visit given prior to discharge from the clinic, and same day calls to patients who failed to keep their appointment for rescheduling. our social worker identified patients with difficulties or problems in consistently keeping appointments and helped address these problems. progress made in this endeavor was shared with parents and caregivers during the "parents' night" held in the fall of 2006. indeed, with consistent implementation of these measures, we have seen improvement in the percentage of patients adhering to guidelines. in our 2005 cf care center data, 68.2% of pediatric patients and 89.5% of adult patients adhered to recommended clinical care guidelines, approximating and in fact for the adults, exceeding, our target of 50% increase in the percentage (48% and 87% increase respectively for pediatric and adult patients). these data were better than the national average for 2005 (63.9% and 48.3% for pediatric and adult patients respectively) but for pediatric patients, still 22% below the national goal of 90%. adults approximated the national goal of 90%. we are committed to continually engage in the process of quality improvement in further improving adherence to clinical care guidelines and to hold on to the gains. weight z-scores and height z-scores were monitored prospectively for all infants, children, and adolescents at a pediatric outpatient cf clinic during an ongoing, multidisciplinary quality improvement (qi) initiative. all cf team members participated in this qi project which emphasized the importance of good nutrition and adequate growth for optimal lung health. specific strategies for improvement included education to families at each visit with a written nutrition action plan, increased emphasis on liquid supplements and enteral feeds as needed, and a consist message from all cf clinic staff in encouraging attention to nutrition. the study objective was to determine if multidisciplinary qi interventions to improve nutritional status had any effect on weight z-scores and/or height z-scores over a 3-year period. the null hypothesis was that z-scores for weight and height would either not improve or worsen. inclusion criterion for this study was attendance at cf oupatient clinic during the first 5 months of the qi initiative (time 1) with a subsequent clinic visit 3 years later (time 2). patients not attending clinic at both time 1 and time 2 were excluded. a cohort of 119 patients (53 male, 66 female) met the inclusion criterion. the mean time 1 to time 2 interval was 3.05 ± .11 years (range 2.70 to 3.40 years) with mean patient age at time 1 of 6.75 ± 4.55 years (range .10 to 15.80 years). pancreatic enzyme replacement therapy was used for 112 (94.1%) of the cohort. paired sample t-tests indicated significant improvements for weight z-score (p <.000, t (118) = 6.16) and height z-score (p <.000, t (118) = 4.49) after the 3-year intervention. at time 1, 89 children (74.9%) were underweight or less than 50th percentile while 30 children (25.2%) were normal-weight, at or more than 50th percentile for age. mean differences in both weight and height z-scores in the initially underweight patients were greater than those for the normal-weight patients over the 3 year interval. no significant differences were found for the initially normal weight-for-age children between time 1 and time 2 for weight z-score ( p = .83) or height z-score (p =.37). children who were not underweight at time 1 grew normally, without acceleration over the 3 yr intervention. therefore, we conclude that a longterm multidisciplinary qi project to enhance nutritional status resulted in significantly improved weight z-scores and height z-scores for those individuals who were initially underweight for age. the most common cause of morbidity and mortality for patients with cystic fibrosis (cf) is pulmonary exacerbations. these exacerbations lead to hospital admissions and treatment with intravenous (iv) antibiotics. all cf patients at our facility are admitted to a teaching service which includes house officers and students. standardized order forms are used to improve accuracy and timeliness of medications and therapies. the efficiency of this process was brought into question in 08/2006, when several parents complained about a delay in the receipt of first dose iv antibiotics after hospital admission. the objectives of this study were to determine what were the actual delivery times of the iv antibiotics to the patients (both retrospectively and prospectively), and areas where improvements could be made. our predetermined target time was < 3 hours. two analyses were performed in order to answer the objectives. the first analysis was done in a retrospective fashion. this analysis included cf patients admitted from 5/1/2006-9/30/2006. a total of 44 patients were included. total median time from admission to receipt of first dose of iv antibiotics was analyzed. a second collection of blinded prospective data was performed between 10/1/2006-12/31/06. only the pulmonologist, pharmacist collecting data, and pharmacy clinical manager were aware of the data collection. a total of 15 patients were included in the prospective project. each part of the process model were recorded and analyzed so that areas of improvement could be realized. the median time from hospital admission to first dose of antibiotics was 7 hours (range 0-31 hours) (retrospective cohort). the prospective cohort median time from hospital admission to first dose of antibiotics was 6 hours (range 67 min-11 hrs). the median time from admission to order entry by pharmacy: 2 hrs, 24 min (range 35 min-4 hrs, 11 min). the median time from the medication order being written by medical practitioner to order entry by pharmacy: 55 minutes (range 3 min-3 hrs, 20 min). the median turn-around time by the hospital pharmacy of order entry until reaching the nursing unit: 19 min (range 3 min-50 min). the median time required by the nursing staff to administer the antibiotic: 2 hrs, 23 minutes (range 29 min-6 hrs). this project was initiated in response to parental concerns. from the results of the retrospective and prospective analyses, it was determined that a delay is occurring in the receipt of iv antibiotics, which translates to hours in the hospital without therapy. this is both inconvenient and uncomfortable to the family, and wasteful of medical resources. the findings of this project were presented to the pharmacy and therapeutic committee of the hospital, to the nursing directors, and to the parents of cystic fibrosis patients. the delay is greater than expected before this project was initiated. as a result of these findings a "fast track" medication order set was developed to expedite initiation of inpatient therapy. four key areas included in this order set are sputum, iv access, diet, and iv antibiotics. we speculate that time to first drug delivery will decrease and patient satisfaction with improve. the pediatric intermountain cystic fibrosis center (imcfc) provides multidisciplinary cystic fibrosis (cf) care to 220 pediatric patients from utah and parts of idaho, wyoming, and nevada. historically the imcfc has embraced early intervention. as new therapies have been added to the cf armamentarium it has become necessary to have a method of keeping track of which patients are candidates for various therapies and also which medications have been prescribed, utilized or even should be discontinued. in order to address this quality issue, the imcfc decided to develop and implement the use of a medication tracker (mt). the imcfc decided to focus on key therapies which were felt to be at risk for variability in prescriptive practice. the five therapies included were: pulmozyme<®>, tobi<™>, hypertonic saline(hs), azithromycin and asthma medications (inhaled corticosteroids and/or leukotriene receptor antagonists). ibuprofen was intially included and later removed from the mt. prior to implementing the mt, the imcfc met to review literature, medication trackers from other centers, and develop consensus among team members. six versions of the mt have been developed between 1/1/2007-4/30/2007 . each mt has been tested in a pdsa cycle and revised. since initating the mt, 188 patients have been seen at least once. there have been 264 mt encounters. the mt is used only after the first visit to the imcfc and reflects changes to established cf care. there have been a total of 76 changes in prescribed therapies in 66 different patients(35% of patients seen). a minority of patients were receiving therapies that did not meet our pre-determined consensus for prescription. the most common therapies in this category were tobi™ and azithromycin. primary reasons cited for prescribing were: chronic suppressive antibiotic use, pseudomonas positive and <6 years old, and significant changes on chest imaging. reasons patients were not prescribed clinically indicated therapies were: patient refusal, participation in clinical trials, cost, and lack of compliance with other therapies. changes in asthma therapy included the discontinuation of medication in 17 encounters and the initiation of therapy in 8. the use of a specific cf patient mt allows consistent practice in selection of therapies. it can be used by all team members and helps to identify discrepancies in therapy. assuring all patients receive information and are considered for new therapies is increasingly important as our treatment options expand. the mt also highlights the need to consider discontinuing therapies. we speculate the mt will become an increasingly valuable tool over time and improve patient care. rationale: recent reports document increasing prevalence of antimicrobial resistance across a range of pathogens in patients with cf. the increase in multidrug resistance of these organisms complicates the therapeutic management of these patients. objective: to describe patterns of antibiotic use among physicians treating cf patients. methods: for the original purpose of evaluating site characteristics that act as effect modifiers on the relative efficacy or cost of novel therapies, we administered a survey to acquire information on practice patterns and characteristics of physicians participating in a clinical study of an investigational drug for patients with cystic fibrosis. chi-square and spearman rank test were used to evaluate associations between physician characteristics and patterns of reported antibiotic use. results: fifty-nine physicians, representing 59 study sites, completed the survey. ninety percent reported a medical specialty of pulmonology and 98% reported practicing at a teaching hospital. on average, respondents reported that about half of their practice consisted of patients with mild cf (defined by fev1 ≥ 75% of predicted normal.). forty-two percent of respondents reported prescribing oral antibiotics and 61% of respondents reported prescribing inhaled antibiotics for maintenance therapy for at least 50% of their mild cf patients. eight-five percent and 53% of respondents reported prescribing iv antibiotics for pulmonary exacerbation and pulmonary 'clean out', respectively, with about a third of these cases being prescribed for outpatient iv use. physicians that prescribed iv antibiotics for pulmonary clean out were more likely to pre-scribe oral (p= .04) and inhaled antibiotics (p=.02) for maintenance therapy than physicians that did not prescribe iv antibiotics for pulmonary clean out. conclusions: antibiotic use for maintenance therapy and iv antibiotic use for pulmonary exacerbation and pulmonary clean out acted as 'complements' rather than 'substitutes' as there was a group of physicians who reported less use of antibiotics. our findings warrant further research as well as additional exploratory analyses with a larger and broader sample to examine associations between physician characteristics and patterns of antibiotic use. understanding the relationship between physician characteristics and antibiotic use has the potential to influence the approach to management of cf patients in an environment of increasing antimicrobial resistance. introduction: hypertonic saline (hs) has been shown to increase mucus clearance and improve lung function in cf, and is now increasingly being used as part of routine therapy. one of the goals of our cf center quality improvement (qi) initiative is to increase use of hs among all eligible patients. objectives: to assess adherence to treatment with hs, parental opinions on treatment, perception of side effects, and the effect of initiating hs on the use of other nebulized medications in pediatric cf patients started on hs therapy during 2006. methods: 86 patients under 18 years of age were identified as initiating hs therapy during 2006. a telephone survey of their parents using a 10-item multiple-option questionnaire was administered in april 2007 as part of our cf center qi initiative. results: 56 parents (65%) completed the survey: 39 (70%) patients were still using hs. of those still reporting use of hs, 16 (41%) were using it twice a day; 20 (51%) once a day; and 6 less often than daily. among the 17 patients who discontinued hs, 8 (47%) stopped following recommendations from their pulmonologist; 5 (29%) stopped due to side effects; 3 (18%) felt hs took too long to administer; and 3 (18%) felt hs solution was too difficult to mix. the most common side effect reported was cough in 33 patients (59%); 19 (34%) reported no side effects. the majority of patients (64%) spent 10-30 minutes per day on hs therapy. thirteen (23%) patients discontinued another nebulized medication when they initiated hs; 11 (85%) of those stopped pulmozyme. reasons for stopping pulmozyme included: time to nebulize both medications was too long (54%), perception that hs was working better for their symptoms (23%); and doctor's instructions (23%). overall, 33 (59%) parents felt hs improved their child's symptoms; 16 (29%) saw no difference; and 2 (4%) believed hs made symptoms worse. there was a significant difference in the proportion of patients that reported improvement of their symptoms in the group using hs twice a day (15/16, 93%) when compared to those using it once a day (11/20, 55%) (p=0.022). discussion: the majority of patients continued to report the use of hs therapy several months after our initial intervention, with the most parents reporting improvement in their child's symptoms. physician recommendation was the most common reason cited for discontinuation of hs therapy. further study of physician opinions on hs therapy, particularly the timing of initiation and discontinuation, is warranted. one-half of patients reported using hs only once a day; however those reporting twice daily use were more likely to report subjective improvement of symptoms. since treatment complexity in cf increasing, determining the effectiveness of once daily hs therapy, either alone, in place of, or in combination with other therapies such as pulmozyme is essential. long-term follow up of these hs-initiated patients will focus on effects on exacerbations, pulmonary function, and hospitalizations. the uab/children's hospital (chs) cf center participated in learning and leadership collaborative ii. as part of the qi journey, the chs cf qi team thoroughly evaluated the current system of care in an effort to identify areas of needed improvement. one area assessed was clinic attendance. it was discovered that 21% of patients failed to attend clinic appointments every week. according to cff care guidelines, patients with cf should be seen a minimum of 4 times each year at the cf center. the cff has also found that those centers that see their patients more frequently have improved outcomes for fev1 and nutritional status. aim to decrease the number of missed appointments at the chs cf clinic. the chs cf center administered a qi survey to identify the reasons patients and families were missing appointments. this survey was a 7 item questionnaire that evaluated: reasons for missed appointments, if the families rescheduled missed appointments, difficulties with rescheduling, the need for reminder calls, distance traveled to the cf center, and insurance status. results 59 completed surveys were collected. 41% of the families reported medicaid as their insurance provider. 74% of families live more than 50 miles away from the cf center. 27% of the families confirmed that they had missed an appointment in cf clinic over the last 12 months. the reasons for the missed appointments were widely variable; however, 15.8% of those who missed appointments reported that they did not have access to transportation to get to the appointment. overall, families did not report difficulties in rescheduling appointments. 68% of those that missed appointments identified medicaid as their insurance which can be correlated with lower income families and 87.5% of those that missed lived greater than 50 miles away from the cf center. 83% of families reported that a reminder call would be helpful for them. interventions after evaluation of the survey data, the center quickly initiated reminder calls for cf clinics. a student employed by the cf center made these telephone calls to the patients scheduled for clinic every week. the majority of the time the student caller left a message for the family. with the implementation of this simple intervention, missed appointments have decreased from 21% to 14% which is a 30% improvement rate. reminder calls are now a routine part of the cf center's system of care. other ongoing interventions include increased involvement of primary care doctors for patients lost to follow up for more than 6 months and a system for quickly rescheduling patients. next steps in addition to reminder calls, the cf team will now determine where patients who routinely miss appointments live. if there are pockets of patients in common areas of the state, the team will attempt to maximize services for those patients such as linking them with community transportation resources, community health advocates, or providing satellite clinics. this will be accomplished through geomapping of the alabama cf population based on zip code. we designed a scoring system to uniformly identify patients experiencing a pulmonary exacerbation. the elements of this pul-monary exacerbation score (pes) have been previously described. at the time this project began the median fev 1 % predicted of the cf patients at our center 6-18 years old was below the national average (2004 cf registry data). our hypothesis was that greater uniformity of identification of pulmonary exacerbations, would improve pulmonary function in the pediatric age group at our center. this abstract describes continued improvement in the pulmonary function of this age group at our center now that the pes has become standardized in our clinical practice. methods: beginning in october 2004, a pes was calculated for all patients presenting to our center age 6-18 years of age. any patient with a pes of > 5 was defined as having a pulmonary exacerbation, and treatment with antibiotics was recommended. the patient's cf physician dictated the ultimate decision for treatment and specific course of treatment. median fev 1 of all patients age 6-18 years was calculated quarterly to determine the effect of the pes use on pulmonary function. in october 2006 the pes was incorporated into our cf clinic medical record and its use was standardized. the use of the pes, the adherence to the recommendation for treatment, and quarterly fev 1 of the population has continued to be monitored. results: from 10/2004 to 9/2006 (the duration of the original qi project), 1067 patient visits occurred, with a pes calculated on 968 (utilization of 86.7%). 327 patients were treated for a pulmonary exacerbation of the 357 patients with a pes of > 5 (adherence of 91.6 %). 64.9% of exacerbations were treated with oral and/or inhaled antibiotics, and 35.1% were treated with intravenous antibiotics. since standardization of the pes in 10/2006, 348 visits have occured. use of the score has decreased slightly (83.4% utilization), however adherence to the score has increased (92.1 % of pes > 5 have been treated). use of oral vs. iv antibiotics has been similar (69.5% oral vs. 30.5% iv). the median fev 1 of our patient population has improved from an average of 83.7 % predicted during the 24 months before implementing the pes to an average of 88.5% predicted during the ensuing 24 months of the original qi project (a 5.7 % improvement). since standardization of the pes in 10/2006, the average median fev 1 of the population has continued to increase to 90.7% predicted (an 8.4% increase from the baseline period). this improvement in median fev 1 is reflected in our cf registry data. summary: implementation of a pulmonary exacerbation score, designed to encourage uniform identification of pulmonary exacerbations in patients with cf, is associated with an improvement in median fev1 in children 6-18 years of age. standardization of the pes has resulted in continued improvement in pulmonary function of this population at our cf center. this work has been supported in part by the cff and akron children's hospital. our cf family council parent and patient advisory group has been involved in the development and implementation of this project. utilizing data from the 2005 cff patient registry, we determined that our center has had nutritional outcomes consistently below the national average for pediatric and near the average for our adult patients. as a result, we developed the global aim to improve and maintain optimal growth and nutrition in all patients at our cf center. our specific aims included: 1)to record growth parameters at all outpatient visits; 2)to identify patients at nutrition risk, categorizing them based on anthropometric data; 3) to develop and implement a specific nutrition plan based on nutritional category; and 4) to educate staff, patients,and families on the benefits of normal growth and nutrition. we surveyed our patients to determine their overall attitudes toward nutrition and nutritional care received at our center. we discovered that many patients and families did not feel that nutrition was addressed at every cf visit nor that they had a nutritional plan at each encounter. this led to the development of an intake form/home-going sheet for our patients to complete that included data elements necessary for assessment of nutrition and that clearly outlined a nutrition action plan. a standardized nutritional assessment score (nas) was devised that incorporated weight, height/length, bmi and weight for length percentiles as well as weight loss, failure to gain or maintain, and failure to remain in the expected growth channel. the nas was developed after examining the medical literature, reviewing work done by other qi teams, and acquiring input from our pediatric gastroenterologist and endocrinologist. we then developed nutritional algorithms to address nutritional intake, malabsorption, evaluation for cfrd, and short stature. these four algorithms were used to develop a nutrition action plan for each individual. beginning in march 2007, a nutritional category (optimal/acceptable, concerning, at risk or failure) was assigned to each patient at each visit based on their nas. an individualized nutrition action plan was developed with each patient. at the time of project implementation, patients age 2-20 at our center had a median bmi %ile of 52.9% with 54.5% of our patients with a bmi <50th%ile. our patients > 20 years had a median bmi of 22.1 with 52.5% having a bmi<50th%ile. after one month of implementation of the nas, 60% of our patients had been categorized with 13% of our patients having individual nutrition action plan developed. to track our progress, we will monitor median bmi, % of patients with bmi <50%ile, and % of our patients in each nutritional category quarterly using portcf. our goal is to have all our patients assessed and a nutrition action plan implemented by the end of 2007. we have developed a nutritional assessment score and nutrition treatment algorithms for pediatric and adult patients with cf as part of a qi initiative to improve nutritional status of patients at our center. the project has been supported, in part, by akron children's hospital. we are indebted to lori lundquist, a parent of two of our cf patients, who has been an integral part of our nutriton qi team. in germany there are about 8,000 cf patients who are usually treated in special centres by a team of trained and experienced health care professionals. the result of this structured approach to patient care, including frequent clinical assessments, monitoring, and aggressive interventions is an improved mean survival and a higher quality of life of pediatric and adult cf patients. however, a recent survey showed that german reimbursement schemes cover only about 50 % of resources used within these centres. objective to assess and evaluate direct costs of outpatient treatment of cf patients in germany. a micro-costing approach was used to record resource use data directly within representative cf centres. results of the evaluation may be used to initiate and support discussions between health care providers and insurances about adequate reimbursement schemes in germany. outpatient care was evaluated in seven different centres for pediatric and adult cf patients. patient reported outcomes, clinical patient data, resource utilisation, and physician related time consumption for treatment was recorded for every patient during one representative month in 2006. cost data for staff, materials, medical equipment, rooms, etc. were assessed within the respective centres. data for 326 cf patients were collected. about half of them were children and adolescents, and half of them were adults. a comparison of the resource uses to the actual remuneration of these services indicates that only about 50 % of the costs are covered by the reimbursement system. correlation analysis identified significant cost drivers like the age of the patient, comorbidities like pancreatic insufficiency and hepatobiliary complications, lung function (% fev1), and the presence of certain pathogens in the lungs. as the actual reimbursement covers only about 50 % of the resource usage costs in germany, the existence of the cf centres and sufficient treatment for cf patients is uncertain in the future. in order to ensure the existence of the centres it would be necessary to agree to a cost-covering reimbursement at minimum. this may be based on a lump-sum payment that could be differentiated for pediatric and adult centres or according to comorbidities or clinical parameters. the data calculated in this study could be used to trigger and support discussions between health care providers and insurances about a new cost-covering reimbursement system for the out-patient treatment of cf patients in germany. the impact of the new us cystic fibrosis foundation (cff) nutrition practice guidelines, i.e., discontinuing the use of percentage of ideal body weight (%ibw) to define "nutritional failure" and setting body mass index (bmi) below the 50th percentile to define "nutritional risk", on evaluating nutritional care practices is unclear. methods: data from 14702 children reported to the 2002 cff patient registry were analyzed to compare the rates of malnutrition among 113 cf centers. results: nationally, eliminating %ibw < 90% as a criterion for underweight resulted in a 6.2% reduction (from 33.1% to 26.9%) in "nutritional failure" rate. misclassification of "nutritional failure" by %ibw < 90% ranged 1.2-15.6% among 113 centers and was greater for centers having a larger proportion of tall patients. one-third of centers switched to a different tertile ranking, after correcting for misclassification by %ibw. use of bmi < 50th percentile led to the classification of 56.8% of patients as "nutritional risk". more than half of the centers had different tertile rankings on "nutritional risk" compared to "nutritional failure". a total of 5.1% (0-12.5% among 113 centers) of patients who had height < 5th percentile but bmi ≥ 50 percentile were not considered at nutritional risk. the cystic fibrosis questionnaire-revised (cfqr) is a disease specific quality of life measure that is currently undergoing clinical validation and may prove to be a useful adjunct to provider assessments. we proposed the routine use of the cfqr in a busy adult cystic fibrosis (cf) clinic would assist in identifying patients in need of limited clinic resources. this performance improvement project focused on new and infrequently-seen patients (i.e. fewer than three outpatient clinic visits/year) to determine the frequency of patients with domain scores that fall more than one standard deviation below our clinic mean. methods: the cfqr is routinely self-administered to all adult cf patients in clinic at least once every year. these assessments occur after vital signs are obtained and prior to any other clinical assessments. patients were deemed to be at baseline if there were no changes made to their pharmacologic or chest physiotherapy regimens. the mean and standard deviation of each domain of baseline cfqr assessments were determined. the frequency of rarely seen and new patients with domain scores less than one standard deviation below the mean was determined. results (202). of these 84 baseline assessments, forty five were obtained from patients who were rarely seen or new to the clinic. 48.9% (n=22) of these patients had one or more domain scores falling more than one standard deviation below the mean. 35.5% (n=16) of these patients had two or more domain scores below these cutoffs. 20% (n=9) of the rarely evaluated patients had domain composite scores (i.e. the sum of the individual domains) that fell below the standard deviation cutoff. the most common outlying domains in these infrequently seen or new patients were body (10), weight (10), physical (7), health perceptions (7) and the composite domain (9). conclusions: the cfqr is easy to administer and score during routine adult cf clinical visits. as many as 35% of our infrequently evaluated patients had multiple scores that were more than one standard deviation below our clinic mean. we conclude that the cfqr is a useful adjunctive measure to provider assessments and can help focus limited personnel resources. aminoglycoside antibiotics are known to be toxic to the inner ear. however, they continue to have a leading role in the treatment of certain infections and in the treatment of pulmonary exacerbations in patients having cystic fibrosis (cf). while there is ample evidence in the literature of ototoxicity, and there are established protocols for monitoring the effects of toxic agents on hearing, less is known about the prevalence of vestibular toxicity among patients who are exposed, and there are no commonly accepted protocols for monitoring vestibular system function. oscillopsia and unsteadiness when standing and walking are the two most commonly reported and disturbing symptoms associated with bilateral loss of vestibular system function. oscillopsia is the perception that viewed stationary objects or surroundings move coincident with head movement. when it is severe, oscillopsia can prevent an individual from having clear vision with even the slightest head movement. the unsteadiness that accompanies vestibular loss can range from mild to disabling. because of the referral of a number of patients to the vestibular testing center (vtc) at the university of michigan in whom severe bilateral vestibular loss was evident secondary to apparent aminoglycoside toxicity and our concern about quality of life issues, we initiated a quality improvement clinical protocol with our cf patients. vestibular system function and hearing are evaluated at the time of admission for pulmonary exacerbations and coincident with the three month follow-up visit with the pulmonologist. our goal is to understand the incidence of ototoxicity in this patient population, and to determine how best to measure incremental changes over time. the ultimate desired outcome is to investigate the efficacy of protective agents for limiting the toxic impact of these drugs on hearing and vestibular function. to date, we have completed vestibular testing on 30 patients with cf ranging in age from 10-56 years, some of whom were referred prior to the initiation of this monitoring program. of the sample, 4 individuals have completely normal vestibular test results, and 2 of the 4 had no prior aminoglycoside exposure. of the remaining patients, 10 have bilateral vestibular loss ranging from mild to severe, two have evidence of a unilateral vestibular loss, and each of the others has non-lateralizing evidence of vestibular involvement. we have also documented change in the three individuals we have seen for repeat testing. specifically, repeat testing has shown incremental decline in vestibular function with repeated exposure to aminoglycosides. interestingly, only 6 individuals in the group have documented hearing loss, and although most report that they do not and have never had any problems with dizziness or balance, many patients have evidence of oscillopsia or abnormal postural control test results. it is clear from our limited data that it is important to monitor vestibular system function whenever potentially toxic agents are used. while monitoring hearing is also warranted, our data suggest that a monitoring program that includes only hearing is insufficient. background: the correlation between bmi, lung function, and longterm outcomes in cystic fibrosis patients has been well documented. based on this data the cf foundation (cff) has established adult patient care guidelines for bmi of 22 or greater for women and 23 or greater for men. in our cf center implementation of nutrition therapies in cf adult patients has been difficult due to lack of acceptance of proposed interventions. due to the importance of optimal nutrition to attain the cff recommendation for bmi, university of cincinnati adult cf center implemented a weight management quality improvement protocol to improve the nutritional status of patients below the cff recommended bmi. our aim is to improve to the nutritional outcomes in our center through patients' acceptance of our nutrition interventions. strategies: as part of the weight management protocol we developed a nutrition action plan, nutrition algorithm, and weight management education record and learning log. the action plan took a systematic approach to weight management, classifying patients into different risk categories based on bmi. patients were categorized as mild nutritional insufficiency (bmi 20-21), moderate nutritional insufficiency , and severe nutritional insufficiency . each category required a specific action to address weight loss or inability to gain weight. areas of focus for the action plan include; intake assessment through computerized dietary analysis, assessment of pancreatic function, pancreatic enzyme replacement therapy (pert) regimen assessment, 72 hour stool collection for malabsorption, assessment of glycemic control, assessment for gerd, and enteral/parental feeding. the dietitian completed a chart review and nutrition assessment with the patients and classified them into one of the three nutritional risk categories. eleven patients with a bmi below 19 were initially identified. nine (82%) of the eleven patients with a bmi of 19 or less have been approached with the protocol and algorithm. patients completed a three day food journal and oral intake was assessed via computerized dietary analysis. oral enteral supplements were recommended 2-3 times per day. g-tube placement was discussed and an educational material on tube feeding was distributed. the protocol and algorithm guided identification and assessment of contributing factors to weight loss. results: of eighty patients seen in the adult center since the beginning of october, thirty-seven of the patients (46%) have a bmi below 22 and eleven patients (14%) have a bmi below 19. out of the nine patients approached with the protocol 55% (n= 5) completed and returned the three day food journal. of the patients that completed their food journal 60% (n= 3) agreed to a peg placement, one of the patients is still contemplating peg placement while sorting through some psychosocial issues, and the other patient has had a differential diagnosis that might negate the necessity for a peg placement. conclusion: we developed a systematic approach to weight management within our adult cf program. this systematic approach to weight management, which includes patient involvement and specific education, has improved the overall acceptance of nutrition interventions in our adult population. the inpatient care of adults with cystic fibrosis can be a challenging given complicated medical regimens, specialized respiratory care orders, and unique dietary requirements. a large proportion of these patients are admitted to academic centers with rotating house staff that may have never treated an adult with cystic fibrosis. to improve the quality of care of our adult patients we have implemented a standardized electronic order set, a recurring educational program, and a "pocket card" to help the house staff. the adult cystic fibrosis program at vanderbilt cares for over 160 patients, with 7-12 inpatient admissions per month. vanderbilt's inpatient order system is completely electronic. we have devised an order set based on our agreed standard of care for inpatients with pneumonia. the order set guides the house staff through most aspects of inpatient care including culturing, antibiotic choice and dosing, dietary consultation, nutritional recommendations, and respiratory care orders. this order set not only educates the house staff on our standard of care but also provides direct access to each order they may wish to enter. we have reiterated this educational process with a pocket sized handout that guides them through the typical issues in adults with cf as well as provide phone numbers to members of the cf team. these resources are presented in short talk given at the beginning of each month to incoming house staff. a preliminary data analysis suggests that over 90% of all adults with cf are admitted using some component of this order set. an informal survey of the house staff also concludes that the pocket card and lectures have been helpful. all three aspects of this quality improvement have been implemented since december of 2006. we plan to formally test the impact of this intervention when a meaningful number of house staff have been through the service. * background: malnutrition is a common complication of cystic fibrosis (cf). the median bmi percentile in cf patients between the ages of 2 and 20 years is 44.8% but ranges between various centers from 23.6 to 65.9 percent.1 previous research has shown a trend toward improvement of pulmonary disease and a significant improvement in weight, height, and bmi z-scores after nutritional supplementation via a gastrostomy tube2; furthermore, fev1 is positively correlated with bmi percentile.1 higher weight at three years of age predicts better pulmonary function at six years of age.3 the effect of gastrostomy tube feedings on pulmonary exacerbation frequency has not been well defined. hypothesis: the incidence of pulmonary exacerbations in children will decrease after the initiation of supplemental gastrostomy feedings. methods: a retrospective chart review of all pediatric patients seen at the cmh cf center, and who received gastrostomy tubes between 1997 and 2007 was performed. data was evaluated in six month time intervals for two years prior and two years after gastrostomy tube placement. we assessed weight, height and bmi percentiles, fev1 percent predicted, number of courses of exacerbation therapy (intravenous and oral) and microbiologic data. results: we identified 20 patients, 14 females and 6 males, with ages ranging from 11 months to 14 years of age (6.82 years mean) at time of gastrostomy tube insertion. all patients had increases in their weight-forage percentile (mean of all patients of 13.83 percentile pre to 31.23 percentile post, p<0.05), height-for-age percentile (16.78 percentile pre to 27.03 percentile post, p<0.05), and bmi percentiles (24.23 percentile pre to 45.98 percentile post, p<0.05). the mean fev1 percent predicted declined overall from 85.5% to 78.1% (2.2 percent predicted per year) over four years in 10 patients. there was a trend of exacerbation reduction after gastrostomy tube placement from 153 to 121 total courses (p=0.06). this decline in exacerbations after gastrostomy tube placement was most dramatic in those patients growing pseudomonas aeruginosa without other organisms, with a decline from 90 total courses to 60 total courses in 10 patients (p=0.05). discussion: weight, height and bmi percentile improved after initiation of gastrostomy tube feedings. lung function (fev1 % predicted) decreased at the rate predicted in spite of gastrostomy feedings. there is a trend toward decrease in exacerbation frequency after improved nutrition with gastrostomy tube feedings, especially in patients infected with pseudomonas aeruginosa. limitations of this study include the small sample size, relatively short follow-up time, and the retrospective nature of the study which did not allow assessment of adherence. conclusion: improved nutrition through gastrostomy tube placement may decrease the frequency of pulmonary exacerbations. evidence of an association of improved pulmonary function with improved nutritional status has motivated aggressive nutritional intervention. percutaneous endoscopic gastrostomies (pegs) are employed increasingly in cystic fibrosis to optimize nutritional status. we reviewed our center data over the past 15 years to assess changes in our peg practice and effects on nutritional and pulmonary outcomes. we hypothesized that our change in practice to earlier peg intervention improved patient's nutritional and pulmonary outcomes. we conducted a case controlled retrospective study of pediatric cf patients spanning the past 15 years divided into two periods: p1 (peg placed before 1996) and p2 (peg placed 1996 or later) compared with their respective age/sex matched controls. we compared peg vs. control cf patients with respect to age, nutritional and pulmonary status. we compared weight, height and bmi directly preceding peg placement and at approximately one-year post peg. we compared available data for best yearly fev1 and fvc before peg and at 1, 5 and 10-years post-peg. we then compared data between the two time periods. results: peg patients in p2 (n=38; mean age 6.6 + 5.1 yrs.) had lower bmi ( z = -1.2 v. -0.2, p<0.001) pre-peg placement compared to controls. by 1 year post peg, the bmi normalized and equaled controls (z=-0.4 v. -0.1, p=0.1). peg patients' fev1 %ile prior to placement was lower than controls (88 v. 94, p=0.2) as was their fvc %ile (97 v. 103, p=0.2) and remained lower at 1 year post-peg. following peg placement, absolute bmi increased (0.9kg/m2, p=0.009), as did bmi z-score (0.6, p=0.01). we did not see significant changes in pulmonary function tests (pft). peg patients in p1, (n= 12; mean age 10.7 + 2.3 yrs.) also had lower bmi (z= -1.6 v. -0.3, p= 0.0001) pre-peg than controls. at 1 year post-peg, the peg patients still had a significantly lower bmi (z = -0.7 v. -0.2, p=0.08). fev1 was lower in peg patients compared to controls in pre-, 1year, and 5-year post-peg data (p<0.05). variability in pfts and small sample size limited our analysis. pre-peg weight, height, bmi, fev1 and fvc were lower in p1 patients compared to the younger p2 patients. at 1-year post-peg, similar bmi zscores (-0.7, p=0.99) was obtained between the two periods. variability in pfts among the patients within each period prevented valid comparisons, although p2 tended to have better pft values. conclusions: peg placement was effective at improving nutritional status in patients with cystic fibrosis. placement in younger patients with better pre-peg nutritional and pulmonary status allowed more complete nutritional recovery. we speculate that improved height z-scores with apparently stable fev1 is consistent with increased pulmonary growth. demonstration of an attenuated rate of pft decline to correlate with this earlier intervention will require a larger population and longer observation period. a multi-center longitudinal outcome study would help determine optimal timing and criteria for peg intervention. 1 1. pediatric gastroenterology and hepatology, university medical center groningen, groningen, netherlands; 2. biochemistery, erasmus medical center, rotterdam, netherlands background: ursodeoxycholic acid (udca) treatment is frequently applied for cystic fibrosis-related liver disease (cfld). it has been hypothesized that udca is beneficial through its choleretic activity, i.e. its capacity to induce bile flow. the hepatic expression of the cftr protein is restricted to the cholangiocytes lining the bile ducts. cholangiocytic cftr is involved in the generation of ductular bile flow. it is not known to what extent the choleretic activity of bile salt treatment depends on expression of competent cftr protein, and whether or not udca differs in this respect from other bile salts. we evaluated the role of cftr in the acute and chronic choleretic effect of bile salt treatment, through comparative studies in cftr-null, homozygous f508del, and corresponding wild type control (wt) mice. methods: bile flow was determined after gallbladder cannulation. bile flow during the first 30 minutes after acute interruption of the enterohepatic circulation was regarded as basal bile flow. after 30 min, taurocholic acid (tca) or tauroursodeoxycholic acid (tudca) were iv administered in stepwise increasing dosages, up to 1200 nmol/min/100g bw, to cftr-null mice and wt littermates fed a standard chow diet. other cftr-null, homozygous f508del mice and their respective wt littermates were fed either the standard chow, or the same diet supplemented with cholic acid (ca, 0.5wt%) for 3 weeks. finally, cftr-null mice and wt littermates were fed the standard chow or the same diet supplemented with ursodeoxycholic acid (udca, 0.5 wt%) for 3 weeks. : upon a regular chow diet, the basal bile flow was similar in cftr-null and homozygous f508del mice, compared to their respective wt littermates (cftr-null, 6.3±1.8 vs. 6.2±0.1; and, f508del(∆/∆), 5.4±2.8 vs. 8.5±10.8 µl/min.100 g bw, resp.; ns). iv administration of tca or tudca to cftr-null mice and wt littermates increased bile flow to similar extents. dietary ca treatment increased basal bile flow significantly less in cftr-null and homozygous f508del mice than in their respective wt littermates (cftr-null, 13.7±3.0 vs. 17.9±1.7, p<0.05; and, f508del(∆/∆), 20.9±5.5 vs. 26.2±6.2 µl/min.100 g bw, p=0.06; resp.). interestingly, dietary udca treatment increased basal bile flow more profoundly than ca treatment, and to similar levels in cftr-null mice and wt littermates (+~500%, 29.0±5.9 vs. 31.0±4.4 µl/min/100 g bw, resp.; ns). conclusion: upon chronic treatment, udca is highly choleretic in mice, independently of the presence of functional cftr. the independence of functional cftr is udca specific, since the choleretic activity of chronic ca treatment is diminished in cftr-null and homozygous f508del mice. we speculate that this specific, cftr-independent choleretic effect of chronic udca treatment could be therapeutically important for cystic fibrosis. most cystic fibrosis (cf) patients have mild to moderate focal portal tract changes (ductal obstruction and cholangitis) and hepatosteatosis. etiology of severe liver disease (ld)with cirrhosis and portal hypertension which occurs in 5-7% of cf patients is unknown. the c57bl/6j congenic cf mice develop progressive cf-like ld. as a useful therapeutic model, we have shown that dietary addition of docosahexaenoic acid (dha) significantly ameliorates inflammation but has little effect on ductal obstruction (am j physiol gastrointest liver physiol 292: g839-g848, 2007) . we hypothesized that treatment with udca,a hydrophilic bile acid will ameliorate portal tract ductal obstruction and have an additive effect with dha in preventing ld. methods: thirty-day old wild-type and cf littermates are fed peptamen and either olive oil or dha, with and without 0.4% udca (8 groups,10 mice/group). mice are killed following 90 days of treatment and bile salts, serum liver enzymes and serum and tissue lipids analyzed. h&e stained liver tissues are coded and assessed blindly by an expert hepatopathologist (jp) for evidence of hepatosteatosis, inflammation, bile duct obstruction, fibrosis and other signs of liver pathology. an arbitrary scale of 0 to 4 is used, with 0 representing no pathology and 4 being the most severe. we present interim statistical analysis performed for pathology results only for untreated mice (wt, n=7; cf, n=6); treated mice with dha, (wt, n=7; cf, n=6), treated mice with udca (wt, n=7; cf, n=7) and treated mice with udca and dha (wt, n=6; cf, n=6). results: mean pathology scores from groups of mice are pooled to examine the effects of genotype, treatment and diet (table) . at present,the bile,lipid and liver biochemical test data are insufficient to perform statistical analysis. udca-treated mice have significantly increased bile duct obstructed scores compared to the untreated mice which subanalyzed (not shown) reveals increased bile duct obstruction in the wild-type littermates and no effect in cf mice. hepatosteatosis and inflammatory scores in the untreated mice (fed olive oil) are increased compared to the mice treated with dha. summary: we anticipate completing and performing final analysis within six months. however, these preliminary data confirm that dha reduces inflammation in c57bl/6j mice livers and suggest that udca therapy has an adverse effect in wt mice and is not effective in ameliorating liver disease in cf mice. udca with dha therapy does not appear to have an additive effect in ameliorating cfld in this mouse model. supported by axcan pharma inc. background the cf mouse intestine has an innate response associated with small intestinal bacterial overgrowth (sibo). a previous affymetrix genechip analysis of the cf mouse intestine showed altered expression of several eicosanoid metabolic genes. eicosanoids are biologically active arachidonic acid metabolites with a variety of pro-and anti-inflammatory actions as well as effects on mucus production, electrolyte transport, and gastrointestinal motility, all of which are of potential importance to the cf phenotype of the intestine. methods wild type (wt) and cf mouse (cftr tm1unc ) littermates congenic on the c57bl/6j background were fed an elemental liquid diet (peptamen). intestinal rna was isolated for quantitative rt-pcr of expression levels of genes involved in eicosanoid metabolism to confirm and extend the genechip analysis. various eicosanoids were measured in small intestinal lavage fluid using specific enzyme immunoassays. results we confirmed microarray results by qrt-pcr that the following genes were differentially expressed in the cf small intestine relative to wt: cyp4a10 (cytochrome p450 4a10; 20-hete synthesis; 5% of wt); ltb4dh (leukotriene b 4 dehydrogenase; metabolizes prostaglandins and leukotrienes; 22% of wt); cyp2c40 (cytochrome p450 2c40; 16-hete synthesis; 25% of wt); ltc4s (leukotriene c 4 synthase; ltc 4 synthesis; 25% of wt); hpgd (hydroxyprostaglandin dehydrogenase 15; metabolizes prostaglandins; 30% of wt); and pla2g5 (group 5 phospholipase a 2 ; potentiates arachidonic acid generation; 500% of wt). non-significant or less than 2-fold changes in gene expression were measured for: cyclooxygenase (cox) genes, pge synthase, pgi synthase, and 5-and 15-lipoxygenases. as measured by their stable metabolic products, pge 2 and pgf 2α were significantly increased in the cf mouse intestine (600% and 1190% of wt, respectively). the following eicosanoids were significantly decreased in the cf mice: 12-hete (20% of wt), 15-hete (19% of wt) and 20-hete (4% of wt). there were not significant changes in the levels of pgi 2 , pgd 2 , ltb 4 , cys-lts, or lxa 4 . the changed levels of eicosanoids are generally consistent with the differential gene expression. conclusions there are significant changes in expression of eicosanoid metabolic genes and certain eicosanoids in the cf mouse small intestine. the increase in pla2g5 and decrease in cyp expression levels are expected to make more arachidonic acid available for eicosanoids except hetes. decreases in degradative genes (hpgd, ltb4dh) will prolong availability of prostaglandins. altered levels of eicosanoids are expected to play important roles in the pathophysiology of the cf intestine. elevated pge 2 may contribute to increased mucus secretion which is characteristic of cf. pgf 2α and pge 2 regulate intestinal motility and small intestinal transit is slower in cf patients and in cf mice. the hetes are mostly known for their effects on vascular tone and electrolyte transport in the kidney but are less-well characterized in the intestine. their dramatic decrease in the cf intestine may affect blood flow, electrolyte transport, intestinal motility, or other gi functions. the specific effects of the altered eicosanoid metabolism in cf remain to be explored. supported by cff grant delisl05g0. for the scandinavian study consortium. background: progressive pulmonary disease, correlating with igg levels, is a major cause of morbidity and mortality in cf patients. recently, immunosuppressive effects of vitamin d and a potential role of vitamin d deficiency in the pathophysiology of autoimmune diseases have been described. we investigated whether vitamin d deficiency could contribute to the chronic proinflammatory state characterizing cf and the higher occurrence of immune-hyperreactivity-related conditions in cf patients. methods: multiple linear regression analysis, taking the confounding factor of age into account, was used to evaluate the relationship between cross-sectionally determined vitamin d variables and: lung function parameters, inflammatory parameters and immune hyperreactivity markers in 898 patients followed at the cf centers in sweden, norway and denmark. vitamin d intake was based on a 7-day precoded dietary food record, calculated in the national food databases. findings: in the population of all patients included in the study (n=898), blood vitamin d positively correlated with fev1 (p<0,01) as well as with fvc (p<0,01). negative correlation was found between blood vitamin d and igg (p<0,001), supplemented vitamin d per kg bw and igg (p<0,001), total vitamin d intake per kg bw and igg (p<0,05). additionally, lower blood vitamin d levels were related to both presence of pathological ogtt (p<0,05) and occurrence of cfrdm (p<0,05). correspondingly, food vitamin d (p<0,05), food vitamin d per kg bw (p<0,05), supplemented vitamin d (p<0,05), supplemented vitamin d per kg bw (p<0,001), total vitamin d intake (p<0,01) and total vitamin d intake per kg bw (p<0,01) all correlated negatively with hba1c values. most of the studied vitamin d variables were lower in patients with endomysial antibodies positivity (n=14) and higher in patients with abpa (n=4) than in the rest of cf population studied (ns). interestingly, in children (n=442) blood vitamin d correlated negatively with transglutaminase levels (p<0,05) and there was also a significant negative correlation between hba1c and vitamin d intake. surprisingly, in adult cf patients (n=456) iga positively correlated with total vitamin d intake (p<0.05) and total vitamin d intake per kg bw (p<0.05). in the population of stockholm cf-center patients (n=165), where it was possible to allow for the influence of both age and compliance, negative correlation between blood vitamin d and igg (p<0.01) was found. after allowing for age, compliance showed significant correlation only with igg (p<0.001) and esr (p<0.05), and with none of the rest of the studied variables. conclusions: vitamin d deficiency might contribute to the continuous shift towards inflammation as well as to the higher prevalence of dm and celiac disease in cf patients. new recommendations for vitamin d supplementation and monitoring of blood vitamin d levels are needed, in order to improve immunological balance and decrease the need for anti-inflammatory therapy in cf patients. supported by swedish heart lung foundation, stiftelsen frimurare-barnhuset i stockholm, norwegian and swedish cystic fibrosis associations, and by an unrestricted grant from solvay pharma. von drygalski, a.; biller, j.a. medical college of wisconsin, milwaukee, wi, usa anemia is associated with increased morbidity and mortality in other chronic diseases, but little is known about anemia in cf. recognition and correction of anemia in cf might lead to better outcomes. chronic inflammation, iron deficiency and malabsorption may be important mechanisms contributing to the anemia in cf patients. we hypothesized that anemia might be correlated with poor lung function. methods: clinical charts and portcf.org visit logs of 218 patients of all ages (1 m to 61 yrs) with cf were reviewed for as many years as charts permitted (range 1-7 yrs). the following data were extracted: cbc, iron studies, pulmonary function tests (pfts), vitamins a, d, and e levels, creatinine, pertinent medical history, and current medications. anemia was defined by age-and gender-specific world health organization (who) criteria. patients were considered anemic if low hemoglobin(hb) was present on two separate occasions at least two months apart, or average annual hb met who criteria. pfts in 176 patients (all >6 yrs) included forced expiratory volume (fev1) and forced vital capacity (fvc) as percent predicted of normal and were considered a reflection of patient performance. the most representative annual pft from at least 4 annual tests was chosen for analysis since acute infection adversely influences daily performance. results: 61 of 218 patients were anemic (prevalence 28%). prevalence of anemia increased with age from 11.5% in patients < age 18 to 58% in patients > age 40 and was significantly higher in patients with vitamin deficiencies. 90% vitamin-deficient patients vs. 59.5% non-vitamin deficient patients were anemic (p=0.02). mean hb was 10.1 mg/dl (range 6.6-12.9). roughly two thirds of patients had moderate and severe anemia (hb <11 mg/dl) with no gender difference in regard to prevalence or severity identified. complete iron studies were available in 16/48 patients. 7 were identified as iron deficiency anemia (ida), 4 as anemia of chronic illness(aci), and 3 as combined; 2 others had renal failure. in 32 patients, iron studies were incomplete, but renal failure, hemoptysis, hematochezia and solid organ transplants were contributing factors in half. pfts obtained in 176 patients ≥ 6 yrs were compared in anemic and non-anemic patients. mean fev1 and fvc at all ages were statistically significantly poorer in the anemic patients (p <0.005). complete iron studies were also available in 9 non-anemic patients with impaired lung function. 8/9 patients had ferritins <35 ng/ml suggesting relative iron deficiency. conclusions: the prevalence of anemia in cf patients is high and increases with age. in patients for whom iron studies were available, ida was the prominent underlying mechanism, followed by aci or a combination of both. in addition, a group of non-anemic patients with poor lung function had relative iron deficiency. the strikingly significant association between anemia and poor pulmonary function as well as the higher prevalence of anemia in vitamin deficient patients uncover anemia as significant co-morbidity in cf. identification of iron deficiency in anemic as well as in non-anemic patients with poor lung function could be important since iron supplementation might allow hb levels to increase as an appropriate response to hypoxia. oral administration of dha to a cf mouse model corrected the lipid imbalance and reversed certain pathological manifestations in tissues affected by cf. the aim of this study was to investigate the metabolism of la through the n-6 pathway, and to determine the effect of dha supplementation on la metabolism in cultured cells with a cf phenotype. methods: sense (wt) and antisense (cf) cftr 16hbe cells were cultured in mem containing 10% horse serum. cells were supplemented with various concentrations of la (ranging from 25 to 200 µm) and dha (10, 20, and 40 µm) for one week. cellular lipids were extracted from confluent monolayers, and fatty acids were methylated and analyzed by gc-ms. metabolism through the n-6 pathway was studied by incubating the cells with 4.6 µm [ 14 c] la for 4 hours, and quantitating its downstream metabolites by hplc. results: the levels of la and dha were significantly decreased in cf cells compared to wt cells (la in wt: 10.8±0.2, cf: 8.2±0.1%, p<0.01; dha in wt: 2.0±0.2, cf: 0.6±0.1%, p<0.01, mean ± sem for each). la supplementation resulted in an increase in la and aa levels in both wt and cf cells. at most la concentrations tested, la levels were significantly lower and aa levels were significantly higher in cf cells, indicating an increased production of aa from la in cf cells. the study of [ 14 c] la metabolites showed an increased production of multiple downstream fatty acids including 18:3n-6 (3.1 fold), aa (4.3 fold), and 22:5n-6 (2.7 fold) in cf cells compared to wt cells, further substantiating an increased metabolism through the n-6 pathway. supplementing 16 hbe cells with dha for 1 week resulted in a significant increase in la levels and a corresponding decrease in aa levels in wt and cf cells. when supplemental dha was added in combination with la, dha inhibited the la-derived increase in aa levels in cf cells and normalized them to the wt values. the formation of [ 14 c] la metabolites was inhibited after 24-hour supplementation with dha. this inhibitory effect of dha was greatest on aa production, and it was more prominent in cf than in wt cells (wt: 3.7 fold, cf: 12.4 fold decrease in aa production). conclusions: decreased la levels in cf are at least in part related to increased la metabolism through the n-6 pathway. the fact that dha supplementation decreased the flux from la to fatty acid metabolites in the n-6 pathway, especially to aa, may partially explain dha's mechanism of therapeutic benefit. these data would also suggest that dha combined with la in the diet may normalize fatty acid metabolism in cf patients. background: decreased levels of linoleic acid (la) and docosahexaenoic acid (dha) have been found in the blood and tissues of cf patients. studies on cultured cf cells and cftr-/-mice have indicated that decreased la levels in cf might be related to its increased metabolism through the n-6 pathway. other potential mechanisms of the observed decreased la and dha levels in cf could be related to altered cellular uptake of these two essential fatty acids. the goal of this study was to investigate the uptake of la and dha into cultured cf cells, and their distribution among the major lipid classes. methods: sense (wt) and antisense (cf) cftr 16hbe cells were cultured in mem containing 10% horse serum. the uptake of la and dha into the cells was studied by incubating the cells with 0.45 µm [ 14 c] la or [ 14 14c] dha for 1 hour and 4 hours, followed by measurement of radioactivity in the supernatant and cell lysate. lipid fractions from the cells (total phospholipids, triglycerides, cholesteryl esters, and free fatty acids) were separated by thin layer chromatography (tlc), and the fatty acid composition was determined by gc-ms after methylation. incorporation of la and dha into cellular lipid fractions was determined by incubating the cells with 4.5 µm [ 14 c] la or 9.1 µm [ 14 c] dha for 1 hour and 4 hours, followed by tlc separation of lipids and measurement of radioactivity associated with the various fractions. (all data are mean±sem). results: the levels of la and dha were significantly decreased in cf cells compared to wt cells (la in wt: 10.8±0.2, cf: 8.2±0.1%, p<0.01; dha in wt: 2.0±0.2, cf: 0.6±0.1%, p<0.01). fatty acid composition analysis of lipid fractions indicated that la levels were significantly decreased in the phospholipid and triglyceride fractions of cf cells (80.9% and 42.3% of the wt values, respectively), and dha levels were significantly decreased in the phospholipid fraction of cf cells (21.2% of the wt values). the uptake of la was significantly higher into cf cells compared to wt cells at 1 hour (wt: 14.5±0.1, cf: 35.5±0.9 dpm/µg protein, p<0.01) and 4 hours (wt: 34.7±2.9, cf: 100.8±5.0 dpm/µg protein, p<0.01). the uptake of dha was also significantly higher in cf cells compared to wt cells at 1 hour (wt: 46.3±8.4, cf: 131.2±8.7 dpm/µg protein, p<0.01) and 4 hours (wt: 140.7±12.9, cf: 276.8±12.0 dpm/µg protein, p<0.01). incorporation of la and dha was increased in the phospholipid, cholesteryl ester, and free fatty acid fractions of cf cells at 4 hours. dha, but not la, incorporation was significantly increased in the triglyceride fraction of cf cells. conclusions: low la and dha levels in cf cells are associated with a decrease in cellular lipid fractions, the largest of which is in total phospholipids. there is an increased uptake of la and dha into cf cells, and into most lipid fractions of these cells. the decreased levels of la and dha in cf cells, despite their increased cellular uptake and esterification in lipid classes, indicate that there is increased mobilization of these fatty acids in cultured cf cells. perez, a.; issler, a.; davis, p.b. pediatrics, cwru, cleveland, oh, usa even though lung disease continues to be the primary cause of death in cystic fibrosis (cf), the considerable increase in life expectancy of the cf patient over the last decades, has made management of the diverse gastrointestinal and nutritional complications in the adult cf increasingly important. malnutrition and fatty acid deficiency has been a hallmark of cf and its impact in long-term lung health has been established. data obtained by us using affymetrix mg-u74av2 arrays from ileum and liver of pristine 8-week-old male mice bearing the y122x cf mutation and their normal littermates, indicates that there is a profound disturbance in genes associated with lipid metabolism, many of which are regulated by the peroxisome proliferator-activated receptor-gamma (pparγ), a ligand-regulated transcription factor that regulates lipid metabolism and possess anti-inflammatory properties. we hypothesize that lack of cftr function results in altered expression of pparγ regulated genes in the gut and liver, leading to the fatty acid metabolism abnormalities observed in cf. pparγ mrna and protein levels were measured in ileum, colon, liver, and fat of 8 week old female mice bearing the cf mutation s489x and its wt littermates (c57bl/6j background), and fabpcftr mice (gut corrected, mixed background) after the administration of ethanol or pioglitazone (a pparγ agonist, 30 mg/kg) by gavage every 24h for 2 days, sacrificed 24h after last dose. highest pparγ mrna levels were found in fat, followed by colon, liver, and lowest in ileum. at basal state, mrna levels were higher only in fabp ileum vs. wt and fabp colon vs. cf. after treatment, higher expression was seen in cf and fabp ileums and cf liver vs. controls and wt treated mice. however, there were striking differences at the protein level, especially in the colon. at basal state, pparγ was localized, mainly at the surface of the epithelia in wt, in crypts in cf, and mainly in crypts but extending towards the surface in fabp mice. upon treatment, pparγ location in wt colon moved almost exclusively to the crypts, and in the cf and fabp mice, although expression remained mainly in crypts, pparγ could now be seen at the surface. changes in pparγ protein were also seen in ileum and especially liver, between wt and cf, at basal and upon treatment, but changes were subtle and will require more sensitive methods to further examine them. we also examined mrna and protein levels for several genes involved in fat processing that could be related directly or indirectly to pparγ. mrna and protein levels for aqp1 and cubilin, and protein levels for megalin, apoa-1, and apob-48 were reduced in the ileum of cf mice compared to wt. our data indicates a pparγ dysfunction in the gastrointestinal system of cf mice, which could be responsible for the gastrointestinal manifestations observed in cf. understanding the association between pparγ, cftr, and fat processing may help us to develop new therapies directed towards improving the nutritional status of cf patients that, in the long run, will influence their lung health. cf is characterized by low linoleic acid (la) and docosahexaenoic acid (dha) levels. although this could be due to a primary abnormality in fatty acid biosynthesis, this could also be explained by alterations in phosphatidylcholine (pc) formation. pc are formed by two pathways. the most utilized is the de novo conversion of choline to pc via the cdp-choline pathway, which preferentially forms pc containing saturated and monounsaturated fatty acids. in the other pathway phosphatidylethanolamine (pe) is converted to pc through three methylation steps utilizing the enzyme phosphatidylethanolamine n-methyltransferase (pemt). this pathway preferentially forms pc containing polyunsaturated fatty acids. thus decreased pemt activity would be predicted to result in decreased dha and la levels. this is supported by results seen in pemt knockout mice (watkins et al, j nutrition, 2003) showing decreased dha and la levels, and variable to increased levels of arachidonic acid (aa). furthermore, innis et al have shown that cf patients have decreased choline levels making them more dependent on the pemt pathway. we hypothesized that alterations in fatty acid levels in cf may be due at least in part to decreased pemt activity. methods: cftr -/mice (cf) and wt littermates were fed with either a diet containing choline (control) or, in order to mimic choline status in humans, an otherwise matched choline deficient (cd) liquid diet for 7 days. choline levels were measured using a kit. livers were perfused and microsomes prepared. to measure pemt activity, microsomes were incubated with radioactive s-adenosylmethionine in the presence of dimethyl pe. the pc product was extracted and radioactivity measured. fatty acids from liver and pancreas were analyzed by gc/ms. results: there was no difference in choline levels in serum or liver comparing wt and cf mice. there was no difference in pemt activity in livers from cf mice compared to wt on control diet. cf mice on cd diet had decreased pemt activity in the liver compared to wt mice on cd diet (wt:26145±1893; cf:16515±1240 cpm/mg protein/min; p<0.001 conclusion: induction of choline deficiency in cf mice to mimic the situation in cf patients, leads to decreased pemt activity. this resulted in low la levels compared to wt mice in both the liver and pancreas and indicates that the fatty acid abnormality in cf is at least in part due to defective pc metabolism through the pemt pathway. (4995). urolithiasis appears to be more common in adult cystic fibrosis (cf) patients than in the general population. a variety of urinary findings have been observed which could account for this, including low volumes, raised oxalate and low citrate, but not all the reports are consistent. most cf patients have pancreatic insufficiency and a plausible explanation for an elevated oxalate excretion in these patients is malabsorbed fat in the intestinal tract sequestering calcium and permitting more oxalate to be absorbed from their food. if this were the mechanism for the elevated stone risk in cf patients then we would expect to observe differences in stone incidence and oxalate excretion between cf patients with exocrine pancreatic sufficiency (ps) and pancreatic insufficiency (pi). methods diagnosis of cf is by clinical and genetic criteria and ps or pi status is based on faecal elastase measurement. 39 patients and 14 healthy volunteers (hv) provided 24h urines for analysis of volume, ph, ca, mg, na, k, po 4 , oxalate, citrate, creatinine, urea. supersaturations were calculated using equil2. there is a history of renal stone disease in 20 out of 307 cf patients attending our unit. 286 patients have pi and include 19 of the stone formers. the remaining stone former is amongst the 21 ps patients. no significant differences in urine chemistry or derived supersaturations were found between the cf patients without a history of stones that were ps (n=10) or pi (n=11). nor were any such differences found when stone forming cf patients were included (ps, n=11; pi, n=18) or when the healthy volunteers were included in the ps group (ps, n=25; pi, n=18). amongst the cf patients, only citrate and creatinine excretions were significantly different between stone formers (n=8) and non stone formers (n=21) (medians (mmol/24h), citrate, 1.4 vs. 2.3, p=0.019; creatinine, 9.7 vs. 12.4, p=0.032) . the difference in creatinine excretion can be accounted for by the difference in body mass of these two groups (median (kg) 54.8 vs. 62.8, p=0.045) compared to the hv, the cf patients with a history of stones had significantly lower citrate excretion and higher calcium oxalate supersaturation (medians, citrate (mmol/24h), 2.9 vs. 1.4, p=0.002; 4.4 vs. 8.1, p=0.037) conclusions there is no evidence to suggest that the prevalence of stones is different in the ps and pi groups nor could we detect any difference in excretion of oxalate or any other urine variable related to pancreatic sufficiency. comparing those with and without stones, the only consistent finding was a lower citrate excretion by stone formers. between the hv and stone formers this was also associated with a significantly reduced calcium oxalate supersaturation. our evidence does not support the hypothesis that fat malabsorption by the majority of cf patients contributes to an elevated risk of renal stone disease. we undertook assessment of the use of a hypopharyngeal sensor for detection of laryngopharyngeal reflux in infants and children. gastric reflux in the airway, or supraesophageal reflux, commonly takes a gaseous form that cannot easily be measured using conventional technology. the miniaturized ph sensor at the tip of the dx-ph probe is the only sensor able to measure ph in the airway. fifteen infants and children referred to the pediatric pulmonary center for assessment of chronic cough, hoarse voice, or uncontrolled asthma unresponsive to usual medical intervention underwent 6-48 hour to placement of the dx-ph probe in supplementation of radiologic assessment of gastresophageal reflux. the probe was placed transnasally and visualzed in the hypopharynx with the red light at the tip. drops in ph <5 were correlated with food and symptom diaries. positive results were found in 10/15 patients, leading to surgical intervention in three patients. one patient with cystic fibrosis age 3 months with failure to thrive underwent study for symptoms of cough. upper gi demonstrated no gross gastroesophageal reflux or anatomic obstruction.gastric emptying scan was mildly prolonged. outpatient supraesophageal ph probe demonstrated drops to ph<4 associated with respiratory symptoms. patient underwent nissen and g tube placement with resolution at six week follow-up.the patient returned at age six months with onset cough and underwent repeat study. there were no drops in ph and the cough responded to oral steroid burst. the device was tolerated in 13/15 patients with early removal in one young adult with endstage neuromuscular disease and in an infant with "colic" due to a hysterical parental reaction.as a result of our experience we have incorporated the use of supraesophageal ph monitoring into our practice, with particular attention to infants with persistent asthma and children with histories suggestive of gastric asthma. we have now incorporated it into our vivometrics lifeshirt, an ambulatory physiologic monitoring device which will allow us to examine supraesophageal ph and its relationship to sleep apnea, cough, tachycardia, tachypnea, and hypoxemia. aim: monitoring and adjusting dose requirements of pancreatic enzyme replacement therapy (pert) are an integral part of the dietetic assessment of patients with cf. we investigated characteristics of enzyme use in our adult clinic population and determine the extent to which inappropriate enzyme use contributed to poor nutritional and clinical state. method: information was collected using an annonymous self-administered questionnaire developed to measure patient practice, knowledge and beliefs on pert. exclusion criteria included pancreatic sufficiency, <1500 units lipase/kg/d, and fev1 <30%. results: 49 out of potential clinic population of 72 patients completed the questionnaire (16-54y, 55% male, fev1 31-125%). 67% of participants reported to never miss enzymes with meals; this was considerably lower for snacks (35%). those patients who omit enzymes with meals also missed enzymes with snacks (r2=30%, p<0.001). a more appropriate use of pert was observed in patients with lower as opposed to higher bmi. despite intensive dietetic input 29% of patients missed pert with foods containing fat and 20% took pert inappropriately with food and drink that did not contain fat. the results identified 5 potentially better practices for measuring pert behaviour and knowledge: 1) taking pert with all meals, 2) taking pert with majority of snacks 3) swallowing capsules intact rather than splitting them open, 4) carrying enzymes around with them and 5) the ability to titrate pert in accordance to the fat content of food. patients were scored on the basis of the above criteria to differentiate between prudent enzyme users and those who are compromising therapy. in conjunction with their nutritional status score (bmi and gastrointestinal symptoms) risk for intervention can be assessed. discussion:underweight patients have more optimal enzyme use, suggesting greater dietetic involvement in these patients. schall et al 2006 also found this to be the case in children. the findings emphasised the need for targeted and effective input in patients where problems are less obvious. the questionnaire has been a useful research tool, and has been adapted as a screening tool for dietitians to gain a subjective perspective of patient's enzyme management and identify patients who need support. the combination of patient's pert usage and their nutritional status could help capture and identify risk objectively and quickly and allows resources to be allocated most effectively. schall .2), nutritional failure, measured as bmi<20, (p>0.99), or lung transplant status (p>0.3) between patients with and without colonic wall redundancy. there was no significant difference in cftr function, per sweat chloride values, between groups (p>0.9). conclusion: we report a previously undescribed colonic wall abnormality seen on ct in patients with cystic fibrosis: proximal colonic wall redundancy was found in 39% of adults with cystic fibrosis, but not in children, appeared to be chronic, and was associated with non-∆f508 cftr gene mutations, particularly the g542x mutation. recognition of the ct appearances will prevent erroneous diagnosis of acute colonic disease in this patient population, and stimulate investigation of a biologic basis for this finding as a recognizable feature of adult cystic fibrosis. intro: infants and children with cystic fibrosis, both pancreatic sufficient and insufficient, are at risk for developing hyponatremia due to an excess loss of salt though skin during sweating. these children should receive daily supplemental sodium. children without cf require 2-4 meq/kg of sodium daily; and one could argue children with cf need more. a historical recommendation is 1/8 tsp table salt daily for infants; this contains 11 to 13meq of sodium based on the density of the table salt. parents may measure the salt inaccurately, and it may be difficult logistically to distribute the salt throughout the day. if supplementation is given as sodium chloride solutions distributed through pharmacies, as suggested in a recent consensus conference, co-pays or full payments may be required. our aim is to provide a simple, inexpensive, and more accessible method for accurately giving sodium supplementation. do pre-packaged salt packets contain a precise amount of sodium and thus could be recommended to fulfill the daily requirement of sodium in patients with cf? method: pre-packaged table salt packets were collected with permission from four national fast food restaurants and one commercial salt producer. contents (sodium chloride) of ten packets from each source were weighed individually. measurements were made using a scientific scale with the ability to measure up to 1/10,000 of a gram. weights of sodium chloride from individual packets were converted to meq sodium. means, ranges and standard deviations were calculated for the data using microsoft office excel 2003™ program. see table 1 conclusions: there is some variability in the content of pre-packaged salt packets, both between and within brands, though some brands have less variance than others. measurements showed that packages contained mean values of approximately 11 to 13 meq of sodium. there is a range in the sodium dose recommendations, and spot urine sodium can be used to determine if patients are sodium depleted. pre-packaged salt packets lack the precision of pharmacological dosing but are inexpensive, practical, and supply a reasonably predictable dose of sodium. background: ten to forty percent of people with cf have vitamin d deficiency secondary to pancreatic insufficiency. obtaining optimal vitamin d levels (25-ohd) has been a challenge. for vitamin d deficient individuals, current supplementation strategies (50,000 iu weekly for 2 months) have been mostly unsuccessful. objectives: to identify individuals at risk for vitamin d deficiency and to evaluate the efficacy of a 2 week repletion course of high dose ergocalciferol in children and young adults with cf. methods: as part of a quality improvement initiative, a prospective cohort study was performed from january to april 2007. phase i included querying our cf practice database to identify patients who were due for annual routine blood tests or who had recently documented levels of 25-ohd less than 30 ng/ml. in phase ii, 50,000 iu of daily ergocalciferol was prescribed for a 14 day period as part of routine inpatient or outpatient care. during phase iii, a post treatment 25-ohd level was obtained. baseline subject characteristics were obtained at entry and included age, gender, pubertal status, pancreatic status, and fev 1 %. post 25-ohd levels were classified as sub therapeutic (<30 ng/ml), therapeutic (30-50), high therapeutic (50-100), or potentially toxic (>100). a paired t test was performed to evaluate pre and post intervention differences. the impact of age, gender, and lung function on the response to vitamin d supplementation were also analyzed. all values are expressed as mean ± standard deviation. results: eighteen individuals with cf participated in the study. the mean age was 17 years with a range of 6 to 25 years. 66.7% were male, and 100% were pancreatic insufficient requiring pancreatic enzyme replacement. fev 1 % was 64.9 ± 26.4%. all 18 participants had 25-ohd levels less than 30 ng/dl pre-treatment. 25-ohd levels increased from 20.5 ± 6.2 to 57.8 ± 21.1 (p<0.001). 17 of the 18 participants (94.4%) became therapeutic over the two week interval. an increase of 37.3 ± 22.0 (ng/ml) or 214% was seen in the two week period. no correlation was seen on the extent of increase in 25-ohd and baseline lung function. pre and peri pubertal individuals had a greater increase in 25-ohd levels than post pubertal individuals (55.5 ± 21.7 vs 28.3 ± 16.3, p<0.05) although the magnitude of change did not reach significance. the degree of response also appeared related to age (r=-0.47). no impact of gender was seen. seven individuals achieved normal therapeutic values while 10 were in the high therapeutic range. no participants had toxic levels. conclusions: the results of this study demonstrate that high dosing of ergocalciferol over a 14 day period is an effective strategy in achieving therapeutic levels of vitamin d. it is unclear whether a pubertal effect on the degree of response exists or if this response is merely age related. further research is needed to evaluate whether this strategy is able to maintain therapeutic levels after completion of the intervention. additional studies that monitor compliance and evaluate responders and non-responders are needed. foundation consensus committee established guidelines for optimizing bone health in individuals with cf, including screening for vitamin d insufficiency and a treatment protocol for achieving and maintaining normal serum vitamin d concentrations. however, since the guidelines were published, there has been additional research assessing various vitamin d treatment regimens and their effect on serum vitamin d concentrations. in addition, vitamin d assay variability has recently been well documented. the objective of this study was to evaluate variability of vitamin d surveillance methods as well as treatment protocols used to treat low vitamin d concentrations at cf centers. methods: a survey was created via survey monkey, and was sent to the cf nutrition listserv, which includes over 200 cf healthcare providers. spss version 13.0 was used for statistical analysis. results: fifty-nine listserv members responded to the survey. the majority (72%) of respondents measured 25 oh vitamin d concentrations, and 75% considered patients to be vitamin d deficient when serum concentrations were below 30 ng/ml. however, 27% were monitoring only 1, 25 oh vitamin d or a combination of both concentrations. there was considerable variability in vitamin d supplementation protocols for treating low 25 oh vitamin d concentrations. only 39% were following the consensus guidelines of 50,000 units of ergocalciferol once/week for ages 5 through adults. one third indicated that they followed consensus guidelines for patients less than age 5; however nearly half of the remaining two thirds were not sure what is prescribed or have not observed low vitamin d concentrations in this age group. fifty-two percent of respondents were aware of vitamin d assay variability. despite this knowledge, the majority were unaware of the type of vitamin d assay used to measure serum vitamin d concentrations at their institution. conclusions: there is considerable variability in the measurement and treatment of vitamin d concentrations across cf centers despite guidelines provided via a cff consensus report. the consensus guidelines for measuring vitamin d concentrations and treatment of vitamin d insufficiency should be revisited based on recent studies and expert opinion. children with cystic fibrosis (cf) who suffer from sub-optimal growth are offered overnight enteral feeding via gastrostomy tube. this study aimed to assess the nutritional impact of overnight feeding over a 2 year period using a retrospective study design. data, including height, weight, fev1, pancreatic function were collated for all patients with cf who had a gastrostomy tube placed in the past 4 years. data were collected at baseline and then every 6 months for 2 years post-gastrostomy insertion. data for height, weight and body mass index (bmi) were converted into z scores using centre for disease control 2000 reference ranges and the lms method (1, 2) . a total of 21 patients (7.7% of the clinic population) were identified as having a gastrostomy tube. two were no longer receiving feeds and there were incomplete data for 6 subjects. on placement of the tubes, there were substantial deficits in nutritional status with mean z scores for height -0.9 (sd 1.02), weight -1.43 (sd 0.71) and bmi -1.1 (sd 0.58), with compromised lung function (mean fev1 69%, range 44-86). using a repeated measures anova with fev1 as a co-variate, there were no significant differences in weight, height or bmi from baseline to 2 years (at 2 years post placement: mean z scores for height -1.1 (sd 0.59), weight -1.36 (sd 0.56) and bmi -0.86 (sd 0.75). fev1 remained stable over time. these results indicate that gastrostomy feeding can potentially halt the decline in nutritional status that is a feature of cf. however, patient expectations that over night enteral feeding will lead to an increase in nutritional status need to be sensitively managed. background : cystic fibrosis (cf) is the most common life threatening autosomal recessive disorder in caucasians.following a landmark paper by corrie et al a high fat,high calorie diet has promoted a normal growth pattern.improved nutritional status together with prevention and early treatment of respiratory infections has contributed to improved survival. objective: the aim of this study was to assess the relationship between lean body mass and disease severity in children with cf. methods: the nutritional status and body composition of 108 children with cf was measured using a harpenden stadiometer, calibrated electronic scales and a ge lunar prodigy densitometer. the following indices were calculated; body mass index (bmi), fat mass (expressed as logarithm % total body fat (ln%tbf) and fat free mass (ffm). body composition data were expressed as z-scores using dutch reference values. correlations were performed between indices of nutrition (bmi, height, ffm, tbf) z scores and markers of disease severity (percent predicted fev 1 and shwachman kulczycki (sk) scores).the nutritional status of these children was also compared with 154 healthy controls. statistical analyses were performed using microsoft excel 2000. results: there was a weak but significant correlation between sk scores and height z score and ffm (r =0.41, p<0.0001 & r =0.54, p= <0.0001 respectively). there was also a weak but significant correlation between % predicted fev 1 and bmi and ffm (r=0.39, p <0.0001, r= 0.49, p<0.0001). height and weight z scores were significantly lower in children with cf (-0.42 and -0.59 respectively) than in control subjects (0.25 and 0.41 respectively) with p <0.0001 in both groups. conclusion: this study demonstrates that there is an association between indices of nutrition and disease severity in children with cystic fibrosis. muscle mass (ffm), assessed by dual energy x-ray absorptiometry (dxa) correlates with lung function and with sk scores. the strongest correlation between markers of disease severity (sk scores and % predicted fev 1 ) was with ffm. ffm may be expected to be associated with respiratory function tests.however,sk scores are a more general assessment of well being and less obviously directly related to muscle mass. ffm and growth may reflect long term nutrition and health in contrast to bmi and ln%tbf which may reflect acute deterioration or short term nutritional intervention. therefore ffm may prove to be a useful tool to assess quality of nutrition and may be predictive of respiratory and general decline. body composition assessment has an important role in chronic conditions such as cystic fibrosis, in order to identify nutritional depletion and evaluate nutritional interventions. it is important that reliable and accurate methods are used. the usefulness of non-invasive methods, such as skinfold thickness measurements (sft) to measure change in body composition has not been evaluated in cf. this study aimed to compare changes in fat-free mass (ffm) and fat mass measured using sft and dual-energy x-ray absorptiometry (dxa) in adults with cf. methods: 57 adults with cf (60% male, 88% pancreatic insufficient, mean age at baseline 30.3 (sd 7.9) years, mean fev 1 at baseline 63.2 (sd 21.3) % predicted) were studied. they underwent body composition assessment using dxa and sft, at baseline and a mean of 3.6 (sd 0.4) years later. durnin and womersley equations were used to estimate ffm and fat mass from sft. estimates of change in ffm and fat mass obtained using sft were compared with dxa using paired t-tests, univariate analysis and bland and altman analysis. results: at baseline, mean ffm was 49.5 (sd 9.0) kg; mean fat mass was 10.8 (sd 5.8) kg and mean bmi was 21.2 (sd 2.4) kg/m 2 . mean ffm, fat mass and bmi were not significantly different at follow-up, indicating no change in nutritional status for the population overall. individual change in ffm ranged from -4.3 to +5.9 kg, while change in fat mass ranged from -4.2 to +5.8kg. the table shows mean change (∆) in ffm and fat mass by each method, correlations (r 2 ) between sft and dxa, and the 95% limits of agreements (loa) between sft and dxa for change in ffm and fat mass. mean change in ffm and fat mass estimated using sft did not differ significantly from dxa. mean bias between the methods was small, and overall correlations between sft and dxa were strong for changes in both ffm, suggesting good agreement for the overall population. however the 95% loa between the two methods were wide for change in both ffm and fat mass. this suggests that sft will not accurately predict changes in body composition in all cf patients. conclusion: sft measurements do not accurately estimate change in ffm or fat mass over time in individual adult cf patients compared with dxa. body composition changes measured using sft tended to more closely reflect changes detected by dxa in male patients compared with females. caution should be exercised when interpreting the results of serial measurements of body composition using sft in individuals with cf. analysed were significantly correlated with age-cml or srage levels (age, gender, bmi, presence of cf-related diabetes mellitus (cfrd) or hba1c level). conclusions: serum levels of ages are elevated in adults with cf. elevation is not restricted to those with cfrd. the levels of srage reflect ability to respond to this pathway and are associated with poorer lung function. modification of the diet in cf may reduce this mediator and may have the potential to modify lung and renal injury. mouse models of cf generated by expression of improperly processed cftr (∆f508) or lacking cftr expression (ko) demonstrate selective induction of sult1e1 in the liver (falany et al., biochem. j. 364:115, 2002) . sult1e1 is responsible for the sulfation and inactivation of β-estradiol (e2) at physiological concentrations. the increase in sult1e1 expression is specific to the hepatocyte, whereas cftr is selectively expressed in cholangiocytes. the induction of sult1e1 activity is associated with changes in levels of e2regulated proteins in cftr (-/-) liver (li and falany, j cystic fibrosis 6:23, 2006) . increased sult1e1 activity is correlated with low body weight and decreased igf-1 message levels in livers of cftr(-/-). the mechanism for the induction of sult1e1 in hepatocytes by cholanigiocytes lacking cftr expression, and the mechanism for the inhibition of igf-1 expression by increasing sult1e1 expression was investigated in human cells. human mmnk-1 cholangiocytes in which cftr expression was inhibited by short interfering rna (sirna) induced sult1e1 expression in human hepg2 hepatocytes when cocultured in a permeable membrane separated system. sult1a1 expression was not altered in this model. the data suggests that loss of cftr function in cholangiocytes is capable of selectively modulating sult1e1 expression in hepatocytes. to investigate the ability of increased sult1e1 activity to modulate expression of igf-1, hepg2 cells were stably transformed with sult1e1/pcdna3 to activity levels intermediate to the levels observed in cftr(-/-) mice. the increased sult1e1 activity was associated with decreased igf-1 message expression in the hepg2 cells. since a major pathway for igf-1 regulation involves growth hormone (gh) stimulation of stat5b phosphorylation, the effects of e2 and increased sult1e1 activity on gh stimulated stat5b phosphorylation were examined. stat5b activation was identified using immunoblot analysis with a rabbit anti-tyrosine phosphorylated-stat5b antibody. treatment of control hepg2 with 10 nm e2 prior to the addition of gh increases stat5b phosphorylation. in hepg2 cells expressing increased sult1e1 activity, the stimulation of stat5b phosphorylation by 10 nm e2 was significantly decreased. e2 had an apparent rapid action on stat5b phosphorylation that is not attenuated by the estrogen receptor antagonist, ici 182,780. e2 was effective at increasing stat5b phosphorylation when applied to hepg2 cells 15-30 min before gh. increased stat5b phosphorylation was observed in control hepg2 cells at 1 nm e2 although 10-fold higher e2 concentrations were required in the sult1e1-hepg2 cells consistent with the increased e2 sulfation. no differences were observed in total stat5b in any of the studies. this study demonstrates that human cholangiocytes with low levels of functional cftr are capable of inducing sult1e1 expression in human hepatocytes and the increase in e2 sulfation inhibits the gh stimulation of igf-1 expression via a decrease in stat5b phosphorylation. supported by grants from the cystic fibrosis research, inc. and nih (gm38953). williams, j.e. 1 ; benden, c. 2 ; jaffe, a. 2 ; suri, r. 2 ; wells, j.c. 1 ; fewtrell, m.s. 2 1. mrc childhood nutrition unit, institute of child health, london, united kingdom; 2. portex respiratory medicine unit, great ormond street hospital, london, united kingdom background: patients with cystic fibrosis (cf) are at high risk of malnutrition. to our knowledge, no study so far has employed a reference fourcomponent model(sup)1(/sup)(4cm) to assess body composition (bc) in cf children, which allows accurate evaluation of both fat mass (fm) and the components of fat-free mass (ffm; mineral, protein, water) and most studies have been cross sectional. methods: 53 cf subjects, aged 8-12 yrs, were compared with age-and sex-matched healthy controls for assessment of bc using a reference 4cm. comparison between groups was performed using paired t-tests and general linear models to adjust for age, height and pubertal status. in addition fm index (fmi; fm/height2) and ffmi (ffm/height2) standard deviation scores (sds) were calculated using measurements performed in a reference population of 415 healthy children aged 4 to 19 yrs. repeat measurements were made in 31 children with cf (15 boys) after 2 yrs and the change in fmi sds and ffmi sds investigated. results: for the initial measurement at baseline, boys with cf (n=26) were significantly shorter (mean (se) height sds: -0.7 (0.20, p<0.01) compared to uk 1990 reference data; girls with cf were lighter, bmi sds (-0.6 (0.2), p<0.05). at baseline, comparison of cf children with pair-matched controls indicated that there was no difference in the boys but cf girls had less fm (cf minus control) (-2.6 (0.8)kg, p<0.01) and mineral mass (-0.1 (0.0)kg, p<0.05) after adjustment for age, height and puberty. fmi sds adjusted for age and puberty was also significantly lower in girls with cf (-0.7 (0.3), p<0.01) whereas ffmi sds was not. for comparison with the large reference population there was no difference in the boys at either time point for fmi sds and ffmi sds (paired t-test). for girls however, there was a significant difference at both baseline fmi sds (-0.8 (1.0), p<0.01), ffmi sds (-0.8 (1.1), p<0.05) and 2yrs fmi sds (-0.9 (1.3), p<0.05) and ffmi sds (-0.7 (1.0), p<0.05). paired t-test analysis between baseline and 2 yr followup measurements indicated that fmi sds and ffmi sds were not significantly different at each point in time in either boys and girls. conclusion: at baseline, the bc of cf boys appears to be within normal range, but the cf girls already have lower fm and mineral than their healthy pair matched controls, even when adjustment for size and puberty is made. follow-up measures in 31 cf children indicated that these descriptions of nutritional status of cf boys and girls remained consistent over the following 2 years, with no evidence of any further deterioration in girls. however, nutritional surveillance is important in both sexes in cf at around puberty to address potential deterioration and this study suggests that the 4cm is a useful tool for this purpose. 1fuller background: although lung function (lf) in children with cystic fibrosis (cf) has been reported to be related to their nutritional status, particularly body fat, the latter has usually been assumed from anthropometric measurements and not measured using an appropriate reference method. habal, h. 1 ; al-turkmani, m. 1 ; freedman, s. 2 ; laposata, m. 1 1. pathology, massachusetts general hospital and harvard medical school, boston, ma, usa; 2. medicine, bidmc and harvard medical school, boston, ma, usa background: increased dietary intake of fatty acids is recommended for cf patients. it is unknown if this results in increased incorporation of all fatty acids-saturated, monounsaturated, and polyunsaturated-into cells with a cf phenotype to a greater extent than into non-cf cells. docosahexaenoic acid (dha) supplementation of cftr-/-mice has been shown to reverse the pathologic cf phenotype. aim: to investigate whether the uptake of multiple predominant fatty acids and the release of membrane-bound arachidonic acid (aa) are altered in cultured epithelial cells with a cf phenotype; in addition, to determine whether fatty acid uptake is selectively inhibited by dha supplementation. methods: sense (wt) and antisense (cf) cftr 16hbe cells were cultured in mem containing 10% horse serum. aa release from membrane phospholipids (pls) was studied by incubating the cells with 2 µm [ 14 c] aa for 15 min, and then with serum free medium containing 0.2% fatty acid free human serum albumin in the presence or absence of 100 µm adenosine for 15 min, followed by measurement of radioactivity in the supernatant. the uptake of fatty acids into the cells was studied by incubating the cells for different time points with 0.04 µm [ 14 c] linoleic acid (la), [ 14 c] aa, [ 14 c] dha, [ 14 c] palmitic acid or [ 14 c] oleic acid, in the presence and absence of 10 µm or 20 µm dha, followed by measurement of radioactivity in the cell lysate. results: basal and adenosine-stimulated aa release from membrane pls was significantly higher in cf cells compared to wt cells (basal wt:113.7±1.1, cf:202.4±12.8 dpm/10 6 cells, p<0.01; stimulated wt:207.8±11.4, cf:468.7±49.1 dpm/10 6 cells, p<0.01; mean±sem for each). the uptake of la, aa, dha, palmitic acid and oleic acid were significantly higher into cf cells compared to wt cells. dha significantly decreased the elevated uptake of these fatty acids into cf cells, but not into wt cells. data are shown in the table below. conclusion: in this cell culture model, these data demonstrate that aa uptake and release are increased in cf cells, which may suggest elevated mobilization of this fatty acid by cf cells. our data also show that cftr dysfunction leads to increased uptake of all tested fatty acids into the cells. downregulation of this increased uptake may be one mechanism by which dha exerts its therapeutic benefits. low bmi in cf correlates directly with worsening lung function and is an independent risk factor for mortality. the causes for this decreased bmi are incompletely understood but could be related to levels of the orexigenic peptide ghrelin, and to levels of leptin, an anorexigen. studies assessing plasma levels of leptin in cf have shown contradictory results, while the data on ghrelin in cf are scarce. we assessed both leptin and ghrelin plasma levels in mild (fev1 > 75% predicted), moderate (fev1 45% to 74% predicted), and severe (fev1 < 45 % predicted) adult cf patients. we compared these levels in cf subjects to 20 age matched controls with normal bmi (range 19.5 to 24.5 kg/m2). we conclude that leptin levels are decreased, while ghrelin levels are increased only in cf patients with severe disease and low bmi. plasma levels of ghrelin and leptin did not differ from normals in those with mild and moderate disease. gastroesophageal reflux (ger) is common in cf before and after lung transplant (ltx). laparascopic fundoplication (lapf) is used when patients fail on conservative medical therapy. aims: to review longer term clinical outcomes of lapf in patients with cf in terms of lung function, body mass index (bmi), reflux symptoms, patient satisfaction with surgery and complications. method: retrospective review of patient records and patient questionnaire to gather relevant information. results:thirty-two patients with cf underwent lap.f recently; 15 transplanted (ltxgroup), 8 female (mean age 40 [21-67]) years, and 17 not transplanted (cf group), 14 female (35 ). the median time between ltx & lapf was 654 (414; 1133) days, range 145-1838. endoscopy was undertaken in 27 patients prior to surgery and all underwent 24hr ph monitoring. complete (nissen) lapf was undertaken in 50% patients and a partial (toupet) in the other 50%. total median time in the operating theatre was 143(81-232) minutes. the average length of stay in hospital (in days) in the ltxgroup: pre-op. was 7.81; post-op. was 7.82 days; and in the cf group was: 5.2 and 12.5 respectively. lung function (fev1 and fvc in percent predicted) and bmi were measured at minus(-)100, plus (+)100, +200 and +300 days from lapf. patient satisfaction with lapf was a mean of 95% in the ltx group and 85% in the cf group and improvement in reflux symptoms of 90% in both groups. nocturnal cough reduced markedly in the cf group and there was an overall improvement in quality of life in both groups. complications consisted of 2 port site hernias, 1 failed lapf with re-operation 20 months later; 2 esophageal dilatations and 1 patient with lower esophageal sphincter hypotonia. there were 3 deaths on day 19, 253 and 382 post fundoplication. one was in a non-transplanted patient with recurrent episodes of hemoptysis. she succumbed to a catastrophic hemoptysis episode not related to lapf. the other 2 were in transplanted patients. conclusions: in this relatively small series of patients there was a small but significant drop in fev1 in the ltx group 300 days after lapf, but not in the cf group who remained stable throughout the review period. there was a significant drop in bmi in both groups after lapf, but the cf group returned to pre lapf values at +300 days. patient satisfaction with the procedure was high. fundoplication is an expensibe complicated procedure not without risk. a long term prospective study is warranted. historically this phenomenon has been attributed to intestinal malabsorption and cachexia as a consequence of chronic lung infection. however, cf mice exhibit similar growth and weight deficits but are not afflicted with malabsorption or lung infection, suggesting other mechanisms are participating as well. we hypothesize that small size is in part a result of increased β-oxidation in adipose stores. to test this we utilized a mouse model to study lipid metabolism. our study includes female cf mice with a null mutation in the cftr gene. these mice are congenic on the c57bl/6j background as well as age matched with their controls (wt) to 50-70 days. liver, adipose and skeletal muscle (calf) were dissected from wt and cf mice in a fed state. tissues were immediately flash frozen in liquid nitrogen and used for rna and protein lysate harvest. quantitative pcr (qpcr) was performed using primers for the lipogenic isoform of acetyl-coa carboxylase 1α (acc1α) in the liver and adipocytes, whereas the non-lipogenic isoform acc2β was assessed in the skeletal muscle. primers for acyl coa oxidase (aox), carnitine palmitoyltransferase 1 (cpt1) and long acyl-coa dehydrogenase (lcad) were used as gene markers for β-oxidation steps in the peroxisome, carnitine shuttle and mitochondrion, respectively. western blots were performed to detect acc (active) and phospho-acc (inactive) forms. the results show that there is a decrease in the total amount of acc protein in the fat as well as the skeletal muscle of the cf mice and the ratio of acc:phospho-acc is substantially lower in the cf tissues. phosphorylation and/or decreased expression of acc results in decreased expression of malonyl coa, leading to a cascade of events that increase β-oxidation. in addition, increased expression of aox and lcad, two downstream β-oxidation genes, is also observed. hyperactivity of the β-oxidation pathway could account for the decreased adipose stores seen in cf mice compared to the wt. such patterns of expression are reminiscent of starvation. caloric intake and intestinal absorption of dietary lipid were measured and cf animals consumed approximately 110% the calories of wt animals (per gram body weight) and lipid absorption was not significantly different (94% for cf, 97% for wt), indicating the cf animals have sufficient access to nutrition. we will continue to investigate fatty acid synthesis and degradation pathways during both the fed and fasted states to delineate the mechanism. this research was supported by grant hl 68883. effect of gender on dha levels in p and rbc with lower values for male cf patients. acknowledgments: this work was supported by the belgian cf association (ablm), the french cf association (vlm) and by a grant from frs (ucl). data are expressed as means ± sd. * and indicate a significant difference with controls and cf ps patients respectively, p<0.022. background: deficit of antioxidant systems and increased oxidative stress have been demonstrated in cf patients with respect to healthy controls. the origin of this imbalance is known to be multifactorial. objective: the aim of our study was to assess changes in antioxidant systems and oxidative stress parameters in cf pediatric patients in presence or absence of pancreatic insufficiency material and methods: we recruited 70 patients (37 with pancreatic insufficiency pi and 33 with pancreatic sufficiency ps) attending the cystic fibrosis centre of turin (2005) (2006) . during the annual routine control visit were performed antropometric measurements, pulmonary function tests (fev1), and were collected fasting blood samples and last 3 days food record. vitamin e, a, c, selenium, glutathione (gsh, gshpx, gssg), dehydroascorbic acid, pcr were assessed. data were analysed using the two sample t-test with equal variances. results: there are not statistically significant differences between two groups for sex, age and fev1. 33/37 of pi patients and 8/33 of ps patients took oral vitamin supplementation. serum levels of alpha-tocopherol and beta-carotene resulted within the local reference range either in pi or ps patients showing no significative differences. the mean serum levels were respectively suboptimal and optimal for vitamin e and a (biesalsky '97). pi and ps patients did not show significant intergroup differences of water soluble and enzymatic antioxidants levels as well. serum levels of vitamin c, selenium, erythrocyte reduced glutathione (gsh) and glutathione peroxidase activity (gshpx) resulted within the local reference range. plasma vitamin c concentrations resulted slightly inferior to the optimal values (biesalsky '97) in both groups. oxidative stress parameters (erythrocyte glutathione disulfide-gssg, dehydroascorbic acid) and inflammatory parameters (pcr) did not differ significantly, presenting values within the local reference range, in both groups. nutritional data revealed significantly reduced zscore for weight (p<0.05) and height (p<0.01) in pi patients vs ps patients. our data did not evidence significant differences in total energy (kcal/kg) and macronutrients (% of total kcal) intake in both pi and ps patients. dietary intake of antioxidant vitamins resulted slightly superior the rda (larn-1996) presenting no significant intergroup differences. conclusions: this study shows that pancreatic insufficiency do not influence oxidant/antioxidant balance in cf patients at least in paediatric age. until innovative supplementation guidelines will be proposed for application, nutritional education and monitoring and correct dietary habits are useful to optimize antioxidant status in cf patients. there are more than 1400 cf mutations that are responsible for the spectrum of disease severity in cystic fibrosis (cf). the most common, f508del mutation, is associated with pancreatic insufficiency (pi), and a classic phenotype. . yet even within a group of f508del homozygotes, there is a small minority of patients (2.3%) who are not taking pancreatic supplements, and are therefore presumably pancreatic sufficient (ps) (data from 2002 * cff registry). this observation prompted us to look more closely at enzyme use in cf patients with non-f508del homozygous genotype. we reviewed enzyme use in cf patients who were fully genotyped and whose enzyme use was documented in the 2002 * cf registry. heterozygotes with one or two non-f508del mutations were included in this analysis. using the assumption that the milder mutation is dominant, if the patient was ps and heterozygous with one f508del mutation, the second mutation was classified as ps; if the person was pi, the second mutation was classified as pi. we also reviewed the pancreatic status and genotype of patients participating in the wisconsin newborn screening study. in this randomized control trial (rct), pancreatic status was determined by 72hour fecal fat analysis. enzyme use was extracted from the database for the year 2002. there were 14494 patients in the 2002 cff registry with a total of 27 different mutations. twenty-one of these mutations were associated with pi. this finding correlated well with the per cent of patients who were on enzyme supplements (range of per cent patients on enzymes by mutation 100-88%). however when we looked at the six mutations associated with ps, the distribution of patients on enzymes vs. not, was much closer. see table below. possible reasons for enzyme supplement use by patients with ps mutations include a) treatment for recurrent pancreatitis, b)the fact that this is a cross-sectional analysis and patient may be on enzymes temporarily, c) patient may truly be pi, d) overuse of enzymes due to unreliable test to measure pancreatic status in patient, e) patients who are on enzymes are older, and have changed from ps to pi phenotype. this analysis suggests a need for better assessment of pancreatic status in patients with cf. * this analysis was originally done by request for the ecfs conference at garda on cftr genetics, and focused on the suggested "33 main mutations", and will be updated with the 2005 cff registry data. (supported by nih grants dk072126, dk34108, mo1 rr03186, mo1 rr00058) number of patients in rct with each mutation: 7,3,1,1,3,0 respectively. dutta, a. 1 ; woo, k. 1 ; fitz, j.g. 2 ; feranchak, a.p. 1 1. department of pediatrics, ut southwestern medical center/children's medical center, dallas, tx, usa; 2. medicine, ut southwestern, dallas, tx, usa extracellular atp is an important signaling molecule contributing to bile formation by the liver through binding biliary epithelial cell (cholangiocyte) membrane p2 receptors and stimulating cl-efflux. importantly, atp appears to work through cl-channels unrelated to cftr, suggesting it may be a potential way to modulate bile flow in cf liver disease. however, the signaling pathways linking receptor binding to channel activation in cholangiocytes are unknown. consequently, the aim of the present study was to identify the pathways responsible for atp-stimulated increases in [ca2+]i and membrane clpermeability in a human biliary epithelial cell model. studies were performed in mz-cha-1 human biliary cells; [ca2+]i was measured by fura-2 and membrane cl-currents by patch-clamp techniques. results: exposure of cells to atp (100 µm) resulted in a rapid increase in [ca2+]i to 1054.23 ± 244.90 nm (n=6) which was abolished by prior depletion of intracellular ca2+ by thapsigargin (37.94 ± 14.50 nm; n= 7), but unaffected by removal of extracellular ca2+ (egta, 819.00 ± 211.94 nm; n= 7). in parallel studies, atp (50 µm) increased current density from -1.99 ± 05 pa/pf to -13.20 ± 2.00 pa/pf (n= 19) . currents reversed at 0 mv (ecl-), were outwardly rectifying, and were inhibited by the cl-channel inhibitor nppb (-1.81 ± 1.71 pa/pf, n= 6), but not the cftr inhibitor cftrinh172. atp failed to activate currents after depletion of intracellular ca2+ stores by bapta-am (-0.70 ± 0.72 pa/pf, n=3). the p2y receptor antagonist suramin inhibited atp-stimulated increases in [ca2+]i (50.04 ± 8.17 nm; n= 7) and cl-channel activity (-2.25 ± 0.80 pa/pf; n= 6). however, the p2x receptor antagonist brilliant blue g did not affect the magnitude of atp-stimulated cl-currents (-14.63 ± 1.81 pa/pf, n=4). together these findings suggest that atp activates cl-currents primarily through p2y, but not p2x, receptors in human biliary cells. since p2y are g-proteincoupled receptors and modulate [ca2+]i by phospholipase c (plc) generation of inositol 1,4,5-trisphosphate (ip3), a series of experiments were designed to directly assess the effects of ip3 receptor inhibition or stimulation on atpstimulated cl-currents. first, the plc inhibitor, u73122 blocked atp-stimulated cl-currents (0.86 ± 0.60 pa/pf; n= 3). second, the specific ip3 receptor blocker 2-apb, inhibited both the atp-stimulated increase in [ca2+]i (34.36 ± 7.33 nm, n= 4) as well as membrane cl-currents (-0.13 ± 0.55 pa/pf, n= 6). lastly, intracellular dialysis with purified ip3 during whole-cell patch clamp activated cl-currents with identical properties to those activated by atp (-9.40 ± 1.23 pa/pf, n=4). together these studies demonstrate that extracellular atp stimulates ca2+-activated cl-channels in cholangiocytes primarily through p2y receptor binding and plc/ip3-dependent release of intracellular ca2+ stores. thus, the p2y-ip3 receptor signaling complex represents a potential target to increase cholangiocyte secretion, and hence augment bile flow, in the treatment of the cholestatic liver disease associated with cf. supported by grants from the nih niddk and the cff to a. feranchak. background: supplemental pep therapy is used to treat cf patients with epi to assist with digestion, food absorption. this open-label, multipledose, single-treatment, multicenter study evaluated efficacy and safety of eur-1008 for malabsorption treatment in young patients with cf and epi. eur-1008 is a novel, zero-overfill, highly-stable formulation of porcinederived pancreatic enzymes, and was specifically developed for use in very young children allowing the product to be sprinkled on food. methods: eligible patients were < 7 years with cf and epi, in acceptable nutritional status, needing peps and clinically stable. patients consumed a standard cf recommended diet, could not take drugs affecting gastric ph or motility. the trial involved a 7-day dose stabilization period and a 7-day treatment period. patients switched from their baseline enzyme treatment without washout. the optimal dose of eur-1008, determined during dose stabilization, was used during treatment. primary endpoint compared malabsorption of fat after eur-1008 vs. previous treatment. clinical symptoms were also evaluated, as was physicians' and parents' judgment on the control of malabsorption signs and symptoms. safety measures included aes, physical exams, vital signs and changes in clinical laboratory findings, including cholesterol levels and fat-soluble vitamins. results: nineteen patients (12 male) enrolled and completed all study phases. mean age was 3.9 years (range 1-6). percentage of responders (patients without steatorrhea (<30% fecal fat content) and without signs and symptoms of malabsorption after 1 and 2 weeks of treatment) was 52.6 at baseline, 68.4 (p=0.375 vs. base) after stabilization and 57.9 (p =0.999 vs. base) at study end. frequency and oily stools showed a significant decrease vs. baseline at study end. the mean number of stools per day was 1.82 stools at screening and 1.45 stools during treatment (p<0.001). the mean proportion of stool samples with visible oil or grease at screening was 11.10% and 4.73% during treatment (p=0.001). a statistically significant improvement was seen in the incidence of moderate bloating. physicians characterized 37% of patients as "improved" in the control of epi signs and symptoms vs. previous therapies and 63% as "unchanged" at the end of the treatment phase, while parent assessments were 47% "improved" and 53% "unchanged." vitamin k absorption (measured through pivka) improved with eur-1008 treatment over prior pep treatment measured at baseline. eur-1008 was safe and well tolerated, with minimal aes. no aes led to discontinuation of study drug interruption. no incidences of uric acid toxicity or fibrosing colonopathy following eur-1008 treatment. no trends were seen in lab parameters, physical examination, or vital signs. conclusion: eur-1008 is effective, safe, well-tolerated in the treatment of epi in young cf patients. treatment controlled malabsorption and clinical symptoms of epi in young patients with cf. safety and efficacy of eur-1008 in this trial is consistent with the pivotal phase iii trial. there is an association between the presence of cf and alterations in fatty acid metabolism, consisting of a decrease in linoleate (18:2n-6). more recently, a deficiency of docosahexaenoate (22:6n-3, dha) has arisen as a prominent fatty acid abnormality in cf. an established breeding colony of exon 10 cftr knockout (cftr-/-, cf, n=14) and wild type (wt, n=7) mice was used for this study. all mice were weaned at 23 days of age and then raised on peptamen and water until 30 days of age, and then continued for 10 days with 15 ml/day of peptamen to homogenize the nutritional status. after this period, some mice (n=3 per group) were subjected to dha treatment (40 mg/day) for one week. after sacrifice, pancreatic tissue was collected, homogenized and extracted. lipid classes were separated by sequential elution in aminopropyl columns. the eluted fractions were transmethylated and injected into a hewlett-packard 5890a gas chromatograph with a flame ionization detector for quantitative analysis. the vast majority of fatty acids were mainly distributed in phospholipids, except those with 18-carbons. wt and cf mice were studied a gender gap in the survival of cystic fibrosis (cf) is well-documented. however, little is known whether there is a gender différence in pubertal growth pattern in cf children. delayed and attenuated pubertal growth are commonly observed in adolescents with cf. however, characterizing height velocity (hv) pattern is very challenging because of errors associated with calculating hv, e.g. interpolation and extrapolation between 2 heights measured in irregular intervals, as well as difficulties in determining the location and the magnitude of peak height velocity (phv). in this study, we applied a new statistical method, namely, the semi-parametric shape-invariant model, to characterize the hv pattern of cf children. this method is based on the assumption that all individuals' growth curves have a common shape. therefore, this method attempts to shift and stretch (squeeze) individual's growth data until they can be modeled by the common shape curve. the semi-parametric shape-invariant model has the advantage of fitting measured heights directly, thereby eliminating the errors associated with calculating hv. the study population included 996 cf children who had height measurements between ages 2 to 18 years documented in the 1986-2002 cff registry. because fitting each individual height curve is computing-intensive, 100 boys and 100 girls were randomly chosen for this preliminary analysis. our results showed that girls exhibited a typical growth deceleration during late preadolescence, followed by accelerated hv and a single phv during adolescence (figure) . unexpectedly, cf boys exhibited a notable hv peak during late preadolescence in addition to the typical adolescent phv (figure) . overall, the mean magnitude and age of phv was 7.4 cm at 14.6 years for boys and 6.6 cm at 11.9 years for girls. when compared to phv of non-cf children based on tanner's reference (pediatr. 1985, 107:317) , cf boys showed a greater delay in the age of phv while cf girls showed a greater attenuation in the magnitude of phv. further analyses are being performed to characterize the hv patterns of all 996 children to confirm these results. (supported by nih-dk02891). anelli, m.; foresti, r.; peloso, l.; ortenzi, g. medical affairs, eurand, pessano con bo, italy background: given their inherent instability, the dose units of all pancreatic enzyme preparations (peps) have always been "overfilled" to compensate for enzyme degradation over time and assure at least 90% of the label lipase content at the end of their shelf life. this overfill issue was first examined by whitehead who reported overfills ranging from 14 to 39% after analyzing several commercially available peps (whitehead am. pharm weekbl sci. 1988) . the usp presently allows for pep overfills up to 65%. thus, the actual active lipase content of a pep capsule labelled at 10,000 iu might vary from 9,000 to 16,500 units according to its "freshness." as a result, patients taking peps receive a product with variable potency, which can lead to efficacy issues, increased pill burden, unnecessary drug interactions, and product switching. the fda has also noted the potential safety risk posed by overfilling and, in a recently published guidance, it imposed a zero overfill requirement for all peps to be marketed in the usa after april 2008. methods: over a period of two years, we ran a series of tests similar to those conducted by whitehead to evaluate overfill on 16 commercial peps currently available in the u.s. and europe. results: no finished product was formulated to 100% of the labelclaimed lipase enzyme activity. overfill on commercially available peps ranged from 7% to 47%, with a median value of 32%. the peps analyzed were 10 to 12 months old on average; therefore, the actual amount of overfill is likely underestimated. conclusion: our findings confirm those reported earlier by whitehead and demonstrate that none of the currently available peps complies with the zero overfill requirement of the fda guidance. these findings highlight a potential cause of suboptimal therapy with currently available peps. references: -whitehead am. study to compare the enzyme activity, acid resistance and dissolution characteristics of currently available pancreatic enzyme preparations. pharm weekbl sci. 1988 feb 19; 10(1): 197-201. -food and drug administration. guidance for industry: exocrine pancreatic insufficiency drug products -submitting ndas. april 2006. heubi, j. 1 ; boas, s.r. 2 ; blake, k. 3 ; nasr, s.z. 4 ; woo, m.s. 5 ; graff, g.r. 6 ; hardy, k.a. 7 ; amaro-galvez, r. 8 ; latino, m. 9 ; lee, c. 10 1. children 's hospital medical center, cincinnati, oh, usa; 2. chicago cf care, glenview, il, usa; 3. nemours, jacksonville, fl, usa; 4. michigan u, ann arbor, mi, usa; 5. children's h, los angeles, ca, usa; 6. penn state h, hershey, pa, usa; 7. children's h, oakland, ca, usa; 8. texas u, tyler, tx, usa; 9. eurand, milan, italy; 10. eurand, vandalia, oh, usa background: supplemental pep therapy helps prevent maldigestion and malabsorption in cf patients. eur-1008 is a novel, zero-overfill, highly-stable formulation of porcine-derived pancreatic enzymes developed to treat epi in cf patients. this phase iii, randomized, doubleblind, placebo-controlled, two-treatment, crossover, multicenter trial evaluated the efficacy and safety of eur-1008 to treat epi in cf patients. methods: patients with confirmed cf and epi, age ≥7 years, good nutritional status and weight ≤ 70 kg were enrolled. no drugs affecting gastric ph or motility were allowed. after open-label dose titration, patients were randomized to receive eur-1008 or placebo over a week-long period. following another open-label normalization, all patients were crossed over to the alternative treatment arm. primary endpoint was change in coefficient of fat absorption (cfa) following oral administration of eur-1008 vs. placebo. secondary endpoints were change in coefficient of nitrogen absorption (cna), cholesterol, fat soluble vitamins, body weight, bmi and epi symptoms following oral administration of eur-1008 vs. placebo. safety endpoints included aes, clinical laboratory parameters, physical exams and vital signs. results: thirty-four patients were enrolled (17 female); 32 were evaluated. mean age was 15.4 years (range 8-23). eur-1008 treatment was associated with statistically significant (p<0.001) increases in both cfa and cna vs. placebo. eur-1008 treatment was associated with decreases in pivka ii (an indicator of vitamin k status). epi symptoms consistently improved during eur-1008 treatment, including a statistically significant reduction in stool frequency and fewer soft and watery stools. treatment with eur-1008 improved the signs and symptoms of malabsorption even in the small subset of patients with cfa values greater than 80% under placebo treatment. there were no notable changes in serum lipids, vitamin a and e values, mean weight or bmi in patients who received eur-1008. eur-1008 was safe and well-tolerated. there were no unexpected or significant differences in the frequency or type of aes between eur-1008 and placebo, and no trends were seen in lab parameters or vital signs. no patient discontinued from the study due to an ae. two serious aes were considered unrelated to the study drug and both resolved. conclusion: in this randomized, double blind, placebo-controlled study, eur-1008 was safe, well tolerated and effective in the treatment of cf patients with epi, with clinically and statistically significant improvements in cfa, cna, and epi signs and symptoms in the absence of any concomitant treatment affecting gastrointestinal motility and ph. the therapeutic benefit of eur-1008 was seen even in patients with very high cfa values (>80%). the relative infrequency of cystic fibrosis related diabetes (cfrd), until recently, means little is known about hypoglycaemia ('hypos') associated with its treatment. insulin is frequently taken by those who have cfrd, for whom the risk of hypoglycaemia may be high due to dramatic changes in insulin sensitivity that can occur with pulmonary exacerbations and a decreased ability to secrete glucagon. a number of studies involving people with type 1 diabetes mellitus (t1dm) have reported on their experiences of hypoglycaemia, but whether results from such investigations apply to cfrd is unclear. an exploratory investigation was conducted to compare the type, frequency and severity of hypoglycaemic symptoms experienced by patients who had cfrd with patients who had t1dm. method a cross sectional study was conducted, involving adults (18-60 years) with t1dm or cfrd, recruited from two hospitals in england. a questionnaire, sent to 265 patients with t1dm and 145 with cfrd, included investigator-developed items about knowledge and frequency of hypoglycaemia and the edinburgh hypoglycaemia scale (ehs), a standardised measure that assesses autonomic (e.g. sweating, hunger) and neuroglycopenic (e.g. confusion, poor co-ordination) symptoms associated with hypoglycaemia. questionnaire data were entered into spss for analysis. comparisons were conducted using t-tests, mann whitney tests or chi square tests as appropriate. results questionnaires were returned by 60 patients with t1dm and 52 with cfrd. five of those with t1dm were over 60 years of age and, therefore, excluded from analysis. almost all participants had experienced a 'hypo' (98%). comparisons between the two groups are reported in table 1. conclusion patients with cfrd reported fewer and tended to have less severe episodes of hypoglycaemia compared to those with t1dm. fewer neuroglycopenic symptoms in the former could be related to their shorter duration of diabetes or to how cfrd was managed; patients with cfrd were given shorter acting insulin, making them less prone to 'hypos' during the night, and had a high sugar diet to ensure weight maintenance. differences between the groups could also be related to specific characteristics of these two forms of diabetes. table 1 : data from questionnaire insulin resistance. the purpose of this study was to investigate the relationship between pem and the risk of developing glucose intolerance and/or cfrd and the effect on lung function in children ≤21 years of age whose history suggests pem. patients whose diagnosis of cf was suggested by failure to thrive(ftt) and whose growth parameters remain <10% for weight and height for age were identified as having incurred pem. a retrospective analysis of our cf registry database was performed. 173 cf patients ≤21 years old were identified. 32 of 173 patients(18.5%) had cfrd. 103(59.5%) of these plotted <10% for weight and height (pem)and 23(22.3%) were diagnosed with cfrd. further review of the 103 patients showed 35 whose diagnosis of cf was suggested by ftt and 10 (28.6%) of those 35 had cfrd. statistical analysis did not show a clinical significance, but a strong relationship is implied. pulmonary function, fev1%, was significantly lower in the pem/cfrd patients (mean fev1 63.5% ± 24.36%) than the pem only group, (mean fev1 88.17% ± 22.14%)(p=0.0001). this review provides further support that patients who have pem are at increased risk of developing cfrd. a significant risk was not established; however a strong association is suggested. assessment of a larger cohort of patients may show a stronger relationship. findings from this study would suggest that further research into the physiological relationship between pem and cfrd should be initiated. cfrd screening should be considered at a younger age than the current cff recommended age of >14 years. diagnosing cfrd/glucose intolerance early may contribute to improve growth parameters, preserve lung function, and improve survival. based on this review, our center is initiating cfrd screening for all patients ≥ 10 years, and considers screening for all pem patients ≥6 years. background: patients with cf are thought to have a rapid postprandial rise in plasma glucose which may be followed by a delayed and prolonged insulin response and hypoglycemia. we sought to estimate the prevalance, cumulative one year incidence, and factors associated with hypoglycemia during oral glucose tolerance testing in non-diabetic adults with cf. we also compared the prevalence of hypoglycemia to a geographically matched cohort of young adults without cf. methods: we performed a prospective study of 161 individuals aged over 16 years with cf followed in clinic providing population-based, specialized care in cambridgeshire, england. we excluded patients with previously-diagnosed diabetes or taking anti-diabetic medications. we evaluated 108 individuals who underwent 75 g 2 hour oral glucose tolerance testing (ogtt) in 2004. of these, 65 individuals had neither new diabetes nor hypoglycemia in 2004 and were retested using ogtt in 2005. we testing the association between biochemical and anthropometric factors measured in 2004 with the prevalence of hypoglycemia in 2004, and incident hypoglycemia in 2005. biochemical hypoglycemia was defined as a blood glucose between <4.0 mmol/l (72 mg/dl), but greater or equal to 2.5 mmol/l (45 mg/dl) and severe hypoglycemia as a value lower than 2.5 mmol/l on either the fasting or post glucose challenge value. for comparison, we used data from the cambridgeshire-based young ely study of 183 individuals age 30 -48 years who underwent 75 g ogtt testing in 1994 -1995 . results: in 2004 , in patients with cf, the prevalence of fasting hypoglycemia was 5.5%. no patients had fasting severe hypoglycemia. following glucose challenge, 20.2% and 2.8% of patients had glucose values consistent with hypoglycemia and severe hypoglycemia respectively. there were no diferrences in age, hepatic, pulmonary or renal function, but patients with hypoglycemia at two hours were five times more like to be male (odds ratio 5.1, 95% ci 1.7 -15.6) and more likely to have a higher bmi. the prevalence of a 2 hours value of < 4.0 mmol/l in patients with cf at 22.9% (25/109) was not different than in controls 20.7% (38/183). of patients who did not have hypoglycemic readings in 2004, at one year, 1 patient developed fasting hypoglycemia and 14 individuals developed post-challenge hypoglycemic for a cumulative incidence 21.5%. of these 6.2 % (4) had values in the range of severe hypoglycemia. there were no differences between age, sex, bmi, liver, renal, or pulmonary function in those who did and did not develop hypoglycemia. conclusions: hypoglycemia appears common in patients with cf but is also common in a healthy population. male sex and bmi were associated with post load hypoglycemia in patients with cf in cross-sectional analyses only. further research needs to assess whether these low blood glucose values are associated with symptoms. it is well recognised that cystic fibrosis related diabetes (cfrd) is a poor prognostic indicator in cf, and therefore early recognition is imperative to allow effective treatment in an attempt to prevent the associated decline in pulmonary function and nutritional status. although the oral glucose tolerance test (ogtt) is the accepted method of detecting diabetes mellitus in non cf individuals, glucose intolerance and lack of insulin can be variable in cf patients such that the reliability of ogtt in the diagnosis of cfrd has been questioned. under these circumstances, other methods of monitoring glucose intolerance, such as serial post prandial glucose monitoring (sgm), may be more appropriate. to look at this further, we prospectively compared ogtt with sgm in a group of adult cf patients. fourteen patients with no previous history of cfrd ( 127], 9 df508 homozygous, 9 male) admitted for acute pulmonary exacerbations were evaluated. all were exocrine pancreatic insufficient, and 1 was on long term oral steroids. eleven patients received 30mg/day oral prednisolone in addition to iv antibiotic therapy for the duration of their admission. ogtt was performed in the standard fashion (ingestion of 1.75g glucose/kg body weight [maximum 75 g] within 5 minutes, after an overnight fast). sgm was performed 2 hours post prandially and before bedtime over the period of the admission. the local ethics committee approved the study and informed consent was obtained from all patients. results sgm revealed elevated postprandial blood glucose values in all patients. there were 83 episodes (mean 6 per patient [0 to 18]) with glucose >11.1 mmol/l (frank diabetes, who criteria) and 86 episodes (mean 6 per patient [1 to 13] ) with glucose 7.8 to 11.1 mmol/l (impaired glucose tolerance, who criteria). however, ogtt revealed impaired glucose tolerance in only one patient (2 hour glucose value 9.8 mmol/l). in the remaining patients, ogtt revealed normal 2 hour glucose values (mean 5.0 mmol/l [range 3.8 to 6.9]). fasting plasma glucose values were also normal (mean 4.5 mmol/l [3.5 to 5.9]) in all patients. we have shown a high prevalence of hyperglycaemia in our adult patients admitted for pulmonary exacerbations that was only detected using serial glucose monitoring. this study suggests that sgm may be more sensitive at detecting early cfrd than the ogtt. further work needs to be carried out to look at the best methods of diagnosing cfrd, to facilitate early treatment and thereby improve the prognosis. the diagnosis of cystic fibrosis related diabetes (cfrd) is associated with a decline in pulmonary function and nutritional state and is a poor prognostic indicator: the risk of its development increases with age. furthermore, the development of cfrd is due to a gradual loss of insulin production, and the disease may therefore be preceded by a period of glucose intolerance. despite this, the incidence of hyperglycaemia in cf patients is unknown. to study this further, we monitored the blood glucose profiles of adult cf patients admitted for the treatment of a pulmonary exacerbation. we looked at 60 consecutive cf patient admissions for iv antibiotic therapy to our large adult unit. of these, 28 had established cfrd and were excluded. the remaining 32 patients (mean age 23 years [range 17 to 54], 22 male) formed the study population. none were on long term oral steroids, but 28 received 30mg/day prednisolone as part of their routine exacerbation treatment. all patients underwent blood glucose measurement 2 hours after each meal and before bedtime as part of a standard assessment protocol in our clinic. thirty patients (94%, 26/28 on steroids) demonstrated post prandial hyperglycaemia: 14 exhibited values between 7.8 and 11.1 mmol/l (impaired glucose tolerance, who criteria) and 16 >11.1 mmol/l (frank diabetes, who criteria). there were no significant differences in age, genotype, fev1, fvc, bmi, or duration of hospital stay between the two groups. of the hyperglycaemic patients, 28 (93%) had abnormal glucose levels detected before bed time, significantly more than 2 hours post breakfast (23%, p<0.001), post lunch (60%, p<0.002), but not post dinner (80%, p=0.16). this study has shown a high prevalence of hyperglycaemia in adult cf patients treated for a pulmonary exacerbation who were not otherwise known to suffer from cfrd. nearly all our patients demonstrated glucose intolerance later in the day, when the recommended screening test for cfrd (the fasting oral glucose tolerance challenge) cannot be used. post prandial glucose monitoring of such patients during times of stress may therefore be advisable, allowing the introduction of insulin treatment at an earlier stage in the disease process. whittaker, l.a.; tilluckdharry, l.; christian, r. medicine, university of vermont, burlington, vt, usa objective: bone disease is a recognized complication of cystic fibrosis (cf). there are currently no guidelines for bone mineral density (bmd) testing or tools for fracture risk assessment in cf adults. we piloted a survey designed to identify cf-specific risk factors for low bone density and fracture. methods: a 45 question survey was completed by patients prior to measurement of bmd at the hip, spine and radius by densitometry (dxa). the survey was comprised of questions on calcium and vitamin d intake, exercise, family history, medications, reproductive health, ultraviolet exposure, cf related diabetes (cfrd), weight loss, severity of cf lung disease and quality of life. z scores (bmd adjusted for age and gender) were used so that male and female data could be analyzed collectively. patient perceived health (pph) was rated by four questions and totaled for the pph score, which ranged from 4-12 with the highest score indicating the best health. functional status (fs) was rated by three questions that reflect the degree to which cf interferes with school, work or play. the total fs score ranged from 3-9, with the highest score representing the least interference with life. questions on pph and fs were derived from a validated clinical assessment tool, the cf questionnaire (cfq) (1) . results: all cf adult patients (n=46, age 18 and older) were offered participation in the study. dxa of total hip and lumbar spine was performed on 39 patients (85%); 30 (65%) of these patients also completed the survey. 51% of patients had low bone density on dxa (z score <-1). 62 % of male cf adults had low bone density vs 39 % of females. fracture was reported by 57% of patients who completed the survey (64% female and 50% male). lower pph score was associated with lower z score at the hip and spine. lower fs score was associated with lower z score at the hip and spine. low body mass index was associated with low z scores at both hip and spine. patients with bmi below 22 kg/m2 were most likely to have osteopenia. conclusions: osteopenia and history of fracture were common in our adult cf population regardless of gender. bmi, pph and fs predicted bone density. risk stratification by a cf-specific bone health survey may guide bone density screening strategies for cf adults. prospective studies in cf adults are needed to determine whether a cf-specific survey can predict fracture risk as well as bone density. references : as life expectancy in cystic fibrosis (cf) increase, osteoporosis has become more prevalent. vitamin d status is one of many factors that contribute to optimal bone health. previous data has shown, the majority of cf patients are deficient in vitamin d despite being prescribed daily cf specific vitamin therapy. previous research has also shown that the current recommendations for vitamin d supplementation and repletion provided by the cystic fibrosis foundation bone health consensus (2002) are not sufficient to achieve optimal vitamin d levels in the majority of patients. moreover, the recommendations fail to sustain levels in the optimal range for bone health. purpose: we demonstrate a successful supplementation algorithm for individualized high dose vitamin d with continued high dose maintenance therapy which achieves and sustains appropriate levels. methods: all patients followed at the pediatric center are prescribed routine cf vitamin supplementation. vitamin d levels are obtained as part of routine clinical care via mass spectroscopy. if serum vitamin d level is <30 ng/dl, vitamin d therapy with ergocalciferol is initiated one time per week. patients < 5 years of age receive 12000 iu/ dose and > 5 years of age receive 50000 iu/dose. at the next routine quarterly visit, vitamin d levels are re-assessed via mass spectroscopy to determine efficacy of once weekly supplementation. if levels are > 30ng/dl, patients continue high dose supplementation 1 time a week as maintenance therapy. however, if levels remain <30ng/dl, the ergocalciferol is increased by 1 time a week in 3 month increments until adequate levels are obtained. once adequate serum vitamin d levels are obtained, patients remain on the level of supplementation needed to achieve levels >30ng/dl as maintenance therapy. all patients are instructed to take vitamin d and their cf vitamins with enzymes and food to optimize absorption. results: after implementation of the algorithm, 70.5% of patients, with varying levels of supplementation, had vitamin d levels > 30ng/dl (mean=38.4 ng/dl). of the patients that achieved goal vitamin d levels, 58% were able to achieve and sustain adequate vitamin d levels without additional vitamin d supplementation while on standard cf vitamins (mean vitamin d level = 35.9 ng/dl). the remaining patients required chronic maintenance vitamin d: 17% required vitamin d supplementation once per week with a resulting mean vitamin d level of 45.2ng/dl, 15.9% required maintenance vitamin d supplementation twice per week with a resulting mean vitamin d level of 39.9 ng/dl, and, 3.6% required vitamin d supplementation 3 or more times per week with a resulting mean vitamin d level of 42.3 ng/dl. another 4% of patients take vitamin d supplements other than ergocalciferol and were not included in this study. conclusions: individualized vitamin d repletion and maintenance therapy as outlined in our algorithm is a successful mechanism to obtain optimal vitamin d levels in patients with cf. inhaled tobramycin (29%). sixty-two percent reported using at least 5 different types of oral medications on that day including pancreatic enzymes (85%), oral antibiotics (33%), anti-reflux medication (49%), azithromycin (47%), anti-histamine (29%), and a decongestant (10%). twenty percent reported taking at least one medication for pain. forty-nine percent reported performing airway clearance at least twice during the day with 35% using a vest, 18% performing standard chest physiotherapy, and 15% using a flutter or acapella device. there was no difference in the reported number of medications or the reported time needed to complete therapies based on respondent age. although respondents with severe lung disease (fev1 <40% predicted) reported a higher median number of therapies (8 vs. 7, p=0.05), the reported time for completing therapies was not associated with fev1 (p=0.4). conclusion: daily treatment burden and complexity in all adults with cf is high, and is only marginally increased in those with worse pulmonary function. given this already high load of daily therapies, efforts to assess the treatment burden of new cf therapies are warranted. the impact of this high treatment burden on overall health related quality of life for adults with cf needs to be assessed. objective: the cystic fibrosis foundation (cff) has a network of over 115 accredited care centers throughout the united states. data is collected from these care centers to help better understand the disease and describe changes in survival, standards of care, and health outcomes. when the cff was founded in 1955 few children survived to school age. now the predicted survival extends beyond the mid 30's (cff, 2005) . with this change has come the need for age appropriate care in many areas including reproduction. the adult cystic fibrosis center at morristown memorial hospital presently follows 68 adults. of those, 3 women(4%) with cf have had children affected with cystic fibrosis. each woman was diagnosed after her child. the cf data registry does not have any means of identifying if a patient has an affected child. as the average age of survival of cf patients has increased, so has the likelihood of cf patients having cf offspring. what is the estimated incidence of cf patients having cf affected children attending accredited cf centers in the united states? method: questionnaires were sent to care centers and satellite centers via standard mail or facsimile based on information from the cff directory. one month after the last questionnaire was mailed, the cf foundation sent out a reminder email to the nurse coordinators. questions asked were as follows: 1) name of center 2) do you presently care for any patients with cf who have given birth to, or fathered a cf affected child? 3) how many patients at your center have had cf affected children? 4) was the parent diagnosed before or after pregnancy was confirmed? 5) if parent's diagnosis came after conception, was this after child was diagnosed? 6) was the parent's diagnosis a result of prenatal testing? results: a total of 144 questionnaires were mailed or faxed. this netted a result of 50 responses. after email reminder, an additional 68 questionnaires were returned for a total of 118 responses, an 82% response rate. name of center was checked to remove duplicate information. the number of parents with cystic fibrosis who have had children with cf numbered 62. of those, 27 (44%) were diagnosed before pregnancy; 35 (56%) were diagnosed after pregnancy. of those diagnosed after pregnancy, the majority (86%) were diagnosed as a result of their child's diagnosis. only 3 (8%) were diagnosed as a result of prenatal testing. conclusion: based on the responses from accredited cf care centers, 62 patients with cf have children also affected with this disorder. prior to conceiving their child, 44% knew that they had cf. having cf did not appear to be a deterrent to delivering cf offspring. this study did not look at the age of the parent or child; they may have conceived their child before genetic screening was common for spouses. as the median survival of this population increases, the incidence of cf adults with cf children may also increase. the cff may choose to add a question to the cf data registry, identifying cf patients with cf offspring. the potential physical and psychosocial impact can then be tracked. anderson, a.; popli, k.; stewart, j.; heslop, k.; gascoigne, a.; bourke, s. adult cystic fibrosis centre and fertility centre, royal victoria infirmary, newcastle upon tyne, united kingdom as the outcome of patients with cf improves fertility issues become increasingly important. almost all men with cf are infertile because of azoospermia due to congenital absence of the vas deferens. these men can attain genetic paternity by sperm aspiration from the epididymis or testis with intracytoplasmic sperm injection (icsi), but few men with cf seek fertility treatment to assess their knowledge and attitude towards fertility issues a questionnaire was sent to 71 men with cf: 50(70%) responded; 13(19%) declined to give information and 37(53%) replied in full. overall 75% thought that nearly all men with cf were infertile, 25% did not know how many were affected, and 66% could describe the exact nature of infertility. only 50% knew that treatment for infertility was available: 33% of these thought it was rarely successful and 33% were unsure of success rates. although 75% had thought of having children at some stage, only 15% had sought investigation of infertility. two men had children, one by icsi and one by donor insemination. overall 67% feared that the child could have cf and 53% worried that their health would affect their ability to function as a parent. in terms of receiving advice 57% thought that this should be given by a fertility specialist, 27% by a cf physician and 16% by a primary care physician. with regard to fertility treatment 44% thought that decisions should be made by the patients and their partners, 26% by the patient alone, and 30% by a doctor. the reluctance of men with cf to seek fertility treatment appears to be due to many factors including lack of knowledge of available treatments, fears about the child inheriting cf, and concerns that their health would adversely affect their role as a parent. in addiition to information provided by the cf team advice from a specialist in fertility treatment may help to improve the provision of reproductive counselling to these patients. nash, e.f. 1,2 ; coonar, a.s. 3 ; stephenson, a.l. 1, 4 ; delgado, d.h. 5 ; singer, l.g. 2, 4 ; tullis, e. 1, 4 ; chaparro-mutis, c. studies have shown varying degrees of right ventricular (rv) dysfunction in adult cf patients with severe lung disease. left ventricular (lv) dysfunction is much less common with conflicting reports as to its prevalence. pulmonary hypertension (ph) is commonly seen, the severity correlating with fev 1 . one group reported that ph was more prevalent in b. cepaciacolonized patients, albeit in a small sample. the aim of this study was to determine the true prevalence of ph and cardiac dysfunction in cf patients with severe lung disease. we carried out a retrospective cohort study of all adult cf patients referred to the lung transplant program at toronto general hospital from 1987-2007. adult patients referred with non-cf bronchiectasis were included for comparison. demographic data, lung function, 6-minute walk, micro-biology, doppler echocardiography and resting and stress radionuclide ventriculography (muga) results were analyzed by students t-test (parametric) or mann whitney u test (non-parametric). results 244 adult cf patients (61% non-b. cepacia, 39% b. cepacia) and 25 non-cf bronchiectasis patients were included. data from these 3 groups are summarized in the table. we found higher 6 minute walk and bmi values and a higher percentage of males in the b. cepacia compared to non-b. cepacia group. ph was present in 53% of the cf patients in whom pasp could be obtained. b. cepacia patients had lower first pass rvef compared to non-b. cepacia patients. 51% of the b. cepacia patients had an abnormal lvef response to exercise (defined as <5% increase on exercise) compared to 42% of the non-b. cepacia patients. we found a high prevalence of ph as well as rv and lv dysfunction in cf patients with severe lung disease. b. cepacia-colonized patients had worse rv function compared to non-b. cepacia-colonized patients on first pass radionuclide ventriculography. *non-cf bronchiectasis group older than cf groups (p<0.001) b. cepacia cf group higher than non-b. cepacia cf group (p<0.01) b. cepacia cf group lower than non-b. cepacia cf group (p<.001) perobelli, s. 1 ; dorazio, c. 1 ; assael, b. 1 ; tamanini, a. 2 ; castellani, c. 1 1. cystic fibrosis center, verona, italy; 2. molecular biology laboratory, verona, italy cf neonatal screeening (nbs) can identify neonates with elevated irt, normal or borderline sweat chloride concentrations, and one cf mutation plus an additional cftr sequence variant, or, alternatively, two cftr sequence variants. the combination of hypertrypsinogenemia and two cftr gene defects is consistent with the presence of a cftr-related disorder (cftr-rd). although these neonates are usually healthy at diagnosis, the long-term phenotypical consequences may be highly variable. such variability makes impossible to predict the clinical outcome, and provide satisfactory genetic counselling for the family. the diagnosis of such a vague condition could potentially cause considerable family stress. this study aimed at assessing how parents of infants diagnosed with a cftr-rd through nbs perceive their children's health. a questionnaire for parents was designed, consisting of three sections. the first section asked about information given to the family in the neonatal period, and about emotional reactions to the cftr-rd communication of diagnosis. the second section investigated the present perception of the child's health, and the parents level of anxiety/concern. the third section focused on the impact of the diagnosis on family planning, on relationships inside the couple and with the child, and on emotional distress; this section was concerned also with the use of carrier testing in relatives. parents were also asked to fill in a standardized form on child behaviour (achenbach child behaviour check-list). the questionnaire was distributed to two populations, the parents of 18 children diagnosed with cftr-rd through nbs (group a), and the parents of 18 children diagnosed with cf through nbs as the life expectancy of children with cf increases new issues arise that have not been dealt with in the past. in the past,issues related to the support and care of a young adult with cf were rarely raised because only small numbers of children with cf lived into adulthood. however, with an increasing young adult population, young adults without family support have surfaced in significant numbers and their needs should be identified and understood by the cf center care team. these young adults face the same range of issues others with cf face related to health insurance, government benefits, housing, education and employment. however, they do not have a family to help them wiht support, health insurance benefits and care needs. method in october 1998, the cf legal information hotline ("hotline") began to provide free and confidential information to people with cf, family members and healthcare providers in the u.s. the hotline is staffed by attorneys, beth sufian and james passamano(bs and jp) and is accessed by dialing a toll free number or by e-mail. information about the hotline is available at cf care centers, in cf publications and on the web. information regarding the content of each call is recorded on an intake form and includes caller name, address, phone number, age of person with cf, cf center attended, and information related to the caller's question, attempts made to access legal information prior to calling the hotline, relation of access to information to access to care. calls and e-mails are tracked by seven major categories which are divided into subcategories. if a caller has more than one question, the call is tracked using the caller's primary issue. results from november 2006 to april 2007 the cf legal information hotline has received 42 calls from young adults with cf who do not have family support and have issues related to obtaining health insur-ance, government benefits, educational oppornties or employment opportunities. as well as issues related to lack of support for care. the hotline has also received 33 calls from cf care centers team members with questions related to the needs of young adults without family support. over an eight year period the cf hotline received _______calls. the categories for the calls were: social security benefits (1, 324) , health insurance (1, 163) , educational (434), employment (422), long term disability benefits (190), family law (59) and miscellaneous legal issues (95). discussion people with cf face obstacles to care if they are unaware of their legal rights especially in the areas of health insurance and government benefits. healthcare providers are busy addressing the medical needs of patients and are often not well equipped to provide detailed information about the legal rights of their patients. the hotline provides important information to both patients and healthcare providers that can optimize both patient care and outcomes. access to legal information can result in individuals obtaining health insurance, medicaid, medicare or other state coverage that in turn results in better access to treatment. legal information can also lead to important modifications in the educational or employment environment for the individual with cf. these modifications can be critical in allowing those individuals access to important care and treatments. providing information that can help individuals obtain social security or long term disability benefits can result in a monthly benefit and health insurance that then allows the individual to devote more time to their healthcare. many people with cf and healthcare providers lack information about the legal rights of individuals with cf. understanding the legal rights of people with cf can reduce obstacles to obtaining care, optimize education and employment, and increase access to retirement benefits. further work needs to be done to identify ways to make individuals with cf and healthcare providers aware of the hotline. the cf hotline provides a unique service to cf patients and healthcare providers by providing information regarding the legal rights of people with cf. identification of ways to assist young adults without family support should be identified and cf center xcare teams shoudl be given the tools to allow them to help their patients access government assistance and other programs that can help make sure that the young adults have acces to care and support they need to live with cf. the dvd gives healthcare staff, patients and families a rare glimpse of ten couples with cystic fibrosis (cf) interacting at a cf care center-sponsored 'couples support group'. they discuss the impact this forum has in their daily lives for gainingo information about how cf affects their lives as individuals, and as couples, and how the 'non-cf partner' learns from others. o skills at networking and personal coping strategies. o reflections on 'family life and cf', and o observations on ways cf care centers can join them in greater health partnership. lastly, this dvd provides a 'starter kit' for ways to establish such a group. one of the realities of a life-threatening illness can be that of social isolation, and of separation from others who share the same disease. having the opportunity of participate in a support group can significantly reduce that sense of isolation and give the participants an ability to share experiences and give support to others with this disease. cystic fibrosis as an illness has growing ranks of 'survivors' who live longer, fuller lives, thanks to the advances made on multiple-research fronts. historically, adults with cystic fibrosis have not had much occasion to share person-to-person experiences within the structure of their cf care centers. justifiably, with the potential for cross-infection, the cystic fibrosis foundation has urged caution for the cf population in having face-to-face interactions. in 2004, our cf care center recognized this need and sponsoredwith guidance from staff facilitators-an on-going adult support group. prior to the formation of the group pre-and on-site group ic protocols were developed. at the pre-group stage, the physician initially certifies that the patient is free from aggressive pathogens. each time the patient rsvp's permission to attend a group session, the physician must 'recertify' this status. on-site protocols emphasize a number of ic precautions to minimize crossinfection. the center recognized that by developing such a support group, 'patient and family centered care' could be enhanced. a goal in the group's formation was to create 'mutually beneficial partnerships among patients, families, and healthcare providers'. group structure/successes to date: the group is limited to 20 participants. two cf care center staff act as facilitators. this is a support, not a therapy group. compliance with therapies is reported to have increased as one outcome of this forum. formal topics are purposefully not predetermined; the group's success is predicated on the personal responsibility of each participant to bring topics/issues from their daily life experiences. the group has gained identity and trust and has evolved over three years into two separate groups: an individual cf group and a couples cf group. ownership of these groups by those who attend is apparent. both the care center and the couples support group are forums for learning 'how to live with cf'. future goals will call for using identified quality of life tools to measure psychosocial and emotional growth in individuals through their participation over time in the group. sawicki, g. 1 ; asher, d. 1 ; dill, e. 2 ; sellers, d. 2 ; robinson, w. 2 1. children's hospital, boston, ma, usa; 2. education development center, newton, ma, usa background: advance care planning (acp) is an important tool to promote alignment of the patient's care with his or her needs, goals and values, and is particularly useful if the patient's ability to make decision becomes compromised. the purpose of acp is to develop a treatment plan promoting a high quality of life for those near the end of life. such planning is especially appropriate in persons with life limiting disease. minimal research exists on the advance care planning process of adults with cf.<p>methods: in the fifth survey wave of the project on adult care in cystic fibrosis (pac-cf), an ongoing longitudinal panel study of cf adults from 10 cf centers in the us, participants were asked to report on their opinions and experiences with advance care planning. the survey asked about opinions on advance care planning documents, communication about advance care planning with family and clinicians and whether a directive had been completed. clinical data were obtained from the participating centers. preliminary bivariate analyses were conducted to examine factors associated with completing an advance care directive.<p>results: 233/303 (77%) surveys were completed. the mean age was 34 ± 13 years, 63% were female, and the mean fev1 (% predicted) was 69 ± 28. 77% reported that they had spoken to someone about the care they would want if they became too ill to make their own decisions in the future, 65% had thought about whom they would want as their healthcare proxy, and 60% reported having specific wishes about the types of medical treatment they want or would not want if they became too ill to make decisions for themselves in the future. however, only 33% reported completing a healthcare proxy form, living will or any other type of written instructions concerning advance care directives. 80% of respondents reported feeling comfortable talking to their clinician about acp. 28% of subjects said that their cf clinicians have asked them about acp, and only 11% reported that they have discussed acp with their cf clinician. the following characteristics were significantly related (p<.05) to completion of an advance care directive: female gender (38% vs. 25%), age (mean 37 years vs. 33 years), having a college degree (41% vs. 23%), being married currently or in the past (38% vs. 25%), having children (41% vs. 28%), having diabetes (48% vs. 31%), having specific wishes about treatment decisions (46% vs. 15%), and reporting that a clinician had discussed acp with them (59% vs. 24%).<p>discussion: though the majority of cf adults report thinking about, communicating with family and deciding on their advance care wishes, only a minority report completing any legal documentation supporting their decision. additionally, very few cf adults report being asked about acp by their clinicians and even fewer report discussing acp with them. formulating specific wishes and discussing acp with a clinician are strongly associated with completing an advance care directive, suggesting that if clinicians were more active in talking to their patients about acp, patients are much more likely to complete advance care directives. efforts to improve clinician communication with cf adults around the issues of advance care planning are needed. objectives: caregiver involvement in children's cf treatments fosters better adherence and improved health outcomes. several studies have suggested that parents of children with cf report elevated symptoms of depression (quittner et al., 1998) , however, there is no data on the effects of caregiver depression on adherence. adherence to treatment is difficult to measure in patients with cf, and prior research has relied primarily on self-report, which is likely to be inflated. the purpose of this study was to examine the effects of caregiver depression on adherence to enzymes using both parent-reported and electronically monitored enzyme adherence over a three-month period. in addition, we examined the relationship between depression, rates of enzyme adherence and short-term changes in weight. methods: as part of a larger intervention study at 3 cf centers, 89 children with cf ages 1 to 11 and their parents were recruited. parents completed a standardized measure of depression (cesd) at enrollment and were provided with mems caps that recorded the date and time of each bottle opening. three months later, the electronic data from the pill caps was downloaded and parents completed a structured interview reporting their child's adherence to all components of the cf treatment regimen. only the section addressing enzyme adherence was included in this analysis. standard health outcomes, such as weight and height, were also assessed at enrollment and 3 months later. results: preliminary analyses indicated that caregivers reported elevated levels of depression, with 30% scoring in the clinical range. rates of adherence to enzymes were poor (43% at home and 48% at school). caregiver depression was negatively associated with adherence, with depressed caregivers demonstrating lower rates of adherence (11 percentage points). adherence to enzymes was associated with changes in weight, with a 100% adherence translating into 5 percentile points of weight gain. final analyses will also include parent-reported adherence to enzymes. conclusions: a substantial number of parents scored in the clinical range on a depression screening measure. in addition, depression was associated with poorer adherence. rates of adherence to enzymes were surprisingly low, both at home and at school. furthermore, poor adherence was associated with a decrease in weight three months later. caregiver depression appears to be under-diagnosed and these results suggest that screening and intervention may be warranted. adherence to enzymes should also be targeted in clinical interventions. funding was provided by nih grant #hl69736 of non-compliance with medical treatment and greater behavioral and emotional distress. adherence is particularly problematic during adolescence, however, few family-based interventions have been developed to target adherence behaviors in this population. the goal of this study was to compare the effects of behavioral-family systems therapy (bfst), an empirically-supported treatment, to both family education (fe) and standard care (sc). methods: as part of a larger intervention study, 117 adolescents with cf ages 10 to 17 and their parents completed the conflict behavior questionnaire (cbq) and the parent-adolescent relationship questionnaire (parq) developed by robin and foster (1989) . adolescents completed these measures for each parent and both parents, if available, completed them with respect to their teen. the problem-solving, communication, and beliefs subscales from the parq were administered. participants were then randomly assigned to one of three treatment arms: bfst, fe or sc. families in the bfst group received ten 90-minute sessions over a 6 month period, including family problem-solving, communication skills training, and cognitive restructuring. families assigned to the fe group received ten 90minute psychoeducational sessions over 6 months aimed at increasing knowledge about cf and its management. those in the sc group received their usual care at the cf center. the cbq and parq were completed pre, post and 6, 12 and 18 months following the intervention. results: adolescents reported that the bfst intervention, relative to the other two, improved their communication with their primary caregivers (p < .03) and caregivers improved their communication with their adolescents (p < .02). in addition, caregivers reported improved problem-solving with their adolescents (p < .04). on the cbq, bfst reduced both adolescent and primary caregiver conflict and fe reduced caregiver conflict. no significant improvements were found for those in sc. conclusions: preliminary findings suggest that bfst is effective in improving communication skills and reducing conflict in adolescents with cf and their caregivers. future analyses will evaluate the effectiveness of bfst and fe on adherence and other family functioning measures over the course of the study. funding was provided by nih (hl #47064) background: cases of diabetes among people with cystic fibrosis (cf) have increased as life expectancy for these patients improves, yet the impact of this additional illness on daily functioning is under-researched. to explore this issue, a study was conducted comparing the views of patients with either cystic fibrosis related diabetes (cfrd) or type 1 diabetes mellitus (t1dm) about being diagnosed and living with diabetes. methods: qualitative research was used because the study was concerned with understanding diabetes from patients' perspectives. purposive sampling was employed to achieve maximum variation in terms of duration and type of diabetes experienced by participants. data were collected via semi-structured interviews, all of which were taped and transcribed verbatim. recruitment continued until no new ideas or insights emerged from additional participants. a framework approach to analysis was adopted. this involved coding and summarising interview data into charts to explore and develop main themes. initial analysis was undertaken by two researchers (st and cd) and amended after comments from the remaining authors. results: interviews, carried out with 11 cfrd and 12 similarly aged t1dm patients, lasted for an average of 40 minutes (range 20-60 minutes). the following themes were derived from the data collected: evolving vs fracturing identity; diabetes in context; self-management motivators. for patients with cfrd, diagnosis represented a progression in their health status, which called on them to adapt existing treatment regimens to accommodate this additional condition. in contrast, interviewees with t1dm had to re-evaluate their previous sense of self as 'healthy' and adjust to manag-ing a long-term complaint. these individuals were more likely to talk about diabetes in relation to a range of competing commitments (e.g. work or family related) and to describe feeling psychologically low due to diabetes compared to patients with cfrd, the latter depicting demands from their primary illness (cf) as a major obstacle to caring for diabetes. participants with cfrd recalled feeling lucky when told they would not face strict dietary restraints, which they associated with other forms of diabetes, and seemed less concerned about diabetic complications than those with t1dm. for interviewees with t1dm, a desire to reduce future health risks motivated their self-management efforts, whilst those with cfrd were driven by the negative effect poor control of diabetes had on their chest and weight. conclusions: both sets of interviewees found diabetes time consuming and, on occasions, frustrating to accommodate into daily life. findings from the study act as a reminder that patients manage their condition in the real world, against a plethora of other demands on their time and energy. in the narratives of participants with cfrd, these demands were often related to the existence of their primary illness. ing the course of development. the purpose of this study is to examine rates of medication adherence across a wide age span using an objective measure of adherence and to compare adherence to health outcomes. methods: patients with cf age 6 years or older who are prescribed azithromycin, colistin, hypertonic saline, pulmozyme, and/or tobi for at least one year are eligible. recruitment is ongoing. the previous year's medication refill records were requested from participant-identified pharmacies. a medication possession ratio (mpr) was calculated for each drug and was defined as the sum of all days of medication supply received during the 12 months divided by the number of days the medication was prescribed. values were truncated to 100% and averaged across all medications to obtain a composite score. medical records were abstracted to identify the prescribed drug regimen and health outcomes (e.g., lung function, exacerbations, bmi) over the same time period. results: thus far, 100 patients have joined the study (80% participation rate), resulting in 213 pharmacy records requested (86 unique pharmacies). of the first 48 participants with complete pharmacy data, the mean age was 21.5 years (sd=11.9; range= 7-69) and 56% were female. the table presents mpr data for each drug and composite score overall and stratified by child, adolescent, and adult participants. the complexity of the drug regimen and age were significantly correlated with the composite score (rho= -.39 and rho= -.29 respectively; p<.05). future analyses will compare the composite score and each drug's mpr with health outcomes. con-clusions: participants had suboptimal medication adherence similar to that reported in the literature. as mpr provides the maximal possible level of adherence, actual adherence may be even lower. poor adherence spanned the age groups and was associated with regimen complexity. children had the highest adherence, but also the least complicated drug regimen. these results suggest that obtaining pharmacy records is a viable means to objectively assessing medication adherence. moreover, results suggest that interventions targeting medication adherence may be a strategy for improving health outcomes, particularly for adolescents and adults. * fewer than 5 participants within this cell 564 (cf) whose exchange capacities for oxygen and carbon dioxide are already diminished. lack of rem and delta sleep have significant implications for the daytime functioning of pediatric patients with cf, potentially leading to impaired memory and attention which would reduce their ability to complete typical activities, such as school work and disease-specific tasks related to self care and adherence. based on these clinic findings, the authors are beginning a prospective study on the effects of sleep on neuropsychological functioning, well-being and compliance in pediatric patients with cf. future research should examine the relationship between decreased slow wave, rem sleep and psychosocial outcomes, including attention, memory, and health-related quality of life. there are diagnostic challenges related to non-classic cf (knowles and durie, 2002) particularly patients presenting in adolescence or adulthood with obstructive azoospermia, chronic sinopulmonary disease and chronic or acute, recurrent pancreatitis. practitioners are often unable to provide clarity to patients about diagnosis, disease course, and prognosis. potential harms and benefits of relaying a clear or unclear diagnosis may be psychological rather than physical and have long-term implications for mental and social well-being. the aim of the current study is to assess the psychological impact of diagnostic information for adults presenting with a cf phenotype, be it confirmatory (cf or most likely not cf) or unclear. method: we administered self report measures pertaining to psychological state, cognitive appraisal and uncertainty in a welldefined cohort of adult patients. presenting symptoms of patients include chronic sinopulmonary disease, obstructive azoospermia, pancreatitis, and those presenting with more than more cf phenotype. patients completed the self-report measures on two occasions: at the time of diagnostic testing and 6 months after being counseled and notified of the diagnostic results. at the time of the initial assessment, the subjects were unaware of the test results and had not been seen by a cf physician. results: we provide interim observations on 70 patients (sinopulmonary (n=34), obstructive azoospermia (n=20), pancreatitis (n=4), and multiple phenotypes (n=6) who completed the initial assessment. at the time of diagnostic testing, the level of depression, hostility, anxiety and interpersonal sensitivity were elevated in each group (t score > 65); male patients reported significantly greater depression, anxiety and interpersonal sensitivity than female patients. compared to published normative data of patients with mixed chronic illnesses, patients presenting with sinopulmonary or pancreatic disorders reported significantly more uncertainty(p < .05). to date, 26 of these patients have completed the measures 6 months after receiving the diagnostic information. preliminary review of results shows that the level of depression, hostility, anxiety and interpersonal sensitivity remained elevated while the degree of uncertainty decreased. among the 26 patients who completed assessment at 6 months, 17 patients were told that it was unlikely that they had cf, 7 were told that they had mild cf, while 2 were given an unclear diagnosis. those who were told that they did not have cf reported more uncertainty than those who were told they had cf (p < .05). detailed analyses related to outcome diagnosis will be conducted at the time of study completion (october 2007) . when this study is complete, the information will assist in our understanding of the psychological impact of a genetic diagnosis at an older age, identify issues confronting those individuals and help to establish appropriate paradigms for delivering complex information about genetic diseases. supported by grants from niddk [scor] and the canadian cf foundation. (1). in accordance with these guidelines, we report our experience in using our nutrition action plan (nap) in patients with suboptimal nutrition. objective: to determine if implementing our nap would improve the bmi of these patients. methods: our nap is a written contract between the patient, caregiver and cf team. for clarity of nutritional goals, the nap is based on the colors of a traffic light. the red zone indicates a bmi less than the 25th percentile and poor nutritional status. the yellow zone indicates a bmi between the 25th and 49th percentile and fair nutritional status. the green zone indicates a bmi greater or equal to the 50th percentile and desired nutritional status. within each color zone, specific recommendations to improve nutritional status are listed. the ultimate goal for each patient is reaching the green zone. the caregiver, patient and nutritionist review the current nutritional status and a mutually accepted weight gain goal is established. the goal is recorded on the nap which is then signed by the nutritionist and patient. this contract establishes a commitment to achieve the goal by the next clinic visit. a reward system is implemented in conjunction with the nap. for patients reaching their goal, a previously agreed upon prize is given. patients achieving some weight gain but falling short of their goal receive a gift such as candy or small toys that serve as an incentive for continued weight gain. patients chosen for inclusion into this study demonstrated a bmi less than the 50th percentile; those on growth hormone, appetite stimulants or chronic systemic steroids were excluded. results: twenty-one patients, 12 male and 9 female, with a mean age of 11.6 years (range 3 to 18 years) were included in this study. fifteen patients demonstrated an improvement in their bmi with use of the nap: 7 improved by 1-10%; 2 improved by 11-20%; 3 improved by 21-30%; 1 improved by 31-40%; 2 improved by 41-50%. six participants had a negative change in bmi which we attribute to family upheaval, missed appointments with the cf team, exacerbations of disease and non-adherence. conclusion: we intervened in 21 patients with a bmi less than the 50th percentile using a nap and a reward system. the results of this pilot study suggest that our nap and reward system may be of benefit in achieving positive gains in bmi. reference: (1) previously it was rare for women with severe cf to undertake pregnancy and doctors tended to advise against pregnancy if the forced expiratory volume in one second (fev1) was less than 60%. the attitudes of doctors and patients are changing and many women living their lives with cf nowadays chose to undertake pregnancy despite the severity of their health problems. over the last 15 years 102 females (age range 16-56 years) have attended the newcastle adult cf centre. there have been a total of 25 pregnancies involving 20 women with 24 live births (1 ectopic). a further 3 women had termination of unwanted pregnancy. mean age at the time of pregnancy was 21 years. we compared an age-matched cohort of never pregnant women to see if there were differences in key variables making successful pregnancy more likely. the mean fev1 was 61% (range 30-112%) and 58% (range 10-91%) of predicted in the pregnant and non-pregnant group respectively (p=0.36); rates of chronic pseudomonas aeuriginosa infection were 14 (70%) and 12 (57%) respectively (p=0.52). 2 cases of burkholderia cenocepacia were identified in the non pregnant group. there were no cases in the pregnant group (p=0.21); 7 (35%) had diabetes with 5 patients having cf related diabetes prior to pregnancy and 2 patients developing gestational diabetes; this compared with 9 (21%) of non pregnant women (p=0.75); there was impaired nutritional status within both groups with a mean bmi of 18.9 pre pregnancy (range 14-27) and 20.1 in the non pregnant group (range 14-31) (p=0.28); 5 (25%) and 3 (14%) patients had had previous gastrostomy feeding (p=0.45). pancreatic insufficiency was present in 17 (85%) and 19 (90%) patients in respective groups (p=0.66). no child had cf but one child had complex congenital heart disease. there were no maternal deaths but 2 patients subsequently underwent lung transplantation and 2 patients died leaving young children (ages 5 and 7 years). 2 patients in the non-pregnant group have also died. conclusion the profile of women with cf undertaking pregnancy is now similar to the overall cf population. the woman's decision to undertake pregnancy is often not directly related to the severity of her disease. amongst those who successfully completed pregnancy maternal and fetal outcomes are generally favorable. objectives: health-related quality of life (hrqol) instruments provide important information on disease progression and are increasingly used as patient-reported outcomes (pros) for behavioral and pharmacological trials (goss & quittner, in press) . recent advances in medical care have improved the long-term stability of pulmonary functioning, which has made it more difficult to detect small improvements in fev1% predicted. new outcome measures are needed; the cystic fibrosis questionnaire-revised (cfq-r; quittner et al., 2004) , a disease-specific hrqol measure, has shown promise in this regard. the purpose of this study was to examine the longitudinal relationships between fev1% predicted, weight percentiles, and cfq-r scores for adolescents with cf. methods: as part of a larger study evaluating adherence interventions, 117 adolescents with cf, ages 10 to 17, and their parents were enrolled at six centers. parents reported on their adolescents using the cfq-parent version, and adolescents completed the cfq-teen/adult version as a selfreport. measures were completed pre and post treatment and at 6, 12, and 24 month follow-up visits. spirometry and weight data were collected at these same time points. results: empirical bayes estimates were used to examine change over time. preliminary results indicated that adolescents' scores on the cfq-r respiratory scale were significantly and positively correlated with changes in pulmonary functioning. parents' scores on the cfq-r vitality scale were also correlated with changes in pulmonary functioning, with higher cfq-r vitality scores associated with better lung function. in addition, parents' scores on the cfq-r weight scale were significantly and positively correlated with changes in adolescents' weight. conclusions: these results support the validity and sensitivity of the cfq-r to changes in both pulmonary functioning and weight for adolescents with cf. additional analyses will be conducted to determine the relative stability of hrqol scores over time and to examine other cfq-r scales that may reflect positive changes in adolescent functioning (e.g., reduced treatment burden background: development of cf specific patient symptom tools as outcome measures is a critical step toward the development of potential new therapies for cf. we have developed a novel instrument to assess respiratory symptoms in cf patients. methods: we conducted 25 in-depth qualitative interviews using the day reconstruction method and 9 cognitive debriefing interviews (3 adults, 4 youth, and 2 parents) at two cf programs, the university of washington and children's hospital and regional medical center. interviews were conducted until no new symptoms were mentioned by interviewees (i.e., data saturation). the interviews were audio-recorded, transcribed, coded and analyzed for themes. results: six pulmonary symptoms were identified in the interviews: cough, sputum production, wheeze, chest tightness, difficulty breathing/shortness of breath and fever. in addition, the most commonly cited activity and emotional impacts identified in the interviews were also included on the questionnaire. these emotions included: frustration, sadness/depression, irritability, worry, difficulty sleeping, and activities included time spent sitting or lying down, reduction of usual activities, and missing school or work. in all, eight symptom items were selected for inclusion on the self-administered questionnaire (see table) . as a result of the cognitive debriefing interviews, we changed initial language and adjusted the response options on some of the items in order to create greater distinctions between them. no important issues were felt to be omitted by patients who were interviewed. conclusions: using qualitative inductive methodology, we have created a novel patient reported outcome measure for cf. further study will be required to assess validation of the instrument. or more signs and symptoms occurred for more than two days, antibiotics were ordered with physician input and phone follow-up in 3 days. if symptoms occurred less than two days, chest clearance was increased with phone follow-up in 24 hours. the initial algorithm (ia) was piloted by 2 nurses on 25 patient calls on the emr and reviewed for adherence. subsequently, the cf phone note was revised, expanding the symptoms to include gi issues, last visit date and last hospitalization. the revised algorithm (ra) was used for a one month trial period with the entire nursing staff. a total of 22 patient calls relating to pulmonary exacerbation were again reviewed for adherence to the algorithm. results: the review of 25 phone notes following the ia found 14 (56%) of the phone notes to be adherent to the assessment and treatment plan on the algorithm. several exceptions included calls received for gi issues, medications, complaints of general pediatric symptoms, and lab results. the phone follow-up within 24-72 hrs was also difficult to accomplish secondary to nursing responsibilities and difficulty locating the patient. the review of calls following the ra found 19 (86%) notes were adherent to the algorithm. discussion: the revised algorithm and cf phone note for pulmonary exacerbation improved consistency in nursing assessment of signs and symptoms, evaluation of the current plan of care, and triggered appropriate interventions based on symptoms and length of illness. this improvement was particularly pronounced among the part-time rns who found it easier to obtain a more accurate history of patient's illnesses without asking for assistance from other cf team members. since the algorithm and cf phone note are newly implemented, tracking for consistency with the cf phone note will continue, revisions will be made as needed, and phone notes for conditions other than pulmonary exacerbation may be developed. methods: in 2000, helen devos children's hospital integrated research coordinators (rc) from the spectrum health research department into the cf care team. this model incorporates two key aspects: 1. a shared resource of experienced, centrally educated and trained research coordinators (rc) with three rc's having a primary cf focus 2. incorporation of rc's into helen devos cf care centers' multidisciplinary team for weekly contact with patients. using the cf registry consent as a starting point of research discussion with the patients and families, the rc's present new and upcoming opportunities and answer questions about research. with attendance at local and national cf meetings, rc's have expanded their cf knowledge, networked with other rc's and explored new research trials. our site also integrated education about various aspects of research into both the clinical staff and cf family education meetings. a review of the research database was completed to assess our cf center's research program. results: over the 7 years reviewed, participation in clinical research trials has seen significant growth and patient participation has exceeded 250 patients enrolled into clinical studies. conclusion: utilizing a team of dedicated rc's from the research department has proven to be effective in advancing our cf center's clinical research program. discussion: to meet the growing demand of patients needed to participate in clinical trials, cf centers will need to provide research education to both the patients and families and the clinical team, improve on skills needed to identify and enroll our patients into clinical trials and include the rc's into the multi-disciplinary cf patient care team. centralized research represents an excellent model for cf centers that want to excel in clinical research. integration of rc's into the cf care team, along with education initiatives for both clinical staff and patients and families has led to successful participation and enrollment in clinical trials. further studies need to be done to better identify barriers that patients and families have with participation in research. the goal of "cystic fibrosis transition care" is to ensure that youth with cystic fibrosis (cf) are adequately prepared to participate in the management of their health condition into adulthood and as they graduate to the adult health care system. in canada, it is unknown if, or to what extent, cf transition care is practiced in individual clinics. we therefore surveyed all canadian pediatric cf clinic coordinators (n=27)using a brief standardized questionnaire; 23 surveys (85%) were completed and returned for analysis. findings: all 23 responding clinics transfer their pediatric patients to a distinct adult cf clinic, however almost half of these share one or more team members between the two clinics. transition care is recognized and practiced by 74% of responding clinics: 22% follow a formal transition program, and 52% follow an "informal program". "informal" practices vary widely from one clinic to another. formal programs were created by 5 individual clinics, and share the common properties of being based on current research, having set goals, and utilizing a tool to document the process. the average age of transfer to the adult clinic is 18 years. most clinics have "rare exceptions only" to delaying the age of transfer, the most commonly cited reason being "very ill/palliative patient". a small number of clinics also cited "intellectually challenged patient" and "reluctant patient" as reasons to delay transfer. only 30% of pediatric clinics hold a formal "transition" or "graduation" clinic, which allows adult team members to be introduced to youth before the actual transfer of care takes place. conclusions: although sharing team members between pediatric and adult clinics may ease the change from pediatric to adult care, it can also create barriers to the transition process. therefore, it is encouraging to learn that the majority of canadian pediatric cf clinics view adult care as distinct from pediatric care, and that there is a consistent age of transfer across the country. transition preparedness is also recognized as an important component of care, as evidenced by the number of clinics following a formal or informal transition process. it remains undetermined, however, whether in general there is adequate preparation for adult care. with only 22% of clinics following a formal transition process (and 26% not following any type of transition program), further assessment seems warranted and may reveal inadequacies requiring remediation. future direction: to strengthen cf transition care in canada, a "patient readiness to transition" questionnaire has been developed and will be administered to canadian cf patients transferring to adult cf clinics in 2007. analysis of this questionnaire will provide insight into how well canadian cf clinics are preparing their youth for transfer to adult life, and may provide directions for further improvement. nearly all females with cf now survive into adulthood, and advice regarding pregnancy and contraception is becoming increasingly important as part of their sexual health education. we set out to identify the level of knowledge of these issues in patients attending our large adult cf unit. using a structured questionnaire, we surveyed 49 consecutive cf females (age range 17 to 42 years) attending routine cf clinics. we asked about knowledge and usage of contraception, and issues relating to pregnancy and its possible effect in cf, ensuring the opportunity for a one to one discussion with the individual on completion of the questionnaire. only 29 (60%) claimed to have had previous advice relating to contraception and 23 (47%) pregnancy education. of these, 23 (81%) stated that this was from adult cf nurse specialists, 17 (57%) from adult cf physicians and the remainder from other sources (general medical practitioners, written literature, and the internet). only 7 patients (16%) recalled that they had been given information in the paediatric sector before transition. the majority of all advice was given verbally. of the 29 (60%) who were using some form of contraception (10 [33%] condoms, 9 [30%] combined pill, 3 [11%] depot injection, 2 [7%] a coil, and 6 [19%] the mini pill); 10 (33%) had experienced problems, and in some cases patients had been misinformed about the reliability of their contraceptive choice. as regards pregnancy, 2 (4%) had undergone previous termination and 8 (16%) already had children. thirty six (73%) had considered becoming pregnant but 46 (93%) said they would discuss this with a member of the cf team beforehand: 35 (71%) were aware this might impact on their health. of these, 32 (91%) believed a reduction in lung capacity to be the biggest possible problem. other problems identified were alteration of medication, tiredness, and weight loss or gain. overall, 29 (60%) felt they did not have enough information regarding pregnancy in cf and 25 (51%) that insufficient contraceptive advice was given, expressing a wish for further knowledge. conclusion this survey has shown that a significant number of adult females with cf require further education about contraception and pregnancy. in some cases the advice they had already been given may not have been appropriate for patients with cf. furthermore, few patients appeared to have had effective counselling in the paediatric sector, despite the risk of pregnancy. we are working with patients and paediatric colleagues to improve the education in this important area for our women with cf. the state of minnesota added cystic fibrosis to the newborn screening panel on march 1, 2006. one-year follow-up to date reveals 286 infants have screened positive, of the screen positive, 23 have been diagnosed with cystic fibrosis, 30 infants are pending sweat chloride tests and evaluation. of this population 8 infants are being followed by the minnesota cf center. the university has a comprehensive, interdisciplinary team available to provide care to infants, children and adults with cystic fibrosis. in response to newborn screening for cystic fibrosis, the center has implemented a prophylactic care program to include clinical care through early intervention and education within the first two weeks of life. benefits of collaborative care have been previously identified in the literature, showing improved quality of care with increased patient satisfaction, lower mortality, and improved outcomes. infants who are identified by newborn screening and receive earlier treatment have the potential for improved physical health and development. the inclusion of comprehensive genetic consultation as an integral component of cf education, allows for informed reproductive decisions. the newborn screening intervention plan is supervised by a nurse practitioner and consists of initial evaluation, initial therapy implementation, and a teaching/consultation schedule, which covers a minimum of four appointments scheduled over 2 weeks. follow-up visits are scheduled within 24 hours of referral and then at one week, 10 days and 2 weeks post referral. the schedule is adjusted based on infant acuity and family needs. initial evaluation consists of the confirmatory sweat chloride testing and/or genetic mutation analysis, serum laboratories, chest x-ray, stool for fecal elastase, and nasopharyngeal culture. general, supportive teaching with evidence from the literature is provided. preliminary results to date demonstrate that our infants have had no pulmonary exacerbations, no pulmonary hospitalizations with pulmonary functions obtained in over half of the infants. nasopharyngeal cultures obtained at the time of ascertainment demonstrate newborn screening patients have positive cultures for staphylococcus aureus, haemophilus influenzae, escherichia coli, streptococcus pneumoniae, klebsiella pneumoniae, acinetobacter junii and alcaligenes faecalis. one infant was culture positive for pseudomonas aeruginosa at the first out-patient clinic visit. this demonstrates colonization of organisms prior to clinic exposure. through minnesota's early intervention and education plan, the families will be more adherent to prescribed care, which will result in improved cf infant outcomes as monitored by number of exacerbations, pulmonary functions and potential complications. a detailed outline of medical intervention, education, and outcomes will be presented. cf-pediatric centre we developed a malabsorption blood test (mbt) using 2 odd-chain length fatty acids, pentadecanoic acid (pa) and heptadecanoic acid (ha), to assess fat absorption in subjects with cf. pa is a free fatty acid, while ha requires hydrolysis of triheptadecanoic acid (tha) by pancreatic enzymes for absorption. objective: to determine the mbt reproducibility in healthy subjects and subjects with cf. methods: subjects with cf ingested a liquid test meal including 2 fats (pa and tha) on 3 occasions at least 5 days apart. for each subject, a standard dose of pert (80,000 lipase units) or the subject's usual dose (if higher) was given with each test meal. serum was analyzed for pa and ha levels at baseline and then hourly for 8 hours. a non-compartmental pharmacokinetic analysis was performed using pa and ha concentration-time data from subjects with cf. c max was directly observed from the individual subject profiles, and auc was calculated using the linear trapezoid rule. summary statistics (mean±s.d.) were calculated for all parameters. reproducibility of the mbt was also assessed in healthy adult controls tested on 3 occasions. between subject (bsv, %) and within subject variability (wsv, %) was calculated for pa, ha and the ha/pa ratio. results: bsv (%) and wsv (%) of pa and ha absorption are presented in the table for 9 subjects with cf (age 17.8±3.9 y, 4 females) and 6 healthy adult subjects (age 25.8±3.8 y, 4 females). as expected, the mbt had greater reproducibility in healthy controls than in subjects with cf. in healthy controls, the reproducibility for pa and ha absorption was comparable; there is less wsv than bsv, and the ha/pa ratio has the best reproducibility. in subjects with cf, pa and the ha/pa ratio show less wsv than ha. conclusion: wider variability in fat absorption in subjects with cf reflects the complex interactions of the biliary, pancreatic and intestinal factors background: the international, multi-center gene modifier study of cystic fibrosis liver disease (cfld) initially enrolled 133 patients with severe liver disease (i.e. cirrhosis and portal hypertension). for the initial population despite severe liver disease with portal hypertension, many of these patients had normal liver biochemical function tests and/or inr (24-68%), depending on the test. the mean age of enrollment into the study was 20 years, with the mean age of cfld diagnosis of 11 years. candidate gene testing revealed an increased prevalence of the alpha-1 antitrypsin z allele in cfld patients, particularly in females, as compared to cf patients (>15 yrs.) without cfld, and an association with tgfβ1 variants (-509 and codon 10) in males smoothing reference centile curves: the lms method and penalized likelihood hepb) and inactivated hepatitis a (hepa) vaccines in cystic fibrosis (cf) patients chronic liver disease (cld) remains the second mortality cause in cf. we evaluated the immunogenicity of hepa-and hepb-vaccines in cf-patients because it is described lower in patients with cld. patients and methods: blood samples of 153 cf-patients aged 0.2-57.8y (mean 13.5y) were tested twice (time interval 1y) by chemiluminescent microparticle immunoassay to asses the presence of hbs-ab and ha-igg, and their vaccination status was recorded. seronegative patients for hbs-ab and/or ha-igg were vaccinated in between tests with engerix®, havrix® or twinrix®. results: 115/120 (95.8%) patients tested positive for hbs-ab. 59/115 cld (9 steatosis and 10 cirrhosis). 3/115 tested positive at first but negative at present data are incomplete for hbs-ab and ha-igg for 33 and 70 patients. conclusions: 1. immunogenicity of hepa-and hepb-vaccines is comparable in cf-patients and healthy subjects. 2. cf-patients are at risk for cld and seroconversion must be checked after vaccination. 3. vaccination records are important as numbers of antibodies may decline beneath the detection limit in time methods: lf was assessed in 53 children with cf, aged 8-12 yrs, using forced expiratory volume (fev(sub)1(/sub)% predicted). body composition was measured using a reference four-component model(sup)1(/sup) (4cm) allowing accurate evaluation of both fat mass (fm) and the components of fat-free mass dual-energy xray absorptiometry (dxa; lunar prodigy, g.e. medical systems) was used to measure fat, lean (non mineral) and bone mass and skin-fold thicknesses (bicep, tricep, sub-scapular) were measured. strength of relationship was assessed using pearson's correlation coefficient (r) and significant body composition components were fitted into a regression model. difference in fev(sub)1(/sub) between the sexes was assessed with an independent t test. results: boys with cf did not differ from the reference population conclusion: our results confirm that body fat, but not ffm or bone mass, are related to the severity of impaired lf in children with cf. it is likely that the significant association between fev(sub)1(/sub)and fat in girls but not boys reflects the poor body composition of the girls with cf. longitudinal follow-up of these children should indicate whether this sex difference persists after puberty. given the fact that prognosis is worse in girls the sex difference we've identified merits more attention. 1fuller nj et al. four-component model for the assessment of body composition in humans: comparison with alternative methods and evaluation of the density and hydration of fat-free mass conclusions: these results show that pancreatic status plays an important role in relation with pufa status in cf patients and particularly in (the initial fatty acids in the n-6 and n-3 pathways) in neutral lipids, nonesterified fatty acids, and phospholipids this may, in some way, account for the fact that 18:2n-6 is more readily metabolized, and therefore depleted, in cf animals than in wt mice. conclusion: dha supplementation increased the localization 539 does an integrated clinical and nutritional approach prevent this case control study examines the impact of clinical approach on pre and post-cfrd clinical course. 48 patients with cfrd (mean age 25.9+5.9yrs) matched to 48 cf controls (25.9+6.3yrs) for age, sex and pseudomonal status had parameters of clinical status and nutritional intervention recorded annually from six years pre diagnosis of cfrd to two years post. weight and body mass index (bmi) were lower at all time points to diagnosis of cfrd (ns) but were stable as a % of control values intravenous antibiotic treatment intensified, peaking at one year post diagnosis 47.7 days/yr (cfrd) v 34.7 (cf) ns but there was no difference in nebulised antibiotic use. an aggressive clinical approach prevents nutritional decline and delays respiratory decline until the year preceding diagnosis of cfrd data show mean (sd) or % 1,2 ; battezzati with advancing age insulin secretory defects and insulin resistance cause glucose intolerance and diabetes in an increasing proportion of cystic fibrosis (cf) patients. prediction of diabetes development in cf is made difficult by unique features: many patients are normoglycemic or even hypoglycemic after overnight fast, and there are repeated changes of glucose tolerance status from normal to diabetes and vice-versa, for many years and for unclear reasons. aim of the study was to detect predictive factors of definitive cf related diabetes (cfrd) development in patients undergoing oral glucose tolerance test (ogtt) evaluations routinarily. methods. starting from 2002, all patients followed at the cf center in milan aged >10 years and without established cfrd undergo ogtt yearly. among those who received their first ogtt between 2002 and 2004, 14 had developed definitive diabetes by 4 µu/ml, and 1.4±0.1 vs 1.4±0.2 ng/ml). glucose (p=0.010) and insulin aucs (p=0.030) were the most important predictive variables, respectively directly and inversely related to cfrd development (pseudo r 2 for the model: 0.581, p<0.001). glycated hemoglobin and baseline glucose concentrations were directly related with outcome at univariate analysis whereas c-peptide concentrations were inversely related. in contrast, no relationship emerged between insulin-sensitivity indexes and outcome. anthropometric (weight, height z scores, bmi) and pulmonary function indexes were also unrelated. conclusions. insulin secretory defects are an important determinant of subsequent cfrd developement response rate was 18/18 in group a and 17/18 in group b. group a included 5 males and 13 females, mean age was 52 months, sd 22.34; group b included 4 males and 13 females, mean age 52 months, sd 20.91. followgroup b parents do ("poor-very poor" health 6% vs 47%, p<0.002), but over time health perception in parents of children with cf improves, and the gap vanishes ("poor-very poor" health in last year 6% in group b). post-diagnosis anxiety, depressive symptoms, sleep disturbances, mood changes, and nervousness are less frequently reported in group a (62% vs 94% p<0.002), and the diagnosis was considered to affect the parent-child relationship more in group b than in a (p<0.01). the two groups did not differ in the assessment scores of internalizing, externalizing and total behavioural/emotional problems. however, group b performed worse than the general control population average (mean 12.65 sd 6.72 vs mean 9 sd 6.6, p<0.04). thirty-five% of parents in group a and 53% in group b changed their family planning projects following the diagnosis physical health and anxiety symptoms: does monitoring mediate the relation? results: physical health, monitoring, and anxiety symptoms were related. children who perceived themselves to be relatively healthy were less likely to have an information processing style characterized by high levels of monitoring (r = -.39, p < .05), and they reported fewer trait anxiety symptoms (r = -.50, p < .01). monitoring, as hypothesized, was associated with increased trait anxiety (r = .52, p = .001). low bmi, a potential indicator of poor nutritional status and physical health among youth with cf, also was related to higher monitoring (r = -.34, p = .05) and trait anxiety (r = -.29, p = .10). tests of mediation indicate that monitoring partially mediated the relation between poor health and trait anxiety for both child report of physical health (goodman test: z = 1.86, p = .06) and for bmi (goodman test: z = 1.67, p = .09), but not when parental report or fev1 were used to assess youth's physical health, or when parental report of youth's internalizing symptoms was used as the outcome. conclusion: youth who tend to generally scan for and are attentive to changes in their environment, which may include their internal physical environment, were found to report more anxiety. moreover, this monitoring style appears to partially mediate the relation between physical health and anxiety among youth with cf. if monitoring is found to be a risk factor leading to an increase in future anxiety symptoms among youth with cf data was analysed using a thematic analysis: "framework" 3 results: young people say that befriending is fun, offers opportunities for new experiences, a confidant outside the family, and gives them a boost psychologically. carers see befriending as confidence building for their children, providing time out for themselves, and helping with the big questions. befrienders see their role as mentoring, broadening young people's horizons and providing a safe place physically and emotionally. challenges include: forming and ending relationships, having multiple befrienders, ongoing support and training for befrienders, maintaining boundaries, sibling rivalry, and cost. conclusions: befriending is a new innovation in cf, and has the potential to make a difference to young people's lives. careful planning at the outset, ongoing support for befrienders and regular evaluation are essential factors in ensuring its success good practice in befriending services for people with learning disabilities qualitative research practice sage london 552ଙ in addition, there was high use of non-cf vitamins (n=102) and protein shakes (n=88) which were not included as a cam therapies. when prayer was excluded (which was used by 63% of respondents), 53.1% of the patients are still using some form of cam. the most commonly used cam therapies in cf were relaxation therapies (n=83), massage therapy (n=61), chiropractor care (n=59), herb/plant product therapies (n=53), homeopathy (n=19), and yoga (n=19). an analysis of the relaxation therapies revealed that deep breathing exercises (n=80) were the most common and frequently combined with other relaxation techniques: meditation (n=43), progressive relaxation (n=31) and guided imagery (n=24). the most common herb/plant products used were: echinacea (n=33), garlic (n=21), selenium (n=20), ragweed or chamomile (n=17) and ginseng (n=14). the use of possible cf specific cam therapies were fish oils/omega fatty acids (n=37), glutathione (n=13), docosahexaenoic acid (n=11) and curcumin (n=9). in conclusion, cam is widely used by the cf population with "prayer for health" being the most common modality, however, many patients are utilizing multiple cam therapies (data not presented) within the framework of a weighted satisfaction model of quality of life, we investigated the importance ratings of adolescent and adult patients regarding disease-specific aspects of living with cf. method: 108 outpatients (aged 15-47 years, m=27.0, sd=7.9; fev1 20-125%, m=62.6, sd=26.2) repeatedly filled in the cf-specific module of the questions on life satisfaction (flz-cf) to measure satisfaction with nine cf-specific aspects of life in relation to the subjective importance of each life domain. a ranking list of the most important life domains across the study group was determined, and intra-individual changes of the importance scores were analysed. associations of importance ratings with changes of pulmonary functioning (fev1%) were examined. results: the most important aspects (% of "very important" or "extremely important" answers) are sleep (96.2%), integration of therapy into daily routine (94.4%), breathing (89.8%), gastrointestinal functioning (86.9%), and eating (86.9%), less important is understanding by others (44%) 1,5 ; durieu, i. 4,5 1. département d'information médicale, hospices civils de lyon dr. von hauner children`s hospital helios hospital, e.v. behring, berlin, germany supported by the cystic fibrosis foundation leroy matthews physician scientist award, the national heart, lung, and blood institute cystic fibrosis foundation therapeutics inc. symptoms 571 patient-reported respiratory symptoms in cystic fibrosis: initial validation children's hospital and regional medical center the first author is supported by a second year clinical fellowship from razvi, s. 1 ; quittell, l.m. 1 ; sewall, a. 2 ; marshall, b.c. 2 ; saiman, l. 1 1. pediatric pulmonary medicine, columbia university, new york, ny, usa; 2. cystic fibrosis foundation, bethesda, md, usa significance: significant improvements have been made in diagnostic and therapeutic strategies for cf patients in recent decades. we hypothesized that these changes could potentially impact cf respiratory microbiology. thus, we examined longitudinal trends in the annual incidence and prevalence of cf respiratory pathogens from 1995 to 2005.methods: cf foundation patient registry data, estimated to include 90% of the cf population in the u.s., was utilized in this analysis. patients were included if results from at least one respiratory culture were in the registry from january 1995 to december 2005. patients were excluded after organ transplantation. to avoid misclassification of incident cases, a retrospective review of registry data from 1985 to 1994 was performed to establish the culture status of included patients prior to 1995. thus, incident cases were subjects with first detection of a given pathogen in a given year. prevalent cases were defined as subjects with at least one positive respiratory culture for a given pathogen in a given year. all submitted culture results were included.results: the number of cf patients submitting registry data increased from 19,735 in 1995 to 23,347 in 2005 . the proportion of subjects meeting inclusion criteria remained relatively constant (85% to 91% per year). the median age of the study cohort increased from 13.1 years in 1995 to 15.1 years in 2005. during the study period, the incidence of hemophilus influenzae remained stable (10.3% to 10.6%) as did the prevalence (15.3% to 17.3%). the incidence of pseudomonas aeruginosa ranged from 20.8% in 1996 to a peak of 24.2% in 2004. the prevalence of p. aeruginosa declined from 60.4% in 1995 to 56.1% in 2005. there is a trend for both an increasing incidence and prevalence of staphylococcus aureus; the incidence increased from 21.7% in 1995 to 33.2% in 2005 and the prevalence increased from 37.0% in 1995 to 52.4% in 2005. the age specific prevalence of s. aureus remained highest in children aged 6-17 years. the incidence of methicillin-resistant s. aureus (mrsa) increased from 0.1% in 1995 to 7.0% in 2005, with a parallel increase in prevalence from 0.1% to 17.2%. the highest prevalence of mrsa was noted in subjects 18 years of age and older. the incidence of burkholderia cepacia complex decreased from 1.3% in 1995 to 0.9% in 2005, while the prevalence remained relatively stable (range 2.9% to 3.6%). both the incidence and prevalence of stenotrophomonas maltophilia increased (incidence: 2.6% to 6.4%, prevalence: 3.5% to 12.4%).this retrospective study aims at assessing the respiratory function and the cystic fibrosis (cf) co-morbid conditions according to the patients' nutritional status.method: data were collected from the french observatory (onm) 2004 (n=4533) (vlm, ined). the last year data for weight (w), height (h), fev1, fvc were extracted from the database (exclusion criteria: transplan-tation); were also analysed the genotype, pancreatic enzymes consumption, pseudomonas aeruginosa (pa) colonization, cirrhosis, diabetes, pulmonary transplantation and mortality (sas 9.1). we used international age-adjusted standards for bmi z-score for children (c) and bmi for adults (over 18 years) (a) (cole tj bmj 2000) to define underw (uw), normal w (nw), overw (ow) and obesity (ob) .results: data are available for 2647 patients in the c cohort and for 1498 patients in the a cohort (36 %). nutritional status subgroups prevalence (%) is: 6.1, 87.1, 5.6 and 1.2 in c and 37.3, 57.7, 4. 3 and 0.7 in a for respectively uw, nw, ow and ob. mean age in a is significantly increasing with bmi (p<.0001). frequency of 508 del/508 del in c and a is lower in ow/ob (p<.02) as well as the use of pancreatic enzymes (p<.001). ow/ob patients have the best fev1 and fvc values whatever the gender and the age, with significantly less pa colonization in a (p<.01). we could identify a positive correlation between the pulmonary function and bmi. the cf co-morbid conditions demonstrated a lower prevalence of diabetes (12/257) and cirrhosis (5/257) in ow/ob. none of the ow/ob c or a are on a transplant waiting list versus 1.7 % in uw/nw (n=69/4042) and none died versus 1 % in uw/nw (n=42/4042).conclusion: the observed prevalence of ow and ob in cf is respectively 5.2 and 1 % whereas the french obepi 2006 study collected 29 and 12 %. our results suggest that increased bmi is associated with better fev1, fvc, lower prevalence of pa, cirrhosis and diabetes. the potential risks of chronically high bmi have not been studied in this population yet, but justify further investigations in this longer life expectancy cohort. materials and methods: we retrospectively identified abdominal ct scans of 38 consecutive patients (19 females, 19 males, mean age of 28 years) with cystic fibrosis and a control group of 38 consecutive patients (21 females, 17 males, mean age of 40 years) scanned as potential renal donors. three readers reviewed all scans and recorded the presence and location of colonic wall redundancy, and the wall thickness of the ascending, transverse, and descending colon. clinical information on cystic fibrosis patients, including cftr gene mutations, was queried from our cystic fibrosis patient registry database. additionally, medical records of all cystic fibrosis patients were reviewed to determine the indication for abdominal ct. results: colonic wall redundancy was seen exclusively in patients with cystic fibrosis, and was noted in 11 of 38 patients each for reviewers 1, 2, and 3 (p<0.05). colonic wall redundancy was seen in 11 of 28 adult patients with cystic fibrosis (39%), but was not seen in any children age 17 or younger (p<0.05). excellent agreement was found for the ct identification of colonic wall redundancy among readers (kappa=0.91, p<0.001). cystic fibrosis patients with colonic wall redundancy had significantly thicker ascending colonic walls (mean 4.0 mm) vs. those without wall redundancy (mean 1.8 mm) or controls (mean 1.2 mm), (p=0.03). three patients with colonic wall redundancy had follow-up ct, and all showed temporal stability (mean of 50 months). among adult cystic fibrosis patients, cftr gene mutations were available for 10 of 11 patients with and 13 of 17 without colonic wall redundancy. while the common ∆f508 mutation was the predominant mutant allele among patients with normal colons at ct (only 3 of 17 patients, or 23%, had an identified mutation other than ∆f508 on either allele), a higher prevalence of less common non-∆f508 mutations was seen patients with colonic wall redundancy (7 of 10 patients, or 70%, p<0.05). the g542x mutation was seen exclusively in patients with colonic wall redundancy (3 of 10 patients, or 30%, p=0.06). there was no significant difference in the proportion of patients with abdominal pain (>0.7), pancreatic insufficiency (p>0.99), diabetes mellitus (p>0.1), history of meconium ileus background: airway inflammation in cf is associated with marked remodelling and bronchiectasis. pro-inflammatory mediators such as advanced glycation end-products (ages) and the soluble receptor for age (srage) may perpetuate this response in the lung and in other organs such as the kidney 1 . the total body burden of ages reflects exogenous sources from the diet, endogenous production by the body, tissue degradation and renal clearance, which may be reduced in renal impairment. the accumulation of endogenous age is accelerated in conditions of high oxidative stress and inflammation, and ages have been implicated in the pathogenesis of diabetic nephropathy. the burden and significance of ages in cf has not been determined, but if elevated, dietary modification of ages may represent a novel anti-inflammatory approach for cf. the aim of this study was to determine serum levels of age and srage, and identify clinical correlates of age and srage levels.methods: adults with cf (n=58, 35 males, 86% pancreatic insufficient, mean age 33.9±7.7 years (range 23 to 62 years), mean fev 1 %predicted 58.7±22.6%predicted (range 20-110)) and healthy adults (n=24, 13 male, mean age 32.7±9.2 years (range 21-55 years)) were studied. cf participants provided 1-3 serum samples each over a six month period, while healthy controls provided a single sample. serum was analysed for levels of advanced glycation end-product (age-cml) and soluble receptor for age (srage) (elisa). for each cf participant, levels in the multiple samples were averaged, and the median for the study sample reported. clinical data, including bmi, presence of cf-related diabetes mellitus (cfrd) and serum hba1c level were collected from the medical record. mann-whitney tests were used to compare cf and control levels, while spearman rank correlations were used to identify clinical correlates of age and srage.results: prevalence of cfrd was 27.6%. mean bmi was 21.8±2.7kg/m 2 and mean hba1c level was 5.9±1.1%. median (iqr) levels for age-cml were 1650 (1158, 1986) background: osteopenia is diagnosed in cystic fibrosis (cf) using dual-energy x-ray absorptiometry (dxa). areal bmd from dxa is subject to error when bones are smaller in volume than reference standards (t-scores). normalization of bone size by use of zscores in cf is controversial and not widely utilized, thus comparing to larger bone areas. reports in cftr-deficient mice (cf mice) reveal osteopenia when measured by dxa. we hypothesized that use of pqct, which eliminates bias of size, would more accurately analyze bmd.methods: femurs were collected at necropsy from 4 cf mice and 4 c57bl/6j (b6) mice at 6 wks, and 7 cf mice and 7 b6 mice at 14 wks (all female) for pqct. time points were chosen to coincide with pre-pubertal and adult ages for comparison to human disease. total mineral, trabecular and cortical densities were measured. student's t-test was used to detect significant differences (p<0.05).results: femur measurements from both cf mice and b6 mice are listed as mean ± sd in the table (below). length was greater in b6 mice compared to cf mice at 6 and 14 wks. total area at the metaphysis and diaphysis were greater in b6 mice at 6 and 14 wks. data are consistent with larger bones in b6 mice. total mineral density 1 , trabecular density 2 and cortical density 3 , however, were greater in cf mice compared to b6 ( 1 6 & 14 wks, 2 14 wks, 3 6 wks).discussion: our study demonstrates greater bmd in cf mice when volumetric data are analyzed and size differences are accounted for by use of pqct. these findings persisted at adult age, suggesting a normal deposition of bone. in clinical management, dxa imaging is readily available compared to pqct, and can be used as a predictor of bmd and fracture risk. correct reference ranges must be utilized to minimize erroneous values secondary to size. z-scores allow correction for differences in bone area and size, even in adults. more studies into understanding bone mineral deficit and tools of measurement are needed in cf. meanwhile, bone measurements by radiographic imaging must be taken in context of overall health, pubertal progression, and size of individuals. gainesville, fl, usa; 2. nemours children's clinic, orlando, fl, usa; 3. suny upstate medical university, syracuse, ny, usa; 4. case western reserve university, cleveland, oh, usa; 5. genentech, san fancisco, ca, usa background: greater growth rates, bmi and fat-free mass are associated with improved lung function in individuals with cf (pedreira, et al. pediatr pulmonol. 2005) . current treatments for weight gain have focused on nutritional supplements and appetite stimulation, and not underlying issues of catabolism or chronic disease. we hypothesized that anabolic effects of gh would not only improve weight and height, but lbm as well.methods: sixty-seven prepubertal children with cf and height less than or equal to the 10th percentile were randomized to daily gh (n=35) or observation (n=32) for a period of 1 year, followed by an off-treatment observation period of 6 months. children randomized to gh received somatropin injections daily at 0.3 mg/kg/wk. height and weight were measured every three months. height was evaluated as height standard deviation score (sds) to control for differences in age and sex. lbm was measured by dexa scan at time zero, at 6 and 12 months, and at the end of the 6-month observation period. in addition, for lbm, change from baseline was calculated for subjects for whom the same dexa equipment was used at both time points. the preliminary results of the first 12 months of the study are presented here.results: data for 27 subjects in the observation arm and 29 subjects in the gh arm, who completed 12 months, are available. as shown in the table below (listed as mean ± sd), gain in height, weight and lbm were significantly greater in the gh treated group than in the observation group over the 12 month period.discussion: gh improves growth in prepubertal children with cf as measured by height sds. in addition, gh significantly improves weight gain and this gain is, in part a result of the significantly greater increase in lbm in the gh-treated group than in the observation group. the relationship between the improvement in lbm and other outcomes in cf deserves further exploration. the national cystic fibrosis(cf) registry database notes cystic fibrosis related diabetes mellitus(cfrd) as a complication in 15.6% of cf patients at all ages. cfrd is associated with poorer nutrition and increased pulmonary morbidity. the cause of cfrd is not completely known but insulin resistance may be associated with fibrotic damage to pancreatic islet cells due to chronic inflammation. other studies suggest protein energy malnutrition(pem) in early life leads to impairment of insulin secretion and cystic fibrosis-related diabetes (cfrd) accounts for increased morbidity and mortality in patients with cf and occurs in approximately 30% of patients by the age of 30 years. cfrd net (network for epidemiology and trials) is a consortium of four large uk cf centres caring for 1052 adult patients with cf. the aim of this group is to undertake research into the diagnosis, investigation and management of cfrd. in this abstract, we estimate the prevalence of diabetes in a screened population of adults with cf. methods annual review data were collected on attending patients with cf aged 16 and above during 2006. 75g ogtts were performed after fasting for at least 8 hours on patients without known diabetes. each ogtt was categorised as either normal (2 hour glucose 3.5 -7.8mmol/l), impaired (2 hour glucose ≥ 7.8mmol/l and < 11.1mmol/l) or diabetic (2 hour glucose ≥ 11.1 mmol/l). all patients with a diabetic ogtt were followed up with serial bm monitoring to determine whether or not they had cfrd. fasting plasma glucose (fpg) was considered to be elevated ≥ 7.0 mmol/l, isolated impaired fasting glucose (igf) was defined as a value between 6.1 mmol/l and 7.0 mmol/l and hypoglycaemia was defined as a blood glucose <3.5mmol/l. in addition, we obtained information on age, sex and prescription of anti-diabetic medications subsequent to ogtt. in three of four centres, hba1c was routinely performed on all patients. of 1052 patients (median age 26 years, 57% male), 344 (33%) had established diabetes and were therefore excluded from further screening. of the remaining 708 patients, 392 (55%) underwent formal ogtt testing. in this latter group 34 (9%) had a diabetic ogtt, 58 (15%) an impaired ogtt and 49 (13%) had evidence of reactive hypoglycaemia at 2 hours. of the 34 patients with a diabetic ogtt, 28 (82%) only had an abnormal 2 hour value, 4 (12%) only had an elevated fpg and 1 (<1%) had both. all patients with diabetic ogtts underwent blood glucose monitoring and 20 (59%) went on to treatment with hypoglycaemic agents in the calendar year. none of the 4 patients with an isolated elevated fpg had diabetes.hba1cs were available in 23 of 34 newly diagnosed patients with diabetes (in the centres which performed hba1c). in this group median hba1c was 6.1%. this study confirms the high prevalence of diabetes among screened patients and the growing burden of diabetes management in adult cystic fibrosis clinics. it further highlights the importance of performing screening for diabetes in this population; the majority of patients were identified on the basis of abnormal 2 hour values. despite this only half of all suitable patients underwent ogtts in the four centres committed to screening. these patients merit further study, as previous work suggests that the decline in clinical status occurs several years before diabetes becomes apparent. cystic fibrosis related diabetes (cfrd) occurs in up to 40 % of patients with cystic fibrosis (cf), the incidence rising with increasing age. the likelihood of developing long-term complications secondary to diabetes increases with poor glycaemic control and duration of diabetes. as survival has improved for people with cf those who develop cfrd may live with diabetes for several years. aim: to establish the frequency of diabetic complications in patients with cystic fibrosis related diabetes.method : patients with cfrd attending the adult cf service at the royal brompton hospital between april 2006-march 2007 were screened for diabetic complications and cardiovascular risk factors. a total of 78 patients (male/female: 44/34) including 10 post transplant patients were screened. mean age was 33.1 years (17-52), mean hba1c 8.0% (5.6-16.2% ) and average duration of diabetes was 7.3 years (<1-38 years).as immunosuppressive therapy can also cause many of the complications associated with diabetes the results are presented separately for nontransplant and post transplant patients (table 1) . 9 patients in total had retinopathy: 7 background retinopathy,1 proliferative retinopathy undergoing laser therapy and 1 maculopathy. the average duration of diabetes in those with background retinopathy was 5.8 years with only 1 patient having diabetes for >10 years. the patient with proliferative retinopathy (non-transplant) had been diabetic for 15 years. a raised creatinine level was identified in all transplanted patients with microalbuminuria but none of the non-transplanted patients. 1 patient (nontransplant) had macroalbuminuria. 2 post transplant patients were on treatment for hypertension and a further 6 patients (2 post transplant and 4 nontransplant) had elevated blood pressure (>140/90) at screening requiring follow-up. none of the patients had evidence of cardiovascular disease or stroke.conclusion: macrovascular complications were not seen. microvascular complications occurred but were less common than the reported incidence in type 1 and type 2 diabetes. this may reflect the relatively short duration of diabetes (mean 7.8 years) of patients in this study. a further study comparing cfrd patients with a non-cf diabetic control group of similar duration of diabetes is warranted. cystic fibrosis (cf) is a disease that leads to serious disturbances in nutritional status and bone calcification. comparison of two methods for assessment of bone mineralization: dexa and hand radiograms in diagnosing osteopenia or osteoporosis. study was performed in a group of 26 cf patients (10f, 16m), aged 7-30 yrs. nutritional status was assessed using bmi, cole's index and bmc. radiograms of non-dominant hand were assessed according to normalized optical density comparing to aluminum standard. bone density was also assessed using dexa. for statistical analysis, backward stepwise binary logistic regression (wald's test) was used. analysis of data revealed that using bmc, cole's index and hand radiograms markers we can diagnose bone mass disturbances (z score <1sd) with precision up to 84.62% comparing to dexa. sensitivity and specificity of this method was respectively 86.67% and 81.82%. false negative results were obtained in 2 patients and false positive were also in 2 patients. hand radiograms method could be an alternative for dexa in screening of bone density disturbances in cf patients. the study was partly supported by grant of ministry of science and higher education no 2 p05e 041 28.in a very recent publication(1) a high rate of fasting (13%) and reactive hypoglycaemia (15%) was described in a group of n=129 cf patients older than 8 years who received an oral glucose tolerance test. reactive hypoglycaemia was related to the 120minute glucose concentration in the ogt test. we were surprised by the high percentage of asymptomatic hypoglycaemic situations in cf patients. as a part of a prospective intervention study in cf patients with cfrd, we screened more than 1400 patients 10 years or older with cf for cfrd. in this multi-centres population ogt was performed in the morning after overnight fasting according to who standards. using the same definition for fasting hypoglycaemia (glucose < 60mg/dl; 3.3mmol/l) and reactive hypoglycaemia (glucose < 50mg/dl; 2.8mmol/l at 120minute during ogt test)as in the other study we calculated the percentage of cf patients with fasting or reactive hypoglycaemia in our cohort. results: ogt were done in 1495 patients with cf. age(mean±sd)19,6±8,6 years; bmi z-score -0.73±1,1;height z-score -0,8±1,1 and weight z-score -1,0±1,3. ogt was categorised according to ada criteria. normal ogt (n=782 all ; fasting hypoglycaemia (fh) n=21(2,7%), reactive hypoglycaemia (rh)n=18 (2,3%),ifg (n=329 all, fh n=0, rh n=5 (1,5%), igt(n=131,fh n=2(1,5%), rh n=0),fgt (n=75 all, fh n=0,rh n=0) and diabetes mellitus (n=187 all, fh n=3(1,7%)). fh was observed in 26 (1,7%) and rh in 23(1,5%) out of 1495 ogt tests. there were no difference related to age, bmi z-score, height z-score and weight z-score comparing those patients with fh or rh to those without fh or rh in all categories of ogt tests.in this large multi-centres cohort of cf patients we were neither able to confirm the high percentage of fasting nor of reactive asymptomatic hypoglycaemia which was reported recently on a small group of cf patients. nutritional status measured by bmi z-scores, height and weight z-scores were unaffected by hypoglycaemia in our cohort as noticed also in the small group. as the ogt tests in the other study were done in a single centre it might by that a centre specific situation influenced the frequency of hypoglycaemia in that small group. we conclude that large numbers of investigations might be needed to come up with firm conclusions related to frequency of specific aspects of glucose disturbance in cf patients.fh does not exclude diabetes mellitus or igt. as survival from cf improves, surveillance to identify and treat complications associated with longevity is an important component of management. renal disease has been reported in adults with cf. risk factors may include cf-related diabetes mellitus (cfrd) and use of nephrotoxic medications. diabetic nephropathy has been observed in the absence of cfrd 1 , highlighting the need for screening for renal impairment. this study aimed to measure renal function in a sample of adults with cf, and to compare estimated glomerular filtration rate (egfr) and urinary creatinine clearance (urcrcl) as markers of renal function.methods: adults with cf aged 25 years or over (n=29, 66% male, 83% pancreatic insufficient, mean age 36.7±9.1 years) underwent screening for renal impairment. of these patients, 15 (9 males) had cfrd, and 14 (10 males) had no history or clinical features of cfrd. 24 hour urine collections were analysed for urcrcl, with renal impairment being defined as <90ml/min. serum creatinine level was used to calculate egfr (mdrd formula), which was classified renal function as normal or mildly impaired (>60ml/min); moderately impaired (30-59 ml/min) or severely impaired (<30ml/min). sensitivity of egfr for identification of renal function was calculated using urinary creatinine clearance as the reference method. unpaired t-tests were used to compared cfrd with non-cfrd patients.results: mean urcrcl was 105±35ml/min, and 8 eight patients (27.6%) had renal impairment (<90ml/min). of these, only 3 had egfr suggestive of renal impairment. the sensitivity of egfr for identifying renal impairment in this sample was 38%. a further 2 patients had moderately impaired egfr, but normal urcrcl. five of the 8 patients with impaired urcrcl had cfrd. age, gender, cfrd, fev 1 %predicted and hba1c level did not correlate with urcrcl. the table compares cfrd patients with non-cfrd patients. hba1c level was higher in cfrd patients. there were no other significant differences in clinical or renal function parameters.conclusion: renal impairment, is common in the adult cf population and is not confined to patients with cfrd. screening using egfr is poorly predictive of impaired urcrcl in this population. these results suggest that surveillance to monitor renal function is indicated in the adult cf population, including in patients without cfrd, and that determination of urcrcl should be included in the screening process. cystic fibrosis (cf) patients suffer from pancreatic insufficiency resulting in malabsorption of fat soluble vitamins including vitamin d. many fac-tors contribute to low bone mass and fractures in patients with cf; however, chronic vitamin d deficiency plays a major role. the prevalence of vitamin d deficiency has been reported as high as 81% in some specialized care centers. in addition, reports of occult vertebral fractures have been reported as high as 25%. we sought to determine the prevalence of vitamin d deficiency (defined as 25-hydroxyvitamin d (25(oh)d) < 30 ng/ml) and of vertebral fractures at our cf center. we obtained irb approval to review the records of all patients seen at our cf center during [2004] [2005] . we collected information related to bone health including 25(oh)d, bone mineral density and lateral spine or chest x-rays to examine for the presence of a vertebral fracture. we reviewed the records of 185 subjects who were seen at our center during the study period. the mean age of the subjects was 29 ± 9 years. subjects had a mean bmi of 21.2 ± 3 and an fev1% predicted of 64.4 ± 25%. the percentage of subjects having an annual 25(oh)d level checked was only 57% and 62% in 2004 and 2005, respectively. the mean 25(oh)d was 22.7 ± 10 and 24.9 ± 10 ng/ml in 2004 and 2005. the prevalence of vitamin deficiency was 78% and 73% in 2004 and 2005. about one quarter of the subjects had bone mineral density testing with half of the tests showing osteopenia or osteoporosis. twenty-seven percent of subjects had vertebral abnormalites detected on lateral chest x-ray. we sought to determine whether any factors were associated with vitamin d deficiency. we found that taking a multivitamin did not significantly protect against vitamin d deficiency. however, not taking a supplement containing vitamin d other than a multivitamin was associated with a 54% risk of vitamin d deficiency (p=0.04). subjects having 25(oh)d levels determined in the winter or spring was associated with a 30% risk of vitamin d deficiency (p=0.05). in summary, we found annual testing for vitamin d status was inadequate in our cf center and that when 25(oh)d level was determined, over 70% of subjects were vitamin d deficient. nearly one quarter of our adult patients already had evidence of vertebral compression fracture seen in lateral chest x-ray. we urge greater screening for vitamin d deficiency in the cf population. effective protocols to prevent and/or treat vitamin d deficiency are urgently needed in the cf population. improved vitamin d status in cf patients is one factor that may reduce the high prevalence of vetebral fractures. support for this study was provided by proctor and gamble pharmaceuticals background: more cf patients are surviving into adulthood, in part due to the increasing use of new therapies and more aggressive management of chronic respiratory and gi disease. as a result, the recommended daily treatment regimens for most cf adults are both complex and time consuming. objective: to assess the self-reported daily treatment burden of cf therapies in a cohort of adults with cf. methods: in the sixth survey wave of the project on adult care in cf (pac-cf), an ongoing longitudinal panel study of adults with cf from 10 us cf centers, respondents were asked to report the type of medications, inhaled therapies, and airway clearance therapies they used during the day prior to completing the survey, as well as estimate the time generally required to complete each type of therapy.results: of the 204 respondents (response rate 69%), the median number of therapies reported was 7 (iqr 5-9) and the median reported amount of time usually spent on treatments was 90 minutes per day (iqr 50-150 minutes). forty-eight percent reported using 5 or more different inhaled therapies or using inhaled therapies 5 or more times during that one day. the most commonly reported inhaled therapies were a bronchodilator (61%), pulmozyme (49%), an inhaled steroid (32%), hypertonic saline (30%), and objective: caring for a child with cf is stressful and often leads to adverse effects for both children and caregivers. support from family and friends can help reduce the adverse effects of chronic stress, however, there is little research documenting types of support provision as well as sources of support. moreover, social support may be related to important health outcomes. this study described the types of support provided to caregivers of children with cf and examined relationships between support and health outcomes.method: as part of a larger intervention study to improve adherence to medical regimens, 89 children with cf ages 1 to 11 and their caregivers were enrolled across 3 cf centers in florida: university of florida, nemours children's clinic, and all children's hospital. caregivers completed the norbeck social support questionnaire (nssq; norbeck, 1980) in addition to other measures at baseline and follow up. this questionnaire provides information about the number and type of people providing support, as well as its type and quality.results: caregivers received the majority of support from their families, spouses and friends and characterized these relationships as longer in duration, with more frequent provision of support. other individuals who provided support included the cf team, religious leaders, counselors or psychologists, and work associates. overall, caregivers rated family members and spouses as providing the greatest amount (i.e., "quite a bit") of emotional support. spouses received the highest ratings for tangible support; they were perceived as providing "quite a bit" of support in comparison to families (i.e., moderate) and friends (i.e., a little). in terms of size of the support network, caregivers in this study had similar networks to those of a normative sample of healthy adults. in addition, there was no difference in amount of emotional or tangible support received by these caregivers. preliminary baseline results indicated that social support was positively related to both adherence, children's growth (i.e., height and weight percentile), and family income.discussion: caregivers of young children with cf reported similar support networks as other healthy adults. however, there was variability in the amount of support provided by source, with spouses providing the greatest amount. social support appeared to be a protective factor, and lack of support was related to negative health consequences for their children. more attention should be focused on the potentially beneficial effects of social support.funding was provided by nih grant #69736 befriending is reported to be valued by those who have been befriended, offering opportunities for social activities and new experiences and can impact positively on self confidence and self esteem 1. whilst government policy supports befriending, few schemes collect evidence to demonstrate the effectiveness of these services 2. young people with cf may suffer social isolation due to chronic illness and treatment demands and a befriend-ing service was offered to those considered socially vulnerable in the edinburgh area.this project aims to evaluate the effectiveness of this befriending service on young people with cf and their carers. methods liverpool, united kingdom; 2. child mental health, university of liverpool, liverpool, united kingdom; 3. child health, university of liverpool, liverpool, united kingdom; 4. psychology, university of miami, miami, fl, usa; 5. psychology, birkbeck college, university of london, london, united kingdom; 6. mathematics and statistics, university of lancaster, lancaster, united kingdom; 7. psychiatry, university of manchester, manchester, united kingdom introduction: existing treatments for cystic fibrosis (cf) are time-consuming and labour intensive and biomedical advances are likely to lead to further novel interventions. there is concern amongst clinicians that a high care burden associated with these interventions may compromise the wellbeing of caregivers, reduce adherence to the treatment protocol and increase the likelihood of inadvertent errors in the delivery of interventions. at present, there is no measure of 'treatment burden' and therefore no way to assess the impact of new and increasingly more complex interventions conducted by lay caregivers at home. furthermore, trials are hampered by the lack of measurable patient-reported outcomes beyond a broad quality of life assessment. to address this gap we have developed a measure of treatment burden for cf using qualitative methods (focus groups, action research, in-depth interviews) with participating parents and a working group of cf team professional staff. this measure addresses the time, effort, meaning and ease of management for caregivers of children with cf up to 13 years and post the first year following a diagnosis. methods: we report here the pilot phase of the validation of this instrument, which involved (i) cognitive interviewing with n=16 caregivers; and, (ii) n=30 caregivers completing the clcf instrument together with a quality of life measure at 2 time points. this yielded data for stability, reliability and coherence of the hypothesized constructs. results: the clcf takes 15 minutes to complete. face validity and acceptability are established. for the first pilot sample the age of the child ranged from 1.5-11.5 years. treatments took, on average 72 minutes per day to complete and an average of 16 minutes of this was devoted to administering enzymes particularly for carers of infants. a prominent concern amongst these parents was their child's height and weight and their efforts were directed at optimising growth. for the child however, management of nebulised medications and physiotherapy were more frequently flagged as a concern. these data suggest this is a reliable, coherent and useful, although complex tool. conclusion: the caregiver challenge for cf cannot be understood simply in terms of time and effort involved in delivery of treatments. it also involves contextualising interventions for their meaning at multiple levels of explanation for the individual concerned. structural equation modelling would be an appropriate way to proceed with the main validation when such complex relationships occur between latent variables and would serve as a confirmatory factor analysis for the hypothesised relationships. sponsored by: royal liverpool children's nhs trust and university of liverpool. funded by: national institute for health research (nihr) research for patient benefit programme.introduction: the french cf practice recommendations, published at the end of 2002 in parallel with the creation of cf reference centres, advise that each patient should be seen at least every 3 months at a cystic fibrosis reference centre.objective: to investigate the impact of these recommendations on the effectiveness of the follow-up of patients at the four reference centres in the rhône-alpes region.methods: all patients with cystic fibrosis attending one of the four cf centres between 1996 and 2005 were retrospectively included. the total number of visits was recorded for each patient and each year of the study period. to determine the evolution slope for each patient followed for at least 3 years, a negative binomial regression of number of visits versus time was carried out (confirmed with a repeated model). to estimate the impact of the recommendations, the analysis was restricted to patients with at least two visits before and after 2003 and a second model was adjusted with a new intercept in 2003 to estimate the change in slope as of this point.results: a total of 650 patients were included in the cohort. the average number of visits per patient rose from 3.68 in 1996 to 4.96 in 2005 (p<0.0001). the proportion of patients with at least 4 visits per year increased from 38% to 70%. the negative binomial regression for the 537 patients having had at least 3 years follow-up confirmed this trend with an average slope of +0.07 (sd=0.18). a total of 352 patients were evaluable for the change in slope. no significant change in trend was observed in 2003: only 34% of patients had a higher rate of growth (this change was significant for only 7 subjects). at the last follow-up visit, patients with increasing rates of number of visits were older (21yrs vs. 17yrs, p<0.0001), had lower %fev1 (68 vs. 75, p=0.02), had a similar average number of visits before 2003 (3.7 vs. 3.9, p=0.33), and a similar weight-for-age z-score (-0.58 vs. -0.46, p=0.50) .conclusion: the number of visits per patient is regularly increasing. since the publication of the practice recommendations in 2003, the growth has tended to slow down: clinicians were already convinced by the need for closer follow-up and had begun to increase the rate of visits. methods: patients > 13 years who presented for routine healthcare voluntarily completed four surveys: 1) the revised eating attitudes test (eat), validated for cf patients by abbott and colleagues, where higher scores reflect worse attitudes towards eating; 2) the rosenberg self-esteem scale (rse), where higher scores reflect better self-esteem; 3) the body image scale (bis), developed for cf patients by wenninger and colleagues, where higher scores reflect better body image; 4) the cystic fibrosis questionnaire (cfq), developed by quittner and colleagues, where higher scores reflect better hrqol. also, fev1%, body mass index (bmi), and pancreatic sufficiency or insufficiency (based on the need for pancreatic enzymes) was recorded. regression analyses controlling for age, gender and bmi were used to examine the associations between the surveys. results: this study included 37 patients with 41 % males and a mean age of 26 years. the eat was negatively associated with the rse (p=0.015, adjusted r 2 =0.127), bis (p=0.001, adjusted r 2 =0.221) and cfq (p=0.015, adjusted r 2 =0.122). the rse was positively associated with the cfq (p=0.001, adjusted r 2 =0.346). the bis was positively associated with the rse (p=0.001, adjusted r 2 =0.529) and cfq (p=0.001, adjusted r 2 = 0.540). neither bmi nor pancreatic function was associated with the surveys (p=ns). fev1% was positively associated with the cfq (p=0.028, adjusted r 2 =0.106) but was not associated with the other surveys.conclusions: more negative attitudes towards eating predict worse selfesteem and body image, while more positive body image predicts better self-esteem. also, attitudes towards eating, self-esteem and body image are significant predictors of hrqol with body image being the most important predictor.clinical importance: hrqol is an important clinical outcome measure in cf. clinicians need to be sensitive to attitudes towards eating, self-esteem and body image in cf patients, because they are important predictors of hrqol. riekert, k.a. 1 ; mogayzel, p.j. 2 ; bilderback, a. 1 ; hale, w. 1 ; boyle, m.p. 1 1. medicine, johns hopkins university, baltimore, md, usa; 2. pediatrics, johns hopkins university, baltimore, md, usa background: existing research suggests that self-reported adherence to all aspects of the regimen is likely suboptimal and objective measures suggest even poorer adherence. there is little empirical data on how much adherence is necessary to achieve desired health outcomes. moreover, there is limited data on how adherence changes dur-objective: cf may be associated with pain attributed to several etiologies. this study evaluates the prevalence of pain symptoms in adult cf patients and the influence on patients` lives.methods: 243 patients of 6 adult cf centers in germany completed a validated, self report questionnaire during a routine clinic visit assessing characteristics of chronic pain (prevalence, duration, location, quality and intensity of pain symptoms). furthermore, the impact of pain on different aspects of life was explored using a numeric scale from 1 to 10 with 10 being the worst. every-day-life was divided into 8 categories: duties at home, recreation, social activities, occupation, sexual life, autonomy, vital activities and cf-therapy. the average intensity of pain within the last 4 weeks was correlated to fev1, bmi and age.results: 243 patients (119 male) completed the questionnaire. the age was 18-65 years (mean 28.13±8.13). 182 patients (75%) aged 18-65 years (mean 28.51±8.23)reported pain within the last 3 months. bmi was 14.7-38.3 kg/m2 (mean 20.4±3.1), fev1 12-142% (mean 54.7±24.5). if asked for pain within the last 4 weeks 174 (72%) reported painful episodes lasting from 1-28 days with a mean of 9.8 days (±7.8). most patients described pain occurring at more than one site with the head being the most localized site, followed by chest and abdomen. concerning the quality of pain 85.4% characterized their pain episodes as attacks whereas 8.8% reported them as continious, 5.8% had continious pain combined with pain attacks. 179 patients desribed the intensity of pain as 5.77 (±2.35) on a scale from 0 to 10 with 10 being the most severe pain. the category of life negatively influenced most of all by pain episodes was recreation, followed by occupation and duties at home. female patients were more limited in their acitivites by pain symptoms than male patients with the highest difference being reported in sexual life. there was no correlation of the average intensity of pain within the last 4 weeks to fev1, bmi or age.conclusion: the prelevance of pain in cf is often underestimated. painful episodes can be the cause of worsening the quality of life for adult cf patients. assessment of pain should be routinely performed as part of care in cf centers. objective. sleep has been examined in a number of pediatric conditions, with impaired sleep resulting in worse neurobehavioral functioning (beebe, 2005) . recent research has shown that slow wave sleep is critical for memory (marshall, helgadottir, molle, & born, 2006) . to the authors' knowledge, no studies have examined slow wave and rapid-eye-movement (rem) sleep in pediatric patients with cystic fibrosis. methods. retrospective medical record review revealed two patients who underwent clinically indicated polysomnography (psg) who reported feeling tired and lacking energy. patients were females with cystic fibrosis, ages 12 and 14 years. results. psg revealed an increase in stage 2 sleep for both patients, resulting in increased risk and observed hypopneic episodes. patient -a experienced 16.3% in stage 1, 65.6% in stage 2, and 0% in stages 3 and 4 combined. this patient also had no rem sleep. patient -b experienced 12% in stage 1, 67.8% in stage 2, and 10% in delta wave sleep, with no rem sleep. respiratory disturbance index (rdi) for patients a and b were calculated at 3.6 and 3.5, respectively. respiratory effort related arousal (rera) contributed to rdi by 1.9 and 1.1 points in patients a and b, respectively. in addition, endtidal co2 for patient a and b were maxed at 50 mm hg and 48 mmhg, respectively. their epworth sleepiness scales were scored at 8 and 10 for patients a and b, respectively. conclusions. structure of sleep was abnormal in both patients with decreased slow wave (delta) sleep in patient b and lack of delta sleep in patient a. neither patient experienced rem sleep. compensatory increased stage 2 (nrem) sleep could cause respiratory related abnormalities, such as hypopnea and apnea in patients with cystic fibrosis background: understanding and advancing the application of tools to measure patients' symptoms is critical to advancing our evaluation of potential new therapies used to treat cf. we performed initial evaluation of the measurement properties of a cf-specific respiratory symptom daily questionnaire. methods: we planned to enroll 60 cf subjects, stratified by age, at three cf programs, the university of washington medical center and children's hospital and regional medical center in seattle and mary bridge children's hospital in tacoma in a prospective assessment of a novel cf specific respiratory questionnaire. cf subjects 2 years and older were eligible for enrollment. patients (parents for children under the age of 12) completed a daily symptom questionnaire during two 7-day periods of clinical stability and one 7-day period when patients were ill for a total of 21 days. the ill state began when patients/parents sought medical attention for respiratory symptoms. the questionnaires were completed using a secure web-based application or via paper for those patients without access to the internet. two health related quality of life measures (generic and cf specific) were completed by the subjects at the end of each 7-day period to assess the relationship between the novel questionnaire and health related quality of life. patients also used pedometers during the well and ill states to assess relationship between symptoms and activity level.results: 52 cf subjects have been enrolled to date. at the time of this interim analysis, 34 patients had completed 384 questionnaires. of this total, only 2 individual questionnaire entries have been missed in 2 patients (<1% of possible questionnaires). interim review of cross-sectional data suggests clear differences in symptom reporting between the ill and well periods. examples include 46% (13/28) noted difficulty breathing and 32% (9/28) noted tightness in the chest in the preceding 24 hours during the initial well period compared to 70% (14/20) noting difficulty breathing and 70% (14/20) noting tightness in the chest during the ill period.conclusions: interim evaluation of this novel instrument demonstrates feasibility of deploying this instrument via the internet with an extremely high completion rate. using a cross-sectional analysis, the instrument can discriminate between the ill and well state. further data will be presented regarding within patient variability and discrimination of the instrument.supported by the cystic fibrosis foundation leroy matthews physician scientist award, the national heart, lung, and blood institute (hl72017-03), national institute of health (rr-00037-39), cystic fibrosis foundation therapeutics inc. background: improved communication in today's health care delivery system is a critically important aspect of patient care. nurses are in an effective position to improve communication due to their close interaction with patients. as a result, they are able to identify potential problems, communicate them to the healthcare team and improve patient care and satisfaction.hypothesis: improving communication between the patient/family and the health care team will improve patient/family satisfaction.method: as a member hospital partnering with the institute for healthcare improvement's (ihi) initiative, transforming care at the bedside (tcab), the children's hospital of philadelphia (chop) introduced the use of daily patient goal sheets (dpgs) as a vehicle to improve communication between members of the healthcare team, patients and families and the dpgs was refined to meet the needs of an inpatient medical unit to which cf patients are admitted. using rapid plan-do-study-act (pdsa) cycles, a multidisciplinary team engaged in small tests of change in different stages. initially, patient goals and discharge criteria generated in daily rounds were posted in each room on easel paper. then large "whiteboards" were placed in each patient room for documenting patient goals, discharge criteria, names of the care team members and questions from patients and families. as a final step in the process, we shifted the health care team rounds to the patient's bedside and implemented bedside shift to shift nurse report and safety checks. patient satisfaction was continuously monitored over this process using the press ganey survey.results: over an eight month period, the press ganey survey results for the unit showed an average increase of 10%. we attribute this increase to better information sharing among patient, family and hospital staff regarding the plan of care for the patient's hospitalization. the health care team also had an improved dialog with the patients/families. conclusion: patient/family and healthcare team communication and satisfaction were improved using an organized and stepwise pdsa process to develop daily patient goal sheets on a medical unit. background: the uab/chs pediatric cf center provides care for approximately 300 patients. the pulmonary division has 5 full-time rns and 3 part-time rns who rotate phone triage for all calls (including cf) of patients seen by the 9 faculty physicians. historically, patient calls received during office hours are taken by the receptionist and put into the emr, then returned by the rn on call. the rn contacts one of the staff md's, gives report of the patient's condition,treatment orders are received, and the patient notified. there are several issues with this phone triage system, which include inconsistencies in rn and md response as well as differences in documentation. global aim: to improve the consistency among rns in phone triage of sick cf patients.method: an algorithm for pulmonary exacerbation phone triage was developed by the cf center director and lead rns to improve consistency in symptom assessment and treatment plan. a specific list of systemic and pulmonary signs and symptoms was formed including length of illness. if three objective: patients with cf often require intravenous antibiotics for treatment of pulmonary exacerbations. patients often receive peripherally inserted central catheter (picc) lines or totally implantable venous access devices (tivads) for venous access. few studies have examined complications of tivad implantation and little published data exists concerning picc line complications in cf patients. this study sought to assess the complication rates of both tivad and picc lines as well as to identify possible risk factors for developing complications.methods: this retrospective study included patients from 3 cf centers in northern new england. data was obtained from each patient's local medical record, port cf, and patient interviews. demographic data was recorded for all cf patients between 1/1/03-6/1/06. for each tivad or picc line, the following information was recorded: type of line placed, history of prior line placement during the study period, patient age, history of use for blood draws, method of line flushing, and status of the line at the end of the study (if still in use). complications were defined as catheter occlusion, vascular thrombosis/stenosis, infection, or other local inflammatory reactions.results: data was collected for 237 pediatric and 155 adult cf patients during the defined study period. seventy-three tivads and 356 picc lines were placed during the study period in 205 patients. the tivad and picc line complication rates were 33% (24/73) and 6.5% (23/356), respectively. in pediatric patients, 27% (6 of 22) tivads and 6.3% (13/205) picc lines had a complication. of the 6 tivad complications, three were systemic infections and three were catheter occlusions. of the 13 picc line complications, there were two venous thromboses, one line occlusion, two systemic infections, and nine minor incidents of localized phlebitis. in adults, complications were recorded in 35% (18/51) of tivads and 6.6% (10/151) of picc lines. of the 18 tivad complications, there were five systemic infections, seven catheter occlusions, and four venous thromboses. of the 10 picc line complications, there were three venous thromboses, three line occlusions, and four minor incidents of localized phlebitis. all adult patients who developed a deep venous thrombosis(dvt) associated with tivad implantation were homozygous for the deltaf508 mutation. the presence of diabetes or burkholderia cepacia complex(bcc) lung infection was associated with dvt with odds ratios of 5.91 (95% ci 1.16-30.1) and 5.11 (95%ci 0.93-28.1), respectively. conclusions: complications of picc lines were uncommon and usually minor. the rate of tivad complication observed was more common over the lifetime of the catheter and was similar to previously published reports. we identified potential risk factors for the development of dvt associated with tivads and picc lines, specifically cf related diabetes, bcc infection, and homozygous deltaf508 genotype. the mechanism by which these factors are associated with catheter complications is unclear and warrants further investigation. key: cord-257167-rz4r5sj7 authors: nan title: abstracts for the 29th annual meeting of the japan neuroscience society (neuroscience2006) date: 2006-12-31 journal: neuroscience research doi: 10.1016/j.neures.2006.04.004 sha: doc_id: 257167 cord_uid: rz4r5sj7 nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns19640. sl2-1-1 frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl3-1-1 neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif1a/kif1bbeta), nmda type(kif17) and ampa type(kif5s) glutamate receptors and mrnas(kif5s) such as camkii ␤ mrna and arc mrna with a large rna-transporting protein complex containing at least 42 rna related proteins such as hn rnp-u, staufen, and fmrps. kif2a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif3 suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif4 controls the activity-dependent survival of postmitotic neurons by regulating parp-1 activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award 2-1 what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in 1-3 months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v1). after complete ablation of v1, the monkeys recover performance of visually guided saccades toward the blind field in 2 months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi 17023055, kakenhi 15029261 sy1-1-01-2 distinct roles of two 7-pass transmembrane cadherins in neurite growth control tadashi uemura 1,6 , yasuyuki shima 1,6 , keisuke sehara 2 , manabu nakayama 3 , shinya kawaguchi 4 , mikio hoshino 5 , yoichi nabeshima 5 , tomoo hirano 4,6 1 graduate school of biostudies, kyoto university, kyoto, japan; 2 school of science, japan; 3 kazusa dna research center at chiba, japan; 4 graduate school of science, japan; 5 graduate school of medicine, japan; 6 crest, jst, japan drosophila flamingo (fmi) and mammalian celsr1-3, which are 7pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian 7-pass transmembrane cadherins celsr2 and celsr3 are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr2 enhanced neurite growth whereas celsr3 suppressed it. our result suggested that celsr2 had a stronger activity as g-protein coupled receptor than celsr3 did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of 7-pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin-1 is preferentially localized in axons; and nectin-3, in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin-1 and dendritic nectin-3 is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi 14104026 sy1-1-01-5 lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa 1 , fumikazu suto 2 1 nagoya university, nagoya, japan; 2 institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca3 pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema6a is expressed in ca3 pyramidal cells and principally suppresses invasion of mossy fibers to ca3. second, the repulsive signal of sema6a is mediated by plexin-a4 expressing in mossy fibers. third, the repulsive activities of sema6a are attenuated by plexin-a2 in the proximal dendritic segments of ca3 pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on (1) working memory maintenance and manipulation, (2) rule representation and task-switching, and (3) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy1-1-09-5 towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy1-1-09-6 neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of −25 genes on chromosome 7q11.23, has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi (17023501) sy1-1-17-3 a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total 21 full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and 18 k songbird cdna array. we used the arrays to reveal a set of 33 genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. -1-17-4 stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca1 pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi 17680033 sy1-2-02-4 role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii ␤ acts as an actin stabilizing molecule to maintain spine structure. camkii ␤ is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii ␤ oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii ␤ oligomer slows down actin dynamics and camkii ␤ is enriched in spines by actin polymerization. the suppression of endogenous camkii ␤ alters spine shape to filopodia-like structures. these experiments suggest that camkii ␤ plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii ␤ detaches from f-actin in an activity dependent manner. we will discuss how camkii ␤ maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using 2-photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., 2006) . furthermore, in response to single spine stimulation using 2-photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration (50 ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by 20 ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy1-2-10-3 basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼20 hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy1-2-10-4 coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f-18 fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos7 cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d1 receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad-1 found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca 2+ sensing protein was activated. synchronies of sv fusion are maintained by a ca 2+ sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori 21st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy1-3-11-3 sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim1. thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka 1 , takeshi baba 1 , sumiko mochida 2 , yoshimi takai 1 1 department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; 2 depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca 2+ -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab3 system, munc-13, munc-18, doc-2, and tomosyn. we have previously reported that tomosyn, a syntaxin-1-binding protein, works as a molecular clamp that controls free syntaxin-1 availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin-1, resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin-1, which increases the affinity of syntaxin-1 for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca 2+ sensor for neurotransmitter release. however, the physiological contributions of its ca 2+ -binding domains (c 2 a and c 2 b) are still unclear. we have studied the roles of aspartate (asp) residues in the c 2 b ca 2+ -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell 79, p. 717) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca 2+ binding to the c 2 b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca 2+ binding to the c 2 b because mutation in the second asp inhibits ca 2+ binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin-3 (nt-3) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt-3 and its low-affinity receptor p75 genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c270t in the 5 noncoding region, bdnf-linked polymorphic region, and val66met), three polymorphisms of the nt-3 gene (g-3004a, microsatellite in intron 1, and gly-63glu) and a missene polymorphism of the p75 gene (ser205leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p75 in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val66met) in the bdnf gene resulting in a prodomain substitution at position 66 from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps10 protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns052819 sy1-3-19-6 processing of bdnf and brain disorders masami kojima 1,2 1 aist, osaka, japan; 2 sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p75 neurotrophin receptor (p75ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); 372 (t/g) and 378 (t/g) of the human bdnf gene, causing amino acid substitution (r125m and r127l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy1-4-04-1 pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣1 leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣1, the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv-1 vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il2ss+fgf-2, and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi (14657350) sy1-4-04-4 hgf gene therapy for the treatment of spinal cord injury masaya nakamura 1 , akio iwanami 1 , kazuya kitamura 1,2 , yoshiaki toyama 1 , hideyuki okano 2 1 department of ophthalmology surgery, keio university, tokyo, japan; 2 department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th10 level in adult rat 3 days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. 1 or 6 weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap43+fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., 1989) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., 1999) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., 1999 . nature neuroscience. nakamura et al., 1989 . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first 1-2 months after stroke, suggesting the presence of a critical period. in the cat, illert et al. (1977) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c3-c4 segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. (1) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c3-c4 propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. (2) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca 2+ responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l1cam(l1) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l1. therefore, these syndromes have been reclassified as l1 syndrome. we performed a nation-wide l1gene analysis and identified li gene mutations in 35 families with l1 syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l1-mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l1 syndrome. research funds: kakenhi (16390424), (16689025) sy1-5-05-4 rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein 2 gene (mecp2) located at xq28 is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp2 was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like 5 gene (cdkl5) manifest as atypical rtt. the mutations of mecp2 are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in 6-ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase-9 are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp150, suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca3, and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣1or ␤-adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣1and ␤-adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context 2-4 days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr1, nr2a, 2b, 2c), glycine transporter 1, and eaac1, by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase 6 ␣ -subunit (pde6c), a component of cone-type pde. since genetic mutations of pde6c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde6 in visual functions. shinichi nakagawa 1 , masatoshi takeichi 2,3 , fumi kubo 1,3 1 nakagawa initiative research unit, riken, wako, japan; 2 riken cdb, kobe, japan; 3 department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt2b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt2b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt2b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi (16570184) sy1-6-06-3 identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea-1 to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea-1 during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea-1 double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea-1-positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea-1 have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx2 and crx play critical roles in retinal photoreceptor development. while otx2 is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi (16790070, 16027257, 17024061) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf19 found in the mesoderm. we find that although fgf19 can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf3 and is actually inhibited by fgf19. for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy1-6-06-6 how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel2, encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel2 in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel2 promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel2 which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein 57e was identified as the gene responsible for this behavioral difference among the species. obp57e forms a gene cluster with obp57d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp57d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp57d/e region obtained from 30 species phylogenetically located between d. melanogaster and d. pseudoobscula. sy1-6-14-4 an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi 1,2 , akinori nishi 1,2 , toshihiko shiroishi 1,2 , tsuyoshi koide 1,2 1 sokendai, kanagawa, japan; 2 national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c57bl/6 (b6) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b6 and msm, and those strains have the same genetic background as b6 except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b6-17msm, which have msm chromosome 17, showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi (16.12064) sy1-6-14-5 cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome 2 (capsq1) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv1) was impaired in kjr neurons in contrast to that of c57bl/6j mice. by searching the candidate genes, eh domain-containing four (ehd4), a trp-binding scaffold protein encoding gene was found. ehd4 binds to c-terminal of trpv1. three mutations were found in ehd4 of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv1. this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy1-6-14-6 comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than 45 different strains of mutant mice with the battery. surprisingly, more than 90% of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least 28 genes and 12 loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt4f caused by prx, and scan1 caused by tdp1. sy1-6-22-2 gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm1, who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm1 can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm2/gd2 synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, 2004) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa1) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi (17109015) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo-1 is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo-1 mediates the inhibition of axonal growth as a component of the ngr1/p75/lingo-1 and ngr1/troy/lingo-1 signaling complex. inhibition of endogenous lingo-1 by soluble lingo-1 or dominant negative lingo-1 can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo-1 treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo-1 treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo-1 mediates the inhibition of differentiation and myelination. loss of lingo-1 function using dominant negative lingo-1, lingo-1 rnai, or soluble lingo-1 or lingo-1 knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo-1 led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo-1 in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a 2d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc005787-01a1 sy1-7-15-2 neuronal avalanches reveal neuronal wirings of layer 2/3 cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi (17700318) sy1-7-15-3 spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi 1 , jean-pascal pfister 2 , kazuyuki aihara 1,3 , wulfram gerstner 2 1 department of complexity science and engineering, university of tokyo, japan; 2 school of computer and communication science & bmi, epfl, japan; 3 aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows 03j11691 and sci. res. on priority areas 17022012 from mext of japan, and swiss natl. sci. found. 200020-108097/1 sy1-7-15-4 timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as 200-300 ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi 1 , ikue otaki 1 , saya suzuki 1 , yasunobu yasoshima 2 , kazuto kobayashi 2 1 laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; 2 institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin-2 receptor ␣ subunit (hil-2r␣) was expressed under the control of dopamine ␤-hydroxylase gene promoter. anti-hil-2r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. -8-16-5 integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy1-8-24-1 molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj-1 is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are (1) leukemia in patients exposed with atomic bomb during the iind world war, and (2) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, (3) many genetic/familial diseases show clinical symptoms at the middle or old age. (4) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. (5) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp58, which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp58 gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp58 mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼100 drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc0/+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi3k/akt signaling, which results in decreased gsk3 activity, has also been shown to ameliorate ami. both pka and gsk3 phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk3 at young ages, declining igf levels during aging may increase gsk3 activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk3 inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk3 causes neuronal dysfunction during normal aging. research funds: kakenhi sy1-8-24-5 molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p53 pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, 5-1-1 tsukiji, chuo-ku, tokyo 104-0045, japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy1-8-24-7 identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue 1,2 1 department of geriatric medicine, university of tokyo hospital, tokyo, japan; 2 research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of 14-3-3 sigma, a negative cell cycle regulator that causes g2 arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr2d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka 1 , hossein esteky 2 , kiani roozbeh 2 , tadashi sugihara 1 , gang wang 3 1 riken brain science institute, wako, saitama, japan; 2 institute for studies in theoretical physics and mathematics, tehran, iran; 3 kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of 670 inferotemporal cells to 1100 object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to 60 • rotation. research funds: kakenhi 17022047 alexander thiele 1 , gene stoner 2 , louise s. delicato 1 , mark roberts 1 1 university of newcastle upon tyne, uk; 2 the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v1 while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy2-1-01-3 context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy2-1-01-4 effects of task demands on color processing in area te of the monkey hidehiko komatsu 1,2 , kowa koida 1 1 national institute for physiological sciences, okazaki, japan; 2 sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development1. monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception2-4. here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy2-2-02-1 dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui 1 , hirotaka sakamoto 2 , katsunori sasahara 1 , hanako shikimi 1 , kazuyoshi ukena 1 , mitsuhiro kawata 2 1 laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; 2 department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi (15207007 and 16086206 to kt) sy2-2-02-2 roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er-␤ genes have revealed that activation of er-␣ and er-␤ differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er-␤ in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er-␤ knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi (17330151, 17051001) sy2-2-02-3 sex steroid receptor function in sexual behavior shigeaki kato 1,2 , takashi sato 1 , takahiro matsumoto 1,2 1 imcb, university of tokyo, tokyo, japan; 2 erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., 2003) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., 2004) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., 2006) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., 2003 . pnas usa 100, 9416. sato, t., et al., 2004 . pnas, usa 101, 1673 . shiina, h., et al., 2006 research funds: probrain sy2-2-02-4 annexin 1: a mediator of cell-cell communication in the neuroendocrine system julia buckingham 1 , helen christian 2 , john morris 2 1 imperial college london, uk; 2 department of human anatomy and genetics, university of oxford, uk annexin 1 (anxa1) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa1 to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca1. the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa1 receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori 1 , yuki takayanagi 2 , masahide yoshida 1 , yoshiyuki kasahara 1 , masaki kawamata 1 1 graduate school of agricultural science, tohoku university, sendai, japan; 2 department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at 5 • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) (14360046) sy2-2-08-1 cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f5); f5 activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m1). local field potential activity in f5 and m1 is also tuned to grasp, and there is strong beta coherence between f5 and m1, indicating reciprocal transmission of information. this is also seen in synaptic responses of m1 neurones to stimulation of f5 (and vice-versa). single pulse stimulation in f5 strongly modulates corticospinal outputs from m1 through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy2-2-08-2 where tactile signals are ordered in time shigeru kitazawa 1,2 1 department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; 2 crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by 30 ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., 1.5 s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy2-2-08-3 decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo 1,2 1 biology/cns, california institute of technology, pasadena, ca, usa; 2 jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. 122-channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha (8) (9) (10) (11) (12) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy2-2-08-4 why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust (061140) sy2-2-08-5 recognizing self actions through externalized eyes atsushi iriki 1,2 1 symbolic cognitive development, riken brain science institute, saitama, japan; 2 cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut1, existing mostly in cortical pyramidal cells, and vglut2, found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut1-expressing terminals (n = 289), but were found in-10% of spines innervated also by vglut2-expressing terminals (n = 444). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy2-3-03-2 gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼44-fold decrease in the potassium-chloride co-transporter 2 (kcc2) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer 2/3 pyramidal cells in visual cortex share common excitatory inputs from layer 4 and from within layer 2/3, when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer 2/3 cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi (17023026,17500208) sy2-3-03-5 local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer 1 of rat visual cortex and layer 2/3 of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer 1, axondescending cells receive excitatory input predominantly from layer 2/3 while neurogliaform cells receive stronger input from deeper layers. layer 2/3 neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer 2/3, while the cr− martinotti cells also receive strong excitation from layer 4. irregular-spiking basket cells also receive strong excitatory input from layers 2/3 and 4, but they often have a gap at the top of layer 4, with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers 2/3 and 4, and only weak input from deeper layers. research funds: nih sy2-3-03-6 physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers (30-50) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r03 ey015273 , mcknight neuroscience of brain disorders award sy2-3-09-1 axon guidance mediated by localized ca 2+ signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca 2+ signals. its turning direction depends on the occurrence of ca 2+ -induced ca 2+ release (cicr) through the ryanodine receptor type 3 (ryr3). the activity of ryr3 is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr3, that serves as a key regulator of axon guidance. attractive ca 2+ signals facilitate intracellular membrane transport to the leading front and subsequent vamp2-mediated exocytosis on the side with elevated ca 2+ . in contrast, repulsive ca 2+ signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp2-mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy2-3-09-2 molecular mechanisms for signaling through plexin-a1 hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema3a acts as a guidance cue for axons through a receptor complex comprising neuropilin-1 as the ligand-binding subunit and plexin-a1 as the signal-transducing subunit. the ferm domain-containing gef, farp2, associates directly with plexin-a1. sema3a induces the dissociation of farp2 from plexin-a1, resulting in activation of farp2's rac gef activity, rnd1 recruitment to plexin-a1 and down regulation of r-ras. simultaneously, the ferm domain of farp2 sequesters pipki␥661 from talin, thereby inhibiting its kinase activity. these activities are necessary for sema3a-mediated repulsion of outgrowing axons. plexin-a1 also functions as a ligand binding receptor of a transmembrane semaphorin, sema6d and contributes to cardiac morphogenesis. sema6d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a1 forms a receptor complex with off-track in the ventricle or with vegf receptor type 2 in the conotruncal segment, which are responsible for the effects of sema6d on the respective regions. research funds: crest sy2-3-09-3 axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins3a (sema3a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema3a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema3a stimulated the transport of neuropilin-1::egfp, plexin-a4::egfp, and fyn::egfp, which are the components of sema3a signaling, but not neuropilin-2::egfp. interestingly, sema3a accelerated the anterograde transport of trka::egfp. these facts suggest that sema3a selectively facilitates the transport of its own signaling components and that sema3a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein-2 (crmp-2) is enriched in growing axon, and play a crucial role in axon specification. crmp-2 interacts with tubulin dimers to promote microtubule-assembly, and binds to sra-1, an effector of rac1 to regulate wave-dependent reorganization of actin filaments. crmp-2 links kinesin-1 to tubulin dimmers and sra-1, and participates in the kinesin-1-dependent transport of tubulin dimmers and the sra-1/wave complex to growing axons. we also found that the par-6/par-3 complex and the ras/pi3-kinase/akt/gsk-3b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk-3b and inactivates its kinase activity downstream of ras/pi3-kinase, thereby increasing non-phosphorylated active crmp-2 which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy2-3-09-5 regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki 1,2 , takashi abe 1,2,3 , shigeyuki kan 1,4 , satoru miyauchi 1,5 1 national institute of information and communications technology, kobe, japan; 2 japan society for the promotion of science, tokyo, japan; 3 graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; 4 kyushu institute of technology, kitakyushu, japan; 5 crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o2-and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p2x4 receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p2x4 receptors produce a reduction of the neuropathic pain behaviour (2003. nature 424, 778-783) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p2x4 causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury (2005 ( . nature 438, 1017 ( -1021 . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi (15209051) sy2-5-05-4 pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the 18 kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk11195, which labeled with carbon-11 can be used for positron emission tomography (pet). using 11c-(r)-pk11195 pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy2-5-10-1 application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past 25 years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy2-5-10-3 brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality (2005 . neuropsychobiology 52, 45), aging (2004 . neuroimage 22, 1715 , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, 2006) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia (2004. biol. psychiatry 55, 501; 2006. neuroimage 29, 172) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every 0.1s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy2-5-10-5 clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy2-5-10-6 measurement of language related brain activities during recovery from aphasia eiju watanabe 1 , yumiko muroi 2 , chizuru nakajima 2 1 department of neurosurgery, jichi medical university, tochigi, japan; 2 department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured 17 aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the 1990s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in 21st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy2-6-06-3 neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy2-6-06-4 neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy2-6-06-5 neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy2-6-06-6 bridging neuroscience and public: neuroethics in cultural contexts osamu sakura 1,2 1 interfaculty initiative in information studies, university of tokyo, tokyo, japan; 2 research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura 1,2 , akira midorikawa 3 , yoshiki kaneoke 4 , shinichi koyama 1 , masato taira 5 , argye hillis 6 1 showa university school of medicine, japan; 2 crest, jst, saitama, japan; 3 national institute of neuroscience, ncnp, tokyo, japan; 4 national institute for physiological sciences, okazaki, japan; 5 nihon university, tokyo, japan; 6 johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi (17022035), crest sy2-6-11-2 impairment of surface perception in patients with ventral occipital damages shinichi koyama 1 , mitsuru kawamura 1 , akira midorikawa 2 , yoshiki kaneoke 3 , masato taira 4 , argye hillis 5 1 showa university, tokyo, japan; 2 national institute of neuroscience, ncnp, tokyo, japan; 3 national institute for physiological sciences, okazaki, japan; 4 nihon university, tokyo, japan; 5 johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient 1 was a 61-year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory 1970) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient 2 was a 63-year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy2-6-11-3 how do pictorial cues influence 3d information processing in the parietal association cortex? masato taira 1,2 1 arish, nihon university, tokyo, japan; 2 department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for 3d perception. basically, it is thought that the parietal association cortex processes 3d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of 3d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of 3d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in 3d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence 3d information processing in the parietal association cortex. research funds: kakenhi 17022038 sy2-6-11-4 case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa 1 1 national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy2-6-11-5 neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v5+ for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi (15500221) sy2-6-11-6 neural correlates of visual attention argye hillis 1 , mitsuru kawamura 2 , akira midorikawa 3 , yoshiki kaneoke 4 , shinichi koyama 2 , masato taira 5 1 johns hopkins university, usa; 2 showa university, tokyo, japan; 3 national institute of neuroscience, ncnp, tokyo, japan; 4 national institute for physiological sciences, okazaki, japan; 5 nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp2 gene, encoding methyl-cpg binding protein 2, cause rtt. mecp2 acts as a transcriptional silencer. abnormal expression of some genes due to mecp2 dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp2 mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp2-downstream genes. using mecp2-null mice (mecp2 −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp2 directly regulates expression of insulin-like growth factor binding protein 3 (igfbp3) gene in human and mouse brains. pathological features of mecp2 −/y have the similarity of igfbp3 transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp3 due to lack of mecp2 may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements-1 or l1 (line-1) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute 45% of the mammalian genome with l1 elements alone representing 20%. the fact that l1 can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l1 on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy2-7-07-6 notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima 1 , jun kohyma 1 , tetsuya taga 2 , masakazu namihira 1 1 grad. sch. biol. sci., naist, ikoma, japan; 2 inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat3. a cpg dinucleotide in the stat3 recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat3 to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat3 binding element in the gfap gene promoter. sy2-8-12-1 neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. (1) which type of brain infarction will be a future target of this therapeutic approach? (2) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? (3) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy2-8-12-2 gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that (1) motor function may recover after minor injury to the primary motor cortex, (2) this recovery is, at least in part, associated with reorganization of cortical motor representation, (3) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and (4) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to 50 repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy3-1-01-3 neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji 1 , toshiya sato 2 , mitsunori yamada 3 1 department of neurology, the university of tokyo, tokyo, japan; 2 center for bioresource-based researches, japan; 3 department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with 129 cag repeats. the q129 mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy3-1-06-1 control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi 17390086, 17023061 sy3-1-06-2 mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima 1,2 1 dept. of anat., keio univ. sch. of med., tokyo, japan; 2 inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day 12.5 mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca 2+ signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca 2+ elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca 2+ and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp1, rho gtpase, mapk and pi 3 k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy3-2-02-1 membranes, water and diffusion denis j. le bihan shfj/cea, france among 1905 einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid 1980s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy3-2-02-2 diffusion tensor fiber tractography using a 3tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display 3d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded 3-t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on 3-t tractography compared to 1.5 t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using 3-t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of 14 patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy3-2-02-4 anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to 15% of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor (12-60 mg/kg) into male wistar rats (from 5 days to 3 weeks of age) significantly caused hyperactivity at 4-5 weeks old. immunohistochemical analyses of the brain tissues at 8 weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy3-2-07-3 non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around 1 mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around 1 mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about 150 -200 meters from a base station. another remarkable observation in our studies is the fact that a significant (p < 0.002) neuronal damage is seen in rat brains 50 days after a 2 hour exposure to gsm at sar values 200, 20 and 2 mw/kg. we have followed up this observation in a study where 96 animals were sacrificed 14 and 28 days respectively after an exposure for 2 hours to gsm mobile phone electromagnetic fields at sar values 200, 20, 2, 0.2 and 0 (controls) mw/kg. significant neuronal damage is seen after 28 days and albumin leakage after 14. our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after 28 and 50 days. sy3-2-07-4 the mitochondrial toxin 3-nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs 2210, france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin 3-nitropropionic acid (3np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of 3np toxicity and show that 3np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by 3np. in summary, the 3np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of 6-ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at 5 days of age showed behavioral hyperactivity at 4-5 weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of 6-ohda-induced neurotoxicity, using pc12 cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of 6-ohda. we also characterized the oxidation of cellular proteins by 6-ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by 6-ohda, ros and p-quinone. sy3-2-07-6 environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥2 subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥2 subunit-containing receptors. disruption of the direct interaction between prip and the ␤ subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥2 subunit-containing receptors in gh3 and hek293 cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥2 subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi (16109010) sy3-3-03-2 involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer 5 pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca 2+ rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k252a at 200 nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k252a application was started soon after hfs or when k252a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi (17300101) sy3-3-03-3 autocrine mglur1 activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb1 receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur1 on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur1-gq/11 transduction pathway as well as being initiated by direct mglur1 activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur1 activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur1 activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy3-3-03-4 regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi 1,2 , tomoo hirano 1,2 1 dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca 2+ -activated protease calpain or by disturbance of association of gaba a receptor ␥2 subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy3-3-08-1 contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than 50 years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the 50th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy3-3-08-2 detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai 1,2 , susumu takahashi 2 1 department of psychology, kyoto university, kyoto, japan; 2 crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl-1s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi (17022036) sy3-3-08-5 impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto 619-0237, japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy3-4-04-1 tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad65 the amount of the tonic inhibition is reduced in ca1 hippocampal interneurons, while unchanged in pyramidal cells. sy3-4-04-2 modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca1 region yoshihiko yamazaki 1 , yasukazu hozumi 2 , kenya kaneko 1 , satoshi fujii 1 , hiroshi kato 1 1 dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; 2 dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca1 region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a1 receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca1 region. research funds: kakenhi (15082201) sy3-4-04-3 carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of 30 m carbachol induces beta oscillations which occur intermittently with the interval of 20-30 s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is 16.9 ± 0.9 hz (mean ± s.e.m.). the oscillations are induced via muscarinic m1, 3, 4 receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations (1-3 hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that (1) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, (2) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca3 and ca1 neurons discharged during both up and down states, (3) gamma and fast (ripple) oscillations were observed in hippocampal ca1 area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: (1) most of the mprf neurons were active during rem sleep; (2) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; (3) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi (17605001) sy3-5-09-1 clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) 18:6 h and ld 6:18 h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per1 and per2 rhythms suggest that light-on resets per1 rhythms in both light conditions, while per2 rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per1 promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld 18:6. the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per1 and per2 showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy3-5-09-3 genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified 169 genes that fluctuated between day and night in the livers, 100 lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose-6-phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other 100 genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy3-5-09-5 neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy3-6-10-1 a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e)10.5 to e14.5, the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy3-6-10-2 live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi (17024040) sy3-6-10-3 visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol 1,4,5-trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy3-6-10-4 rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦2 (glur␦2) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦2 into glur␦2-null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦2 lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi 16650071 sy3-6-10-5 gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi (17300100) sy3-7-11-1 physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi (17022042) sy3-7-11-2 deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid-20th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy3-7-11-3 firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no.454) sy3-7-11-5 characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama 1,2 1 department of neurological surgery, nihon university school of medicine, tokyo, japan; 2 division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate (49.4 ± 31.7 hz, n = 27) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca 2+ -atpase isoform 2 that is located on the 6th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that (1) d2-type dopamine receptors are downregulated presynaptically in the striatum, and (2) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy3-8-05-2 metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr54 (2001. nature 411, 613) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr54 impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat (2005. endocrinology 146, 4431) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca 2+ imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi 15370032 sy3-8-05-5 metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy3-8-12-1 molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi 17510039, 17390060 sy3-8-12-2 thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of 15 isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp1c1 is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t4 and reverse t3, but the transport activity of t3 is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp1a4, the transport activities of t4 and t3 are similar, is also expressed. the uptake of t4 and t3 by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t4 and t3. research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy3-8-12-3 alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi 1 , masaki takahashi 1 , yasuhiro yoshikawa 2 , tomoko tashiro 1 1 department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; 2 department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to 4-hydroxy-2,2 ,3 ,4 ,5pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to 4-hydroxy-2,2 ,3,4 ,5,5 ,6-heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites (2005. dev. brain res. 154, 259) . koibuchi et al. (2004) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. (2004) detected some pcb and oh-pcb congeners in human csf, of which 4oh-cb187 is the highest concentration. to determine its effects on developing neurons, we examined 4oh-cb187 in rodent cerebellar culture cells. interestingly, 4oh-cb187 promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or 5-htp to examine the relevance of serotoninergic function. the treatment of 5-htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., 2005) . reference kawano et al., 2005. j. neurosci. res. 80, 191-202. research funds: kakenhi 16500234 os2a-8-03 neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama 1 , kazumi takahashi 2 , yukihiko kayama 2 1 cluster of science and technology, fukushima university, fukushima, japan; 2 department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic (5ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the 5ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣1 receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os2a-8-04 correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi 1,2 , kazuyoshi hirano 2,3 , yukimasa muraishi 2,3 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, japan; 3 secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l2) and grey matter volume among students aged 13-17, who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l2. os2a-8-05 grammar center activation in honorification judgment of japanese sentences kanako momo 1,2 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f3op/f3t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os2a-8-06 top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui 1,2,3 , kimitaka kaga 2 , kuniyoshi l. sakai 1,3 1 dept. of basic science, univ. of tokyo, tokyo, japan; 2 dept. of otolaryngology, univ. of tokyo school of medicine, japan; 3 crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m140) (neurosci. res. 52, s61). in a subsequent study, we confirmed that m140 was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m140 was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m140 elicited by the original songs and that by the modified ones. however, while m140 without the deviation showed no significant effect of lateralization, m140 with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os2a-8-07 cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto 1,2 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in 16 sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os2a-8-08 hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima 1,2 , naoki fukui 3 , kuniyoshi l. sakai 1,2 1 dept. of basic science, univ. of tokyo, komaba, japan; 2 crest, jst; 3 dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as 130 ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os2a-8-09 individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext (14002005), jsps research fellowship (1611149) os2a-8-10 brain activity of happy facial recognition in mother-daughter relationship jun shinozaki 1 , nobukatsu sawamoto 1 , toshiya murai 2 , takashi hanakawa 1 , hidenao fukuyama 1 1 hbrc, kyoto univ. grad. sch. of med. kyoto, japan; 2 neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os2a-8-11 anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto 1,2 , atsushi maki 1,2 , kuniyoshi l. sakai 2,3 1 advanced research laboratory, hitachi, ltd., tokyo, japan; 2 crest, japan science and technology agency, saitama, japan; 3 dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f3t/f3o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f3t and f3o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (−54, 27, 21) , (−39, 3, 42) , and (−51, 27, −6) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os2a-8-12 effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada 1 , sunao iwaki 1 , mitsuo tonoike 2 1 aist, osaka, japan; 2 chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = 4), and with those from an inner semantic category (middle, d = 2), while others were not changed (normal, d = 0). peak amplitudes of waveforms of the root mean square values on the components of 170 ms (f (2/38) = 4.92, p = 0.013) and 220 ms (f (2/38) = 5.23, p = 0.0098) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the 170 and 220 ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os2p-2-02 mechanisms of p2y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono 1 , xiaoming zhu 1 , takashi tominaga 2 , fumihito saitow 3 , shiro konishi 1,2 1 waseda-olympus bioscience research institute, singapore, singapore; 2 department of neurophysiology, tokushima bunri university, kagawa, japan; 3 department of pharmacology, nippon medical school, tokyo, japan postsynaptic p2y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and 5-15 min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were 160 ± 9.9% and 94 ± 6.1% of the controls, respectively. pretreatment with the protein kinase a inhibitor h-89, but not the calmodulin kinase ii inhibitor kn-62, completely abolished the atp-induced ipsc enhancement. os2p-2-03 activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip1 isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca 2+ permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca 2+ influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein 1 (arip1). activin-induced nmdar activation persists more than 24 h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os2p-2-04 roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr177 by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr177 phosphorylation following ltp induction in rat hippocampal ca1 region. like camkii activation following ltp (fukunaga et al., 1993) , the increased camki-thr177 phosphorylation remained elevated at least for 60 min after ltp induction. the increased camki-thr177 phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca1 region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto-609 significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca1 region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing (5 ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing (20 ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi (17021037) os2p-2-06 mouse brains deficient in neuronal pdgf receptor-␤ develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr-␤ subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-␤. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr-␤ expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr-␤ expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p6) rats that had received a single brdu injection at p5 were substituted with the corresponding region of entorhino-hippocampal slices from p6 rats. after five days in vitro, newborn gcs, detected by brdu and prox1, migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os2p-3-02 cdk5 is required for neuroblast migration in the adult mouse brain yuki hirota 1,2,6 , toshio ohshima 3 , takuji iwasato 4 , ashok b. kulkarni 5 , hideyuki okano 2,6 , kazunobu sawamoto 1,2,6 1 bridgestone lab. keio univ., tokyo, japan; 2 dept. physiol., keio univ., tokyo, japan; 3 dev. neurobiol. riken, tokyo, japan; 4 behavioral gen. riken, tokyo, japan; 5 nih, bethesda, usa; 6 sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase 5 (cdk5) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk5 mice to emx1cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk5 has a cell autonomous function in neuroblast migration in the adult brain. os2p-3-03 colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi 1 , wu sheng-xi 2 , yuchio yanagawa 3,4 , kunihiko obata 5 , nobuaki tamamaki 1 1 kumamoto univ., kumamoto, japan; 2 fourth military medical univ., xi'an, people's republic of china; 3 gunma univ., maebashi, japan; 4 sokendai, hayama, japan; 5 riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng2, plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad67-gfp knock-in mouse neocortex at e18-p0 by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t7 rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s100-␤ expression in the gabaergic neuron progenitors. os2p-3-04 role of ␤-catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi 1,2,3 , masanori sakaguchi 3 , toru yamashita 2,3,6 , yuko fujita 3 , yukiko gotoh 4 , arturo alvarez-buylla 5 , takeshi kawase 1 , hideyuki okano 3 , kazunobu sawamoto 2,3 1 neurosurgery, keio univ. sch. med., tokyo, japan; 2 bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; 3 physiol., keio univ. sch. med., tokyo, japan; 4 inst. mol. cell biosciences, univ. tokyo, tokyo, japan; 5 neurosurgery, ucsf, san francisco, usa; 6 neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of ␤-catenin signaling in the adult mouse svz. ␤-catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated ␤-catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that ␤-catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt2b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt2b. to examine the molecular interactions between c-tsk and wnt2b, we co-electroporated them into the optic vesicle at stage 9-10 chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt2b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt2b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) 9 in developing cortical neurons. an expression vector of gfp-tagged hdac9 was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac9 was primarily localized in nuclear until 1 week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac9 contributes to neuronal differentiation in cortical development. research funds: kakenhi (16700286) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., 2004) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum 2.45 ns) was measured. in high-voltage electron microscopy (hitachi1250m, nips, 2005 , gap junctions (average size 0.86 m long) were present in contacts. in confocal laser-scanning imaging, connexin36 localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be 5180/m 2 and a single channel conductance is 15 ps, the conductance of each junction would be 45 ns. the presence of seven junctions between a pair will lead to estimate a total junction of 315 ns. the measured conductance could allow to estimate a fraction of open channels as 0.8%. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi (17023014), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost 2000 genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os2p-4-04 multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a 60 channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer 4 stimuli and propagated horizontally in the layer 2/3. in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer 4 stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap5 + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os2p-4-05 presence of two phases in the sensitive period of orientation plasticity shigeru tanaka 1 , toshiki tani 1 , kazunori o'hashi 1,2 , jerome ribot 1 1 brain science institute, riken, saitama, japan; 2 graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after 2 weeks of goggle rearing starting around the age of 3 weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of 5 weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os2p-4-06 residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida 1,2,3 , tadashi isa 1,2,3 1 dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; 2 sch. life sci., grad. univ. adv. stud., hayama, japan; 3 crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v1 lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield (80-90% correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v1 lesion is unlike normal, near-threshold vision. research funds: kakenhi 13854029, kakenhi 16700343 and crest, jst os2p-4-07 comparison of the angle representation in macaque visual areas v1 and v2 minami ito 1,2 , hidehiko komatsu 1,2 1 national institute for physiological sciences, okazaki, japan; 2 the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v2 neurons has angle selectivity. here, we studied the angle selectivity of area v1, which is the major source of inputs to area v2. we conducted single-unit recordings from the superficial layer of area v1, while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v1 neurons responded mainly to sharp angles (30 • ), straight lines (180 • ) or right corners (90 • ), but not to intermediate angles (60 • or 120 • angle width). this contrasted with area v2, where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v1 neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v1 neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v2. os2p-4-08 firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to 10 • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os2p-4-10 integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v1, we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os2p-4-12 probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner 1,2 , pei sun 2 , keiji tanaka 2 , david j. heeger 1 , kang cheng 2 1 department of psychology and center for neural science, new york university, usa; 2 laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri (0.75 mm × 0.75 mm × 3 mm voxels) in human v1. we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t2*-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih (1f32ey016260-01) os2p-4-13 relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations (18-350 ms; isi = 1 s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase (350-550 ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase (70-270 ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼50% of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os2p-4-15 the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe 1 , kazuto terai 2 , osamu hieda 3 , shigeru kinoshita 2 1 dept. of ophthal., kyoto second red cross hospital, kyoto, japan; 2 dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; 3 baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from 0.4 to 1.2. the logmar average was 0.31. eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was 0.03, improved from 0.50. the residual five eyes showed less or no improvement. the change of logmar average was from 0.15 to 0.08. conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os2p-5-01 the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada 1 , hideomi tanaka 1,2 , satomi nakayama 1 , miki iwasaki 1,2 , hitoshi okamoto 1,2 1 laboratory for developmental gene regulation, bsi, riken, japan; 2 crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) 4 and migrate tangentially to r6 near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr2 and frizzled3a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled3a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os2p-5-02 disrupted-in-schizophrenia 1 (disc1) regulates the transport of the nudel/lis1 complex to axons via direct interaction with kinesin-1 shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc1 is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc1. disc1 is reported to interact with nudel, which forms a complex with lis1. although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc1 remain largely unknown. here we report that disc1 interacted with kinesin-1, a motor protein of anterograde axonal transport. kinesin-1 interacted with the nudel/lis1 complex through disc1, and these molecules accumulated at the distal part of axons. the knockdown of disc1 by rnai of disc1 induced the delocalization of nudel and lis1 from the axons and reduced axonal growth. the knockdown of kinesin-1 induced the delocalization of disc1 from the axons. taken together, these results indicate that disc1 links kinesin-1 to the nudel/lis1 complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os2p-5-03 role of a novel collapsin response mediator protein-2 interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein 1 (slp1) can mediate the vesicle transport. slp1 is known to associate with rab27. we found that slp1 associates with collapsin response mediator protein-2 (crmp-2), which is a key regulator of axon formation. slp1 could form the trimeric complex with rab27b and crmp-2, and also associate with kinesin-1 through crmp-2. slp1 is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp1 functions as a mediator of recruitment of certain receptor depending on crmp-2 and kinesin-1. os2p-5-04 absolute quantification of mdr1a, mrp1, mrp4 and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie 1,2 , yuki katsukura 1,2 , sumio ohtsuki 1,2 , xiao-kun cai 1,2 , tetsuya terasaki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr1a, mrp1, mrp4, and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between 10 and 1000 fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr1a, mrp4, bcrp were 3.9, 2.7 and 4.8 fmol/g, respectively, while it of mrp1 was under detection limit of standard curve. this quantitative profile suggests that mrp4 and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr1a. os2p-5-05 dominant expression of claudin-5 in highly purified brain capillary endothelial cells sumio ohtsuki 1,2 , hirofumi yamaguchi 1 , saori sato 1 , tmoko asashima 1,2 , tetsuya terasaki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam-1) and magnetic cell sorting. mrna expression of caludin-1-23 was measured by real-time rt-pcr. claudin-5 showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin-1 and -12 were 0.0027% and 0.20% of that of claudin-5. claudin-5 mrna was concentrated in the purified bcecs, while claudin-1 and -12 mrna in the purified bcecs were lower than that in the whole brain. rat claudin-5 mrna was also concentrated in rat brain capillary fraction, but claudin-12 mrna did not. these results suggest that claudin-5 is a dominant tjs protein in bcecs, and expression of claudin-1 and -12, which was reported as tj protein in bcecs, are not restricted in bcecs. os2p-5-06 effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad 1 , a. pauzi m. yusof 1 , p.d. mourad 2 , m. bainbridge 3 , s. ab ghani 1 1 universiti sains malaysia; 2 university of washington, seattle, usa; 3 brody school of medicine, east carolina university, nc, usa h 2 o 2 is the main peroxides produced in mammalian cells that consume o 2 . the main source of h 2 o 2 in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h 2 o 2 towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h 2 o 2 microsensor, we investigated the level of h 2 o 2 in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h 2 o 2 level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h 2 o 2 was injected into the brain. fatality occurs when the injected h 2 o 2 exceeds 250 m. these finding showed that the altered paracellular permeability in the presence of h 2 o 2 is caused by a series of events that happen one after another. research funds: short term grants 304pkimia633134 and 304pfar-masi670008 os2p-6-01 somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t9-11) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t9-11 spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os2p-6-02 responses of renal sympathetic nerve activity and sodium excretion to 3 days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the 7 days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions (0, 50, 154, 308 meq./l nacl) over 3 days. the sodium loading with 308 meq./l nacl suppressed rsna significantly and then it gradually recovered while either 0 meq./l nacl or 154 meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os2p-6-03 cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku 1 , haruko masumiya 1 , yasumasa okada 2 1 dept. physiol., hyogo col. med., nishinomiya, japan; 2 dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c4 ventral root (c4vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as 3d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: (1) a continuous column extending from the para-facial region to the pre-bötzinger complex, and (2) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c4vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os2p-6-04 slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca 2+ -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi 16590180, 16086210 os2p-6-05 the effect of music to sex hormones of elderly person hajime fukui 1 , kumiko toyoshima 2 , kiyoto kuda 1 , katsuhiko iguchi 3 1 nara univ. of edu., nara, japan; 2 grad. school of human sciences, osaka univ. japan; 3 nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and 36 females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a 30-kda protein band in csf of 100% (n = 5) of confirmed and 90% (n = 138) of suspected tbm patients out of 153 tbm patients. the 30-kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv3804c (ag85a) and rv1886c (ag 85b) and one host derived protein as the components of the tbm specific 30-kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q 10 , vitamin d 3 and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os2p-7-02 involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh 1 , boon chock 2 , dennis l. murphy 3 , chuang c. chiueh 4 1 dept. applied pharmacol., univ. toayama, toyama, japan; 2 lab. bioch., nhlbi, nih, md, usa; 3 lab. clin. sci., nimh, nih, md, usa; 4 cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy5y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk1/2 and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy5y cells. consistently, trx increased the expression of mnsod and bcl-2 supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl-2 proteins for protecting against mpp + -induced neurotoxicity. os2p-7-03 pgd 2 induces neuronal apoptosis via 15d-12,14 -pgj 2 tatsurou yagami 1 , noboru okamura 2 , toshiyuki sakaeda 2 1 facul. health care sci., himeji dokkyo univ., himeji, japan; 2 kobe univ. grad. sch. med., japan prostaglandin d 2 (pgd 2 ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd 2 induced neuronal apoptosis in rat cortical cultures. however, a pgd 2 receptor blocker did not suppress neurotoxicity of pgd 2 . little pgd 2 receptor was detected, suggesting an involvement of pgd 2 metabolites in the apoptosis. among pgd 2 metabolites, 15-deoxy-12,14 -prostaglandin j 2 (15d-12,14 -pgj 2 ) caused neuronal apoptosis most potently and rapidly. although 15d-12,14 -pgj 2 is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that 15d-12,14 -pgj 2 induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ 3 h]15d-12,14 -pgj 2 (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd 2 induced apoptosis via 15d-12,14 -pgj 2 in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the 15d-12,14 -pgj 2 -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c6 to t1). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi 17022043 os2p-7-06 compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than 20 longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi (17022027) os2p-7-09 timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae 1 , xiaofeng lu 1,2 , yusuke uchida 1 , toshimitsu takahashi 1,2 , shigeru kitazawa 1,2 1 dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; 2 crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of 16 targets following a gosignal given at a random timing between 500 and 800 ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: (1) those that showed buildup activity during the delay period until the go-signal, and (2) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, 30-200 h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (>9 ms) than larvae (>80 hpf, 3 ms). m-cell activity monitored with confocal ca 2+ imaging during the tail flip (>80 hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (<25%) after 100 hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca 2+ release through ryanodine receptors, triggered by glutamate receptor-mediated ca 2+ influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os2p-8-02 distribution of voltage-gated calcium channel ␣ 2 ␦-4 mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka 570-8506, japan the ␣ 2 ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ 1 subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ 2 ␦-1 to ␣ 2 ␦-4. the distributions of ␣ 2 ␦-1, -2, -3 mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ 2 ␦-4 remains to be determined. we cloned ␣ 2 ␦-4 cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ 2 ␦-4 mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ 2 ␦-4 mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ 2 ␦-4 subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about 34 glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about 6 glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os2p-8-05 hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb334867) depressed the excitation, while bicuculline depressed the inhibition. h 1 or h 2 histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os3a-5-01 activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c57 bl/6 mice. by recording field epsp (fepsp) along 80 m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at 7-8 div, then the amplitudes of fepsps in the ventral side began to decrease at 9-10 div, and dominated in the dorsal area at 14 div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at 7-9 div but the axons terminals in the ventral area were eliminated until 14 div. this synapse elimination from the ventral side was blocked by apv application from 6 div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until 7 div. the number of those ventral axons is reduced before 14 div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co2 incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at 6 div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during 7-11 div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os3a-5-06 transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os3a-5-07 segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r3 receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa2 in r2 is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa2 expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa2 function resulted in selective loss of eph4 expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa2 dependent cues are required to determine the territory for whisker representation in r3 and the assembly of a somatosensory circuit. os3a-5-08 the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p7 and those in the venrtolateral (vl) area were eliminated from p8 to p10 and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p12 and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p12, reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p14 some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os3a-5-09 proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi (17023030), kakenhi (15300107) os3a-6-01 the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine 1 department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m1, s1, sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms (1, 5, 10 hz, 500 stimulations) was compared in 20 dp patients. the pain relief was evaluated with visual analogue scale. high frequency (5, 10 hz) rtms of m1 was the only effective stimulation for treating intractable pain in 10 of 20 patients (50%). the pain relief continued for 3 h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m1. os3a-6-02 involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p2x 3 r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,␤-meatp (p2x 1,3,2/3 rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,␤-meatp with ppads (p2x 1,2,3,5,1/5,4/5 r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p2x 3 rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p2x 3 r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the ␤-endorphin (␤-ep)-ir was decreased 7 days after snl. furthermore, the subpopulations of ␤-ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os3a-6-04 the cantor-like patterns in rat hippocampal ca1 pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca1. this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca1 pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca1 pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after 35 days of age, before which they had been reared with song tutors from birth. when the noise stopped at 102-200 days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization (30 days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d90 of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before 80 days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, 2005) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, 2005) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada (1996) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca1 hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca1. they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca3. this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca3, may be encoded in ca1 in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca1, using 2-compartment model of neuron, and we found the cantor coding of information of time series in the model ca1. it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca1 region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in 15 patients with chronic ischemia due to diverse vascular occlusive diseases (n = 12) and moyamoya disease (n = 3). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in 8 patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os3a-7-02 hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata 1,2 , heng zhao 2 , gary steinberg 2 1 department of neurology, brain research institute, niigata university, niigata, japan; 2 department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia (30 • c) reduced infarct size by 84%. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < 0.05). p-␦pkc (t505) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s643) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, 4-vessle occlusion was induced. fluo-3/am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu-21, yokogawa, japan) equipped with 10× objective lens] was inserted into the brain. 4-vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo-3 increased up to 115%, and persistently increased up to 130% during 20-min reperfusion, indicating the long-lasting ca 2+ increase in the ca1 region. in contrast, in the frontal cortex, 10-min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca1 region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca 2+ in situ. research funds: kakenhi #16390407, #16047212 os3a-7-04 reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto 1 , h. watanabe 1 , y. kumon 1 , t. ohnishi 1 , chi ii 2 , y. imai 2 , j. tanaka 2 1 dept. neurosurgery, ehime university, japan; 2 dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within 36 h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b4 and cd11b recognized with ox42 antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba1+ mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba1+ amoeboid-shaped mg started to accumulate 3 days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os3a-7-05 insulin regulates the hepatic clearance of amyloid ␤ peptide tetsuya terasaki 1,2 , chihiro tamaki 1 , sumio ohtsuki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan the liver is the major organ that eliminates amyloid ␤-peptide (a␤) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein 1 (lrp-1) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a␤ . insulin infusion into the rat portal vein increased lrp-1 expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a␤(1-40), which reached 1.6-fold greater uptake than non-treated control after 10 min treatment. increase of the hepatic uptake of a␤(1-40) by insulin was concentration dependent (ec 50 = 230 pm), and was completely suppressed by rap (2 m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp-1 to the plasma membrane of hepatocytes, leading to increase of a␤ hepatic clearance from the circulation. research funds: sorst, jst os3a-7-06 mr images of intra-arterially administered microglia surrounding ␤-amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a␤-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used 1321n1 astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of 1321n1 cells was inhibited by an inhibitor of ip 3 receptors, 2-aminoethoxydiphenyl borate (2-apb) or an endoplasmic reticulum ca 2+ -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca 2+ was inhibited by 2-apb and cpa. moreover, removal of external ca 2+ accelerated the reversal of thrombin effects. these results suggest that refilling of ca 2+ store by ca 2+ entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age 38 and a 25-year-clinical course. the brain showed severe atrophy (630 g). microscopic examination disclosed that all telencephalic neocortices had nft of more than 100 and sp of more than 10. all limbic cortices and nuclei had nft of more than 100 and sp of more than 10. although there was no sp, various numbers of nft were observed in the following structures: claustrum 85, caudate 9, globus pallidus 6, hypothalamus 22, meynert's nucleus 4, thalamus 74, substantia nigra 6, central gray 12, locus ceruleus 6, purkinje cells 0, posterior root ganglion 0, adrenal medulla 3. this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os3a-7-09 transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with 32plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv4 is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv4 was expressed in soma, dendrites and synapses in the neurons. since trpv4 was firstly cloned as an osmotically activated channel, we hypothesized trpv4 might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv4 expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv4-deficient neurons at 37 • c, and found rmp in wild type was more depolarized by approximately 5 mv than rmp in trpv4-deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv4-deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv4 is involved in regulation of both neural activity and spine motility in hippocampus. os3p-2-04 a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at 100 locations per 200 ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by 2-photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os3p-2-05 d1-like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a 1 receptors (adora1) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora1 in the ng decreased by >90% of that in the non-treated ng, being not accompanied by a change in mrna level for a 2a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase-3 activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf1 in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf1 blockers such as brefeldin a, exo1, and arf1 n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf1 and subsequent pld may regulate the k + -current response induced by da. os3p-3-05 p250gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa 1 , toshihiko kuriu 2 , ayako m. watabe 3 , toshiya manabe 3 , shigeo okabe 2 , tadashi yamamoto 1 1 div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; 2 dept. of cell biol., tokyo medical and dental univ., tokyo, japan; 3 div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p250gap, which interacts with the nr2b subunit of nmdar. within neurons, p250gap was highly concentrated in the postsynaptic density and co-localized with nr2b and an actin-binding protein, cortactin. p250gap promoted gtp hydrolysis of cdc42 and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p250gap. when over-expressed in dissociated neuron, p250gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p250gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os3p-3-06 non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os3p-4-01 spatial frequency tuning of disparity-selective neurons in macaque v4 hironori kumano 1 , seiji tanabe 2 , ichiro fujita 2 1 grad. sch. of engineering science, osaka univ., osaka, japan; 2 grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v4 of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional (2-d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v4 neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using 2-d filtered noise stereograms. the disparity tuning of most v4 neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v4 neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os3p-4-02 predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v4 hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v4 of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v4 are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v4 while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v4 neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v4 neurons. the results suggest that v4 neurons are involved in mediating stereoacuity. os3p-4-03 the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field 2-d ternary dynamic random noise, generally refreshed every 26-40 ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v1/v2 neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v1, and especially areas beyond. research funds: mext(13041033), jsps(13308048), coe21 os3p-4-06 analysis of center-surround organization of v1 neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area 17 (v1) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of 52 cells analyzed, 80% showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ±90 degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os3p-4-07 spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki 1 , izumi ohzawa 1,2 1 grad. school of eng. sci., osaka univ., japan; 2 grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial 2nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with 51 × 51 dots was presented over an area 2 to 4 times larger than the rf of a complex cell. for each dot location, a 2nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of 2nd-order kernels at all locations. 2nd-order kernels had roughly comparable extent as the rf, and contained 3.3 subregions on average (n = 53). among 35 complex cells, whose rf envelopes were elongated, 16 cells exhibited the horizontal elongation. research funds: mext(13041033), jsps(13308048), coe21 os3p-4-08 orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito 1 , osamu sadakane 1 , masahiro okamoto 2 , hironobu osaki 3 , hiromichi sato 1 1 grad. sch. med., osaka univ., osaka, japan; 2 grad. sch. front. biosci., osaka univ., osaka, japan; 3 med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about 19% lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that 97% of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os3p-4-09 temporal dynamics of suppressive receptive field surround in cat v1 satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v1), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes (500 ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v1 cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (<100 ms) but not in middle (100-200 ms) and late responses (200-500 ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from 2.5 deg (early response) to 5 deg (middle response) and 7.5 deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi (15500259) os3p-4-10 spatial-frequency dependent surround suppression in cat v1 ayako ishikawa 1 , satoshi shimegi 2 , hiroyuki kida 3 , hiromichi sato 2 1 grad. sch. front. biosci., osaka univ., osaka, japan; 2 grad. sch. med., osaka univ., japan; 3 grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v1. we used a stationary flash (duration, 500 ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus (50 ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = 0) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os3p-4-11 contrast-dependency of spatial summation property in cat v1 and lgn masahiro okamoto 1 , tomoyuki naito 2 , osamu sadakane 2 , hiromichi sato 2 1 grad. sch. front. biosci., osaka univ., japan; 2 grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v1) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v1 and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by 0.76 and 0.68 times in v1 and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v1, the strength of surround suppression did not affected by stimulus contrast on average. we conclude that 1) rog model describes the surround suppression better than dog model both in v1 and lgn, 2) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida 1 , satoshi shimegi 2 , ayako ishikawa 3 , hiroshi sakamoto 3 , hiromichi sato 2 1 grad. sch. eng. sci., osaka univ., japan; 2 grad. sch. med. sci., osaka univ., japan; 3 grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v1), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v1 neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into 8 flanks (45 • step), and stationary stimulated with an annulus, oppositely-faced flanks (2-fk) or a flank (1-fk) stimulus. the durations of stimulus presentation were 500 ms for crf and 50 ms for srf stimulation. localized srf stimulation with either 2-fk or 1-fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for 1-fk stimulation throughout the crf response. however, 2-fk stimulation of end position to crf had strong and long-lasting suppression on responses during 80-200 ms after onset. os3p-4-13 temporal-frequency dependency of receptive field size and surround suppression in lgn and v1 osamu sadakane 1 , tomoyuki naito 1 , hironobu osaki 2 , masahiro okamoto 3 , hiromichi sato 1 1 grad. sch. med., osaka univ., japan; 2 med. sch., osaka univ., japan; 3 grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v1) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v1 neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v1 neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v1 neurons, suggesting that the subcortical tf tuning property contributes to that in v1. ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. 2001; yang and maunsell 2004) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across 70 daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using 4.7 t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of 50 m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around 1%) was smaller than that of bold (>2%), the temporal profile showed a constant precedence of the diffusion signal by 2.4s. os3p-5-03 new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os3p-5-04 spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international 10-20 system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within 1 cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad (1.33), approaching unity (∼1.02) for oh, and in between for control (1.14). statistical analysis was performed and significant differences were observed for these groups (p < 0.01). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p38 mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p38 mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: (1) resting, (2) staring at a standing human object, (3) thinking of standing, (4) standing with eyes open, (5) standing with eyes closed. regional cbf data analyzed by spm2 were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os3p-6-03 potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono 1,2 , eiji shigetomi 2 , kiyoharu inoue 1 , fusao kato 2 1 dept. neurol., jikei univ., sch. med., tokyo, japan; 2 lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os3p-6-04 effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan 3,4-methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin (5-ht) release and cause degeneration of 5-htergic nerve terminals via 5-ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the 5-htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on 5-ht release and 5-htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant 5-htergic neurons around the raphe nuclei. treatment with a 5-htergic neurotoxin 5,7-dihydroxytryptamine dramatically reduced the tissue contents of 5-ht and its metabolite, which was blocked by a selective 5-ht reuptake inhibitor. mdma and meth (0.1-1000 m) increased 5-ht release, and reduced the tissue contents of 5-ht and its metabolite at higher doses. the mesencephalic slice culture including the 5-htergic raphe nuclei may be useful to examine the mechanisms underlying 5-htergic neurotoxic effect of mdma in vitro. os3p-6-05 studies on drug dependence (rept. 421): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os3p-6-06 prostaglandin d 2 is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d 2 , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp1 receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd 2 by hpgds and that astrocytes expressed both dp1 and dp2 receptors and were activated by pgd 2 . we found that gliosis and demyelination were well suppressed in hpgds-or dp1-null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd 2 is a key molecule of neuroinflammation involved in the demyelination. research funds: 09670806, 12558078 os3p-7-01 on a sodium channel distribution enabling high frequency signal processing go ashida 1,2 , kousuke abe 2 , kazuo funabiki 1 1 grad. sch. medicine, kyoto univ., kyoto, japan; 2 grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to 8 khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below 1khz, while that in the node does up to 10 khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated 140 nl cells from 16 owls and achieved intracellular recordings in 38 of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = 58 ± 17 mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, 13 vision impaired (7 blind and 6 low vision) and 15 sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os3p-8-01 autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada 1 , runa araya 1 , naoto kitamura 1 , yuji mishinsa 2 1 yamada unit, riken bsi, saitama, japan; 2 nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr1a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr1a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr1a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba1-positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os3p-8-04 fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita 1,2 , hideomi tanaka 1,2 , sachiko tsuruoka 3 , hironori wada 1,2 , hitoshi okamoto 1,2 1 riken bsi, wako, japan; 2 jst crest, kawaguchi, japan; 3 riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose 4,6 dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi (17024013) os3p-8-06 initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd95-homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin2 (fas2) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas2 genetically expressed on a muscle, we found fas2 accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas2 is crucial for the postsynaptic localization of fas2. in addition, postsynaptic localization of a scaffolding protein dlg, psd95-homolog, and glutamate receptors was impaired in fas2 mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor 2a (mef2a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef2a that is sumoylated at lys403 promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef2a at ser408 and thereby promoted a switch from sumoylation to acetylation at lys403, leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns4102, ag11085 ps1a-a002 characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., 1999) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified 1152 randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., 1999. mol. brain res., 72, 147-157. research funds: kakenhi (17500252) ps1a-a003 the distribution of snap-25 protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap-25 derived from exon 5 splicing are expressed in brain. we generated two specific antibodies for snap-25a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap-25a and b during early postnatal period. snap-25b was low at the birth and increased remarkably thereafter. by the contrast, snap-25a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap-25b but not snap-25a was recovered in raft-enriched fractions of triton x-100-treated lp1 membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap-25b was widely distributed throughout brain, whereas, snap-25a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap-25 protein are regulated differently in isoformspecific manners, and snap-25a and snap-25b play different functional roles in brain. ps1a-a004 erc(elks/rab6ip2/cast) regulates syaptic short-term plasticity by recruiting bmunc13-2 to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain (2001. mol. brain res. 89, 20-28) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos7 cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt-1, are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt-1, the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt-1(-/-) mice, and a novel antagonist of glial glutamate transporters, (2s,3s)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately 20%) of functional transporters, glast and/or glt-1, is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat-4 gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat-4 mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat-4 vglut. a mutation in avr-14 glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr-14 functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps1a-a008 biochemical characterisation of the vesicular glutamate transporter 1 stephan schenck, shigeo takamori department of neurology and neurological science, 21st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut1 and vglut2 are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut1 and vglut2 has been found. to further characterise the specific properties of the transporters we make use of the vglut1-ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut1 by yeast two-hybrid screening and found endophilin a1. as expected for the interacting molecule to vglut1, endophilin a1 was typically localized to vglut1-positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in 60∼90 min, followed by spontaneously recovered after120 min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of 14c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) 2 was demonstrated in the lactate medium. results suggested that increased mct2 expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu 1 , kazuhito tomizawa 1 , shuang liang 2 , iori ohmori 1 , teiichi nishiki 1 , kohji takei 2 , hideki matsui 1 1 dept. of physiol., okayama univ., okayama, japan; 2 dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca 2+ -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos-7 cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately (27th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm1-43 and fm4-64, and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca2+ channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin 1a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin 1a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin 1a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca 2+ channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca 2+ influx and epscs, suggesting that exocytotic machinery downstream of ca 2+ influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca 2+ or g-proteins. ps1a-b017 spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb1 cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca 2+ channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap-2 subunit can directly bind with synprint site. this interaction was ca 2+ -dependent, being weaker at concentrations higher than 200 nm. in contrast, the interaction of synaptotagmin with synprint was optimal at 100 m ca 2+ , being weaker at lower or higher concentrations. the binding domain of synprint for ap-2 and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca2+ channel synprint site makes ca 2+ -dependent interactions with ap-2 and synaptotagmin thereby contributing to vesicular endocytosis. ps1a-b019 acl-4, an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl-1˜14, and generated their deletion mutants. the mutants of acl-4 gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat-3 mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl-4 expression, suggesting that certain phospholipid species produced by acl-4 are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku 1 , hussam jourdi 1 , akiyoshi kakita 2 , tadasato nagano 1 , hitoshi takahashi 3 , nobuyuki takei 1 , hiroyuki nawa 1 1 dept. mol. neurobiol., brain res. inst., niigata univ., japan; 2 brain resource center, brain res. inst., niigata univ., japan; 3 dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb1) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip1 and sap97. moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb1 receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima 1 , eriko miura 2 , keiko matsuda 1 , tetsuro kondo 1,3 , masahiko watanabe 2 , michisuke yuzaki 1 1 dept. physiol., sch. med., keio univ., tokyo, japan; 2 dept. anatomy, hokkaido univ., sch. med., sapporo, japan; 3 mol. neurophysiol., aist, tsukuba, japan cbln1 is a member of the c1q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln1 is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln2-4, are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln1, cbln2 and cbln4 were secreted as glycoproteins, whereas cbln3 was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd-95 and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by ␤-catenin. s-scam forms a ternary complex with neuroligin and psd-95. more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd-95 at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf4/ra175 (ra175), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca2+independent homophilic trans-cell adhesion activity. ra175 participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra175-/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra175 interacted with par-3 being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par-3 was colocalized in the cell adherent region of p19 embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra175+/+ testis, whereas it was undetectable in the spermatid of the ra175−/− testis. ra175 and jam-c were localized around the head portion of spermatid and ra175 deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra175 and par-3. research funds: 12324122 izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd-95 clusters in cultured hippocampal interneurons. higher density of dendritic psd-95 clusters was observed in interneurons at 11 div. at 18 div, this difference was less prominent, mainly due to >3-fold increase of psds in excitatory neurons. psd-95-gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at 11 div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd-95-gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps1a-c025 two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in 3-7div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac1 in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p2x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, 2001; shigetomi & kato, 2003) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites (2 s, 4-micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p2x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi (17300123) ps1a-c027 ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of 2d-and 3d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps1a-c028 integrative spike dynamics of rat ca1 neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca1 stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca1 circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur6 connects with syntenin at the dendrites and mglur7 with pick1 at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur7 that was known to be ectopicly expressed in the dendrites of mglur6-deficient on cone bipolar cells. we observed no pick1 but only syntenin at the mglur6-deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur7 immunopositivity. these findings suggest that mglur7 connects with syntenin in place of mglur6 that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca 2+ -permeable channels. trpv1, expressed in sensory neurons, is activated by noxious heat above 42 • c, whereas trpv 4, expressed in keratinocytes, is sensitive to moderate temperatures (>34 • c). here we examined the role of trpv1 and 4 in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to 55 • c did not induce the change in bt, desensitization of trpv1 with capsaicin resulted in the increase in bt. on the other hand, in trpv4-knockout mouse, moderate thermal radiation (>43 • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv1 and 4 respectively. research funds: kakenhi (17657052) ps1a-c032 generation and biochemical analysis of a glur␣2 knockout mouse hirotsugu azechi 1 , manabu abe 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, niigata, japan; 2 sorst/jst, saitama, japan glur␣2(glur2) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣2, we generated a conditional glur␣2 knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra2f using c57bl/6(b6) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra2f was used as a target of glur␣2 line. by crossing gra2f and tlcn-cre that expressed cre in germ line cells, glur␣2 null ko mice were produced, but most of them died within 3 days after birth. to overcome the lethality, the glur␣2 mutation was transferred onto b6/129 or b6/cd-1 genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣2 in the distribution of functional ampa receptors in vivo. ps1a-c033 gisp: a novel brain specific protein that binds to the gaba b1 subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific 130 kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b 1 subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b 1 and gaba b 2 from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b 1 protein and also promotes gaba b 1 surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b 1/gaba b 2 complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b 1-binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut1-3) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut1 and vglut2 seem to participate in this glutamate accumulation. in the present study, we found that vglut3 mrna is also expressed in the adult rat pinealocytes. vglut3 immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut1-ir or vglut2-ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito 1,2,3 , kenji nakamura 4 , toshikazu kakizaki 1,3 , satoe ebihara 5 , masakazu uematsu 6 , shigeo takamori 7 , minesuke yokoyama 4 , shiro konishi 4 , masayoshi mishina 3,8 , jun-ichi miyazaki 9 , kunihiko obata 10 , yuchio yanagawa 1,3 1 gunma univ., maebashi, japan; 2 sokendai, hayama, japan; 3 sorst, kawaguchi, japan; 4 mitsubishi kagaku inst. life sci., machida, japan; 5 kumamoto univ., kumamoto, japan; 6 toyohashi univ. tech., toyohashi, japan; 7 tokyo med. den. univ., tokyo, japan; 8 univ. tokyo, tokyo, japan; 9 osaka univ., suita, japan; 10 riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi (17052002) ps1a-c036 postnatal changes in the colocalization of vglut1 and vglut2 immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura 1,2 , akiya watakabe 3 , hiroyuki hioki 1 , fumino fujiyama 1 , yasuyo tanaka 1 , tetsuo yamamori 3 , takeshi kaneko 1,2 1 dept. morphol brain sci., grad. sch. med., kyoto univ., japan; 2 crest, jst, japan; 3 div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut)1 and vglut2 accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut1 and vglut2 in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p7. the cc was higher in area s1 than areas m1 and area v1 throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of 5 amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r1 and r2. the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin (5-hydroxytryptamine, 5-ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several 5-ht receptor subtypes, 5-ht transporter (5-htt) and vesicular monoamine transporter 1 (vmat1) in primary cultured mouse osteoblasts and mc3t3-e1 osteoblastic cells. sustained exposure to fluoxetine, a selective 5-ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc3t3-e1. these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦2 glutamate receptor (glur␦2). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦2 were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho15j, was caused by a new type of intragenic deletion of the grid2 gene, which was indeed translated as glur␦2 lacking 52-amino acids in the n-terminus. mutant glur␦2 proteins were retained in the soma of purkinje cells and degraded. as a result, ho15j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦2 signaling and cerebellar functions. research funds: kakenhi (16200024) ps1a-d040 role of the dry motif in melanin-concentrating hormone receptor 1 in signaling yumiko saito 1 , yoshimi aizaki 1 , mituse nakano 2 , kei maruyama 1 1 dept. pharamacol., saitama med. sch., saitama, japan; 2 international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp140, arg141 and tyr142 in the dry of rat melanin-concentrating hormone receptor 1 (mch1r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec50 value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch1r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a 1 r and p2y 1 r electrophysiologically using xenopus oocyte expression system. when a 1 r and p2y 1 r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/11 pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps1a-d043 dual signaling via metabotropic glutamate receptor1␣ is regulated by a cytoskeletal protein 4.1g michihiro tateyama 1,2 , yoshihiro kubo 1,2 1 department of biophysics and neurology, nips, aichi, japan; 2 sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor 1␣ (mglur1) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur1 are differentially regulated by different types of ligands, glutamate and gd 3+ . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur1. these proteins, such as homer and 4.1g, are also known to change the membrane expression of and modulate the function of mglur1. here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur1. interestingly, the functional couplings of mglur1 to gq and gs pathways were altered by co-expression of 4.1g, but not by homer. deletion of the c-terminal tail abolished the effect of 4.1g, indicating that the interaction of 4.1g with the c-terminal tail of mglur1 regulates the multi path signaling. ps1a-d044 modulation of the eaac1-mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto 1,2 , saori akiduki 1,2 1 age dimension research center, aist, ibaraki, japan; 2 graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein 3-18 (gtrap3-18), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier 1 (eaac1). it contains two potential pkc phosphorylation motifs at positions 18-20 and 138-140. however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac1-mediated glutamate transport activity in c6bu-1 cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine 18 into alanine (s18a) or at serine 138 into alanine (s138a). as compared with wt, s18a had no inhibitory effect on glutamate transport activity under exposure to 100 nm pma, and had increased glutamate transport activity under normal condition. by contrast, s138a had the same glutamate transport activity as that of wt. thus, the eaac1-mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine 18 in addicsin. kaori akashi 1 , manabu abe 1 , toshikazu kakizaki 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, japan; 2 sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur␤1-3(glur5-7) and glur␥1-2(ka1-2) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur␤2ko mice from c57bl/6 es cell line. the glur␤2ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥1 and glur␥2 proteins were significantly decreased in the crude fraction of ca3 region of glur␤2ko. furthermore, subcellular localizations of both subunits were also changed in glur␤2ko. these results suggested that native kainate receptors might function as heteromeric channels (glur␤/␥) and the glur␤2 subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps1a-d046 sema4d/plexin-b1 activates gsk-3␤ via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin 4d (sema4d) receptor plexin-b1 induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b1-mediated r-ras gap activity, leading to growth cone collapse. sema4d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk-3␤. ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk-3␤ antagonist suppressed the sema4d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b1-induced dephosphorylation of akt and gsk-3␤. plexin-a1 also induced dephosphorylation of akt and gsk-3␤ through its r-ras gap activity. thus, we conclude that plexin-b1 dephosphorylates akt and gsk-3␤ through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos7 cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur2. hops was co-immunoprecipitated with grip1 (glutamate receptor interacting protein 1) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur2 via grip1 binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc-1. a neural amino acid transporter snat2/ata2 also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc-1 immunoreactivity (asc-1-ir) was detected in dendrites and somata of pyramidal neurons. the snat2-ir was widely detected in neurons, whose intracellular localization was similar to that of asc-1-ir. deferent from asc-1-ir, snat2-ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc-1 and snat2 to the neuronal uptake of l-serine. snat2 might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps1a-d049 neuronal glutamate transporter eaat4 controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake 1 , si-young song 2 , shiro konishi 3 , keiji imoto 1 1 natl. inst. physiol. sci. (nips), okazaki, japan; 2 mitsubishi kagaku inst life sci, tokyo, japan; 3 tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat4/glt-1 blocker threo-3-methylglutamate, but not the glt-1 blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat4 expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat4 was abundant. the results suggest that eaat4 plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat4 expression in postsynaptic pcs. chitoshi takayama 1 , yoshiro inoue 1 1 department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k-2cl co-transporter 1 (nkcc1) and k-cl co-transporter 2 (kcc2). in this study, we focused on kcc2, which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc2 with special reference to the neuronal development in the cerebellum. kcc2 was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc2. the kcc2-protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc2-expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima 1 , hisashi mori 1 1 molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d1-like and d2-like according to their pharmacological properties. the d1-like receptors consist of d1 and d5 receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d1 receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop (3i loop) domain of d1r fused with egfp effectively reduce camp production mediated by d1 and d5 receptors. interestingly, we also identified that the 3i loop domain of d1r interfere with gs coupled beta2 adrenergic receptor signaling. these results suggest that the third intracellular loop of the d1 receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d1-like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama 1 , yoichi ogawa 2 , hiroki yoshino 1 , masayuki yamashita 2 , toshifumi kishimto 1 1 dept. psychiatr.; 2 dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca1 pyramidal cells. da (100 m) profoundly decreased gaba a receptor-mediated postsynaptic currents to 32% in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to 29% in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d1-like receptor, since skf 83959, a selective agonist for pi-linked d1-like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d1-like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina 1 , jinko konno 1 , sachine yoshida 1 , hideki ohmomo 1 , hitoshi kawano 2 , fumihiro shutoh 1 , haruo nogami 1 , setsuji hisano 1 1 graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; 2 tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut1 or -2 mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day 13 (e13), vglut1 immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e15, vglut1-ir was seen as puncta close to l1-ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l1or tag1-ir in the imz, whereas vglut2-ir was first observed in the sp and upper imz where l1-ir existed. these results show that vglut1ir corticofugal fibers appose to elongating vglut2-ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps1a-d054 developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca1 regions michie kon 1 , yoichi avil 2 , hiroshi tsubokawa 1,2 1 dept. of information engineering, tohoku univ., sendai, japan; 2 grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca1 region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca1 region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under 15 days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca1 region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca1 region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine (2.5-10 mg/kg) produced a dose-dependent cpp. using a 3-day schedule of conditioning, it was found that intra-ca1 administration of nmda receptor antagonist, mk-801 (2 and 4 g/rat) significantly attenuated the morphine (7.5 mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda (0.1, 0.2 and 1 g/rat) significantly potentiated the morphine (2.5 mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk-801 receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura 1 , kouichi nakamura 2,3 , hiroyuki hioki 2 , masanori uemura 1 , takeshi kaneko 2,3 1 dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; 2 dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; 3 crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d1 receptor are expressed in striatonigral neurons and d2 receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d1, d2, d3, d4, d5). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p10-15) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since (1) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, (2) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, (3) depolarizing agent released tpa from isolated neurosecretosomes, (4) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u2st, galnac4-6st, d4st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda 1 , maki ishii 1 , isao nagata 2 , yumiko shimazaki 1 1 dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; 2 dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo-225 epitopes, glca(2s)␤1-3galnac(6s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa 1 , mineko kengaku 2 , nobuaki maeda 1 1 dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; 2 lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka 1 , tohru marunouchi 1 1 division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of 3 m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp1, has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp1 gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp1 or sulffp2 appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of 6-o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of 5-hydroxytryptoamine (5-ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day 16 or 18 rat cerebral cortex was cultured in the presence of 5-ht, na or da. after 4 days, we analyzed dendrite formation using anti-map2 antibody. after 14-21 days, we analyzed synaptogenesis with anti-psd-95, anti-synaptophysin, and anti-map2 antibodies. the addition of 5-ht (100-10000 nm), na (100-1000 nm) or da (10-1000 nm) increased dendritic length of pyramidal neurons. 5-ht (100-1000 nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by 5-ht 1a receptor, ␣ 2a receptor and d 2 receptor, while inhibited by 5-ht 2a and ␤receptors. in addition, synaptogenesis was promoted by 5-ht 2a and 5-ht 2c receptors, whereas inhibited by 5-ht 1a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in 293t cell, singar associated with p85 and p110, the subunits of pi3kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps1a-e068 lrfn2, a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd-95 naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn2 was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn2 protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd-95, a postsynaptic scaffold protein. we examined the physical interaction between lrfn2 and psd-95 by immunocrecipitation and pull-down assay, since lrfn2 contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn2 associated with psd-95/nmda receptor complex in the brain extracts and lrfn2 directly bound to psd-95 via its pdz domain-binding motif. in this study, we suggest that lrfn2 may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt1-8). among the prmt proteins, the prmt3 has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt3 in the rat brain. the prmt3 was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt3 expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt3 relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt3 in the brain, we tried to identify novel interacting proteins with the prmt3 in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps1a-e070 proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi 1,2 , michihiro igarashi 1,2 1 div mol cell biol, grad. sch. med dent sci; 2 trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than 150 identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi 17023019; project-promoting grant from niigata univ ps1a-e071 netrin-1 is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda 1 , keisuke watanabe 2 , kazuhiro ikenaka 2 , katsuhiko ono 2 , hiroyuki yaginuma 1 1 dept. anat., fukushima med univ. sch. of med., fukushima, japan; 2 div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin-1 is known to be a chemorepellent for a subset of axons, the role of netrin-1 for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin-1 in this repulsion. in the mouse embryo at e11, netrin-1 is expressed in the floor plate and the dermamyotome, and the netrin-1 receptor unc5c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin-1 repelled chick e5 drg axons. moreover, using function-blocking antibody against netrin-1, we revealed the fact that netrin-1 plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin-1 to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst 17590166 to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p6 neonates, but the ventral neurons became unresponsive after p9. the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe 1 , hirohide takebayashi 1,2 , kazuhiro ikenaka 1 , katsuhiko ono 1 1 div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; 2 dev stem cell biol. program, ucsf, usa netrin-1 is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin-1 also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin-1 −/− background, using olig3-lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig3 +/lacz ;netrin-1 −/− mice, ventral migration of olig3 cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig3-positive cells. these results suggest that netrin-1 exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g1 and netrin-g2 are vertebrate-specific membrane-anchored members of the unc-6/netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g1 and netrin-g2 proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g1 and netrin-g2 differentially bind to lrrcontaining proteins, ngl-1 and a related molecule nag14, in vitro. ngl-1 and nag14 in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g1 and netrin-g2, respectively. furthermore, in netrin-g1 and netrin-g2 deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang 1 , ryuichi sakai 2 , teiichi furuichi 1 1 lab. molecular neurogenesis, brain science institute of riken, saitama, japan; 2 division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p4-p8, stimulation of parallel fibers produces excitation of purkinje cells lasting for 600-800 ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p5-p7. since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p4 and that they then locate in purkinje cells, in cell body on p5 and in primary dendrites on p6-p7. the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya-1 and mmahya-2) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya-1 protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya-1 is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya-1 for the early cerebellum development. toshitaka morishima 1 , erina fukushi 1 , kazuto kobayashi 2 , naohiro hozumi 1 , sachiko yoshida 1 1 toyohashi university of technology, toyohashi, japan; 2 honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto 1 , hideyuki dekimoto 1 , tomiyoshi setsu 1 , masahiko watanabe 2 , mikio hoshino 3 , yo-ichi nabeshima 3 , toshio terashima 1 1 dept. of anat., kobe univ. grad. sch. of med., kobe, japan; 2 dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; 3 dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf1a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida 1 , tomoko nishiyama 1 , hitoshi tatsumi 1 , masahiro sokabe 1,2 1 department of physiology, nagoya university graduate school of medicine, nagoya, japan; 2 icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately 12 h and the elongation started at approximately 24 h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca3. once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca3 region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca3 toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca3, which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a 37 amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) 16 or e18 rat. dendritic outgrowth of pyramidal neurons was analyzed after 4 days using anti-map2 antibody. synapse formation was analyzed after 2-3 weeks, using anti-psd-95 and anti-synaptophysin antibodies. in the presence of cgrp (10-1000 nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of 5hydroxytryptoamine (5-ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day 18 rat hippocampus was cultured in the presence of 5-ht or na. after 5 days, we analyzed formation of dendrites using anti-map2 antibody. after 21 days, we analyzed formation of synapses using anti-psd-95, anti-synaptophysin, and anti-map2 antibodies. the addition of 5-ht (500 nm) or na (500 nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites 5-ht (100-1000 nm) and na (10-1000 nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ 2a receptor promotes dendritic outgrowth, while ␤ receptor suppress dendritic outgrowth and branching. in addition, 5-ht 2a receptor and ␣ 2a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within 24 h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c4 inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c4 inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di-2 anepeq) and the imaging system (micam02). in dscam-ko mice, the optical signal which precedes c4 inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps1a-f088 hippocampal cells cultured on 3d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after 2 weeks in vitro, neurons formed a dense layer, >0.1 mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, (1) collagenasespecific inhibitor did not affect cell depth, (2) limiting gliosis by antimitotics reduced the thickness of the tgl by 40%, (3), neither glial nor neuronal cell body were found in the tgl by gfap/map2 detection, (4) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after 2 weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca2+ by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama 1 , kazuto kujira 1 , tetsuya kawabe 2 , takao ito 1 , yoshihiro tsuruo 1 1 department of cell biology and anatomy, wakayama medical university, wakayama, japan; 2 department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) (17590600) ps1a-f091 metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama 1 , hitoo nishino 1 , takehiro yahata 2 1 nagoya city university graduate school of medical sciences, nagoya, japan; 2 nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit 1 , anand goswami 1 , nobuyuki nukina 2 , nihar ranjan jana 1 1 national brain research centre, india; 2 laboratory for structural neuropathology, riken brain science institute, 2-1 hirosawa, wakoshi, saitama 351-0198, japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin-3 for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc70. finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of 11,092 ests (5,442 non-redundant sequences). 2858 of them matched known genes of rodents in the genbank databases, but 2584 remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of 0, 2, or 4 weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps1a-f096 transforming growth factor-␤ in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor-␤ (tgf-␤) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-␤. these results led us to postulate that tgf-␤ in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf-␤ in the brain using intracisternal administration of anti-tgf-␤ antibody or sb-431542, an inhibitor of the type 1 tgf-␤ receptor (t␤r1). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf-␤ has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the t␤r1 signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps (2 h restraint; 20 min forced-swimming; ether anesthesia) then they were put in untouchable situation for 7 days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming 15 min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c57bl/6j mice. pups were separated from mothers daily (3 h) on postnatal days 1 through 14. starting at 13 weeks of age they were tested for (1) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; (2) responses to social stimuli in social investigation (sit) and social preference tests; and (3) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of 90 db (so), or were forced to swim (sw). these manipulations were performed for 15 min once a day at 2 weeks after birth (from postnatal days 14 to 18) or 3 weeks after birth (from postnatal day 21 to 25). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of 6 weeks. it was found that the spatial learning activity of both so and sw mice manipulated 2 weeks after birth was impaired as compared to no mice. so mice manipulated 3 weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated 3 weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo 1 , yukiko yamada 2 , mitsuo iinuma 2 , yasuo tamura 2 , fumihiko iwaku 1 , kazuko watanabe 3 , minoru onozuka 4 1 dept. oral anat., asahi univ. sch. dent., japan; 2 dept. ped. dent., asahi univ. sch. dent.; 3 dept. physiol., gifu univ. sch. med., japan; 4 dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp8 mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca3 region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for 12 weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara 1 , yayoi kitamura 1 , junichi tanaka 2 , yukio hattori 1 , yasushi hayashi 1 1 department of human nutrition, notre dame seishin university, okayama, japan; 2 department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c57bl/6j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a 10-min interval, to measure the basal temperature (t 1 ) and stress-enhanced temperature (t 2 ), respectively. the difference ( t = t 2 − t 1 ) gives the sih. in control mice, t was approximately 1 • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t 1 . furthermore, spermidine and spermine also lowered t and t 1 at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox-1 and cox-2. it has been known that, under some stressful conditions, cox-2 is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for 6 h and subjected them to the elevated plus maze task 24 h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox-2 by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps1a-g104 imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded 15o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when 11 male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang 1 , takesi sakurai 2 , yasuitirou fukuda 3 , tomoyuki kuwaki 1,3 1 dept. molec. integ. physiol., chiba univ., japan; 2 dept. pharmacol., univ. tsukuba, japan; 3 dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of 3-8 weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of 5 weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between 3 and 5 weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of 3 weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between 3 and 8 weeks of age, and in the hippocampus at the age of 3 weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi #14760187 ps1a-g108 a systematic analysis of genetic factors associated with behavioral diversity between msm and c57bl/6 koide tsuyoshi 1,2 , aki takahashi 1,2 , toshihiko shiroishi 2,3 , akinori nishi 1 1 mgrl, national institute of genetics, mishima, japan; 2 sokendai, hayama, japan; 3 mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c57bl/6. in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b6-6cmsm, which carries chromosome 6 of msm, showed extreme behavioral differences from c57bl/6. the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga 1 , yu kajiyama 2 , shigenobu shibata 2 , hiroshi kunugi 1 1 department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; 2 department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for 4 days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e2) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e2) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e2 mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri 1 , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps1a-g113 synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi (15500238) ps1a-g114 involvement of nr2b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary 1 , takanobu nakazawa 1 , yuji kiyama 2 , toshiya manabe 2 , tadashi yamamoto 1 1 div. of oncology, ims, univ. of tokyo, tokyo, japan; 2 div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr2b is tyrosine-phosphorylated, with tyr-1472 being its major phosphorylation site. to investigate the role of tyr-1472 phosphorylation, we generated mice with a tyr1472phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr-1472 and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr2b is involved in the control of emotional responses by regulating crf content. ps1a-g115 increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca 2+ signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for 5 weeks, locomotor activity and longinterval (200, 1000 and 5000 ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for 5 weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui 1,3 , yosky kataoka 1,2 , yasuhisa tamura 3 , yasuyoshi watanabe 2,3 , hisao yamada 1 1 department of anatomy and cell science, kansai medical university, osaka, japan; 2 department of physiology, osaka city university graduate school of medicine, osaka, japan; 3 molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at 50-60 times/min), inhibition periods (less than 10 times/min) were often observed 2 h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns-398, a selective cox-2 inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps1a-g119 impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima 1 , naoya aoki 2 , andras csillag 3 1 biology, hokkaido univ., sapporo, japan; 2 agriculture, nagoya univ., nagoya, japan; 3 anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task 1, chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to 3 s. in task 2, chicks chose between small reward located at 20 cm and large reward at −140 cm, where cues signaled the distance of invisible food. task 3 was similar to the task 2, except that cues signaled the food quantity. in task 4, total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi (15370033, 17021018) ps1a-g120 analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe401, which had a missense mutation in the casy-1 gene. several casy-1 deletion mutants also showed learning defects. casy-1 has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x11l/mint2. however, after dissociation of x11l, alcadein is susceptible to cleavage by protease(s). we found that casy-1 was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy-1 can also be proteolytically cleaved. ps1a-g122 insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama 1 , atsushi kuhara 1 , akiko mohri 1,2 , kotaro kimura 1,3 , masatoshi okumura 1 , masahiro tomioka 4 , yuichi iino 4 , ikue mori 1,5 1 div. of biol. sci., nagoya univ., japan; 2 present address: univ. of texas, health sci. cent., usa; 3 present address: natl. inst. of genet., japan; 4 mol. genet. res. lab., univ. of tokyo, japan; 5 inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins-1 encoding insulin homologue caused defective associative learning, mutations in daf-2 and age-1 encoding the homologues of insulin receptor and pi 3-kinase, respectively, suppressed the defect of ins-1, and the mutation in daf-16 encoding forkhead transcriptional factor caused the learning defect. this suggests that ins-1 antagonizes daf-2 insulin-like signaling for associative learning. interestingly, age-1 animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age-1 in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins-1. we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps1a-g123 analysis of ttx-8: novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx-8(nj21) and ttx-8(nj34) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx-8 encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx-8 is similar to ric-3, identified in c. elegans at first and conserved among several species (halevi et al., 2002 (halevi et al., , 2003 . ric-3 is thought to be required for the maturation of acetylcoline receptor (halevi et al., 2002) , so ttx-8 may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps1a-g124 analysis of aho-3 mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio 1 , akiko mohri 1,2 , eiji kodama 1 , atsushi kuhara 1 , mizuho koike 1 , kotaro kimura 1,3 , ikue mori 1,4 1 div. of biol. sci., nagoya univ., japan; 2 present address: univ. of texas, health sci. cent., usa; 3 present address: natl. inst. of genet., japan; 4 inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho-3(nj15) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., 2000) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho-3 gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho-3 is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho-3 gene further. we have demonstrated aversive conditioning in lymnaea using 100 mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b3, located in the buccal ganglion. an associative memory, lasting 24 h, was produced with 20 pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b3 neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b3 neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura 1 , dai hatakeyama 1 , tetsuro horikoshi 1 , etsuro ito 2 , manabu sakakibara 1 1 lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; 2 cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts1 in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno 1 , minoru saitoe 1,2 , tim tully 3 1 tokyo metropolitan institute for neuroscience, tokyo, japan; 2 department of biology, tokyo metropolitan university, japan; 3 cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. 1 day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi 17005050 ke zhang 1 , jian z. guo 1 , ai k. guo 1,2 1 institute of neuroscience, chinese academy of sciences, china; 2 institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal4/uas-shi ts1 system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (<4 min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased 2 months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari 1,2 , kazuo okanoya 1,2,3 1 lab. for biolinguistics, riken-bsi, wako, japan; 2 grad. sch. of sci. and tech., chiba university, chiba, japan; 3 presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day 120, developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps1a-h137 facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi 17023034 ps1a-h138 the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment 1, rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of 0.15 m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment 2, rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi (17730431) ps1a-h139 transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps1a-h140 gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c57bl/6 mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over 80%, while those in the mscand ptx-injected mice decreased to 30%. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦2 that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps1a-h143 post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao 1,2 , takehito okamoto 1 , fumihiro shutoh 1,3 1 lab for motor learning control, riken bsi, saitama, japan; 2 sorst, jst, saitama, japan; 3 dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of 1 h daily training for 1 week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi (16500204) ps1a-h144 modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri 1,2 , takehito okamoto 1 , shin-ichi nishimura 1 , fumihiro shutoh 1,3 , soichi nagao 1,4 1 lab. for motor learning control, riken bsi, saitama, japan; 2 univ of the air, chiba, japan; 3 dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; 4 sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., 2006) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps1a-h145 influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho 1,2 , t kobayashi 1,2 , e hori 1,2 , k umeno 1,2 , t ono 1 , h nishijo 1,2 1 system emotional science, univ. of toyama, toyama, japan; 2 crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of 133 place cells recorded, activity of 40 was significantly modulated by the car speed and/or distance between the car and rat; 21, 12 and 7 cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, 16 place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a 2.0-3.0 mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about 40 ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas 5 and 7, contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area 5 to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca1 performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca1 neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca1 plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca 2+ but is blocked by mg 2+ in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg 2+ binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca1 cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l1 is a component of the ubiquitin system. uch-l1 is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l1 in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl1. gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca1. in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l1 is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps1a-i151 learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage 0). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band (64-120 hz) was strong at stage 0 and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps1a-i152 longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about 1.5 mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of 1 mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal1 ubiquitously expresses in brain. bmal1 functions by forming a heterodimer with either clock or npas2, which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal1 in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal1 function in a forebrain. using a dominant negative mutant of bmal1 (bmal1 r91a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., 2004), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal1 r91a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal1 in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn (0, 70, or 100 db; intermittent white noise). 70 db bgn induced significant decreases in number of error and time to goal in maze compared with 0 and 100 db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, 100 db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn 1,2 , anucha pongpanparadorn 2 , wasana pratchayasakul 2 , anchalee pongchaidacha 2 , nipon chattipakorn 2 1 fac. dent. cmu, chiang mai, thailand; 2 cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were 101 ± 14, 124 ± 19 and 108 ± 22 in the groups administered 250, 500 and 1000 mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was 17.4 ± 6.3, 22.7 ± 6.9 and 16.6 ± 5.0 for 250, 500 and 1000 mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a 2-h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a 2-h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses (10, 60 and 120 min). percentage inhibition of cache activity following a single tde injection at doses 500 and 1000 mg/kg significantly decreased at 10 and 60 min after tde injection, but not at 120 min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣4and ␣7-nachr and kinases involved in pi3k-akt pathway, and increase the levels of phosphorylated akt and bcl-2. these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi3k-akt pathway via ␣4and ␣7-nachrs. ps1a-i160 arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae 1 , yoshiyuki ishikura 2 , shigeaki fujikawa 2 , yoshinobu kiso 2 , manabu sakakibara 1 1 lab. neurobiol. engr., tokai univ., numazu, japan; 2 inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in 20-month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps1a-i161 thimet oligopeptidase co-exists in gfap-and cd11b-positive glia in rat pc/rsc treated with mk-801 takeshi kato 1 , mohammad arif 1 , michiyuki yamada 2 , toshiyuki chikuma 3 , md. mahiuddin ahmed 4 1 lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; 2 grad. sch. integr. sci., yokohama city univ., yokohama, japan; 3 dept. hygien. chem., showa pharmaceut. univ., machida, japan; 4 dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep24.15) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk-801 activated gfapand cd11b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) 3 day after the treatment. mk-801 also increased ep24.15 and prolyl oligopeptidase. immunohistochem data showed that ep24.15 co-localized with gfap and cd11b positive glial cells. since mk-801 causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep24.15 in the pc/rsc. these data suggest that ep24.15 in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps1a-i162 effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) 1 and 2 in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) 1 and 2, code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps1 shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps1 and ps2 with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps2 protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps1 level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps1a-i163 effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloid␤proteins. in vitro, we examined the effects of various form of a␤peptide on the proliferation and differentiation of nsc from svz of 10-week-old adult mice. in this study, a␤42 peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a␤42 peptide. we found that treatment of nsc with oligomeric form of a␤42 peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a␤42 peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a␤ clearance is noted. high mobility group box protein-1 (hmgb1) is a non-histone chromosomal protein. here, hmgb1 was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb1 immunoreactivity was observed around dying neurons in the kainic acid-and a␤1-42 (a␤42)-injected rat hippocampi. hmgb1 was not co-localized with a␤ in transgenic mice which show massive a␤ production without neuronal loss. furthermore, co-injection of hmgb1 delayed the clearance of a␤ and accelerated neurodegeneration in a␤42-injected rats. these results suggest that hmgb1 released from dying neurons may inhibit microglial a␤ clearance and enhance the neurotoxicity of a␤. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps1a-i166 x11-like protein regulates metabolism of app in the mouse brain yoshitake sano 1,2 , tadashi nakaya 2 , shigeyoshi itohara 1 , toshiharu suzuki 2 1 riken bsi, saitama, japan; 2 hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a␤) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x11-like protein (x11l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells (1999 . j. biol. chem. 274, 2243 2003. j. biol. chem. 278, 49448) . there is no evidence, however, that endogenous x11l suppresses app metabolism and a␤ generation in vivo. to examine the physiologic role of x11l in app metabolism in the brain, we generated x11l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the ␤-site and a␤, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x11l suppresses the production of a␤ by inhibiting ␤secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin-1 protein by 2d-electrophoresis and tof-mass delineate that mutant at1 and htt proteins similarly reduce transcriptional factor x1 and x2. immunoprecipitation and pull-down assays support interaction between polyq and factor x1 and x2. immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x1 and x2 into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x1 and x2 ameliorates poly-q pathology in htt-/at1-expressing neurons and transgenic drosophila. these results suggest that factor x1 and x2 are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps1a-j170 ba1-42 was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, 10 rats/group (g). group 1 received e2 at 2.5 g/kg per day; groups 2 and 3 received ycj at 20 ml, and 100 ml/kg day, respectively, once everyday. group 4 received ycj 100 ml/kg plus e2 at 2.5 g/kg day twice a week, all for 5 weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e2 in group 3 was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti-␤-amyloid 1-42 antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer 3 and layer 5 of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group 3 was lesser than ovx and control groups. amyloid deposition was correlated with e2 serum at r = −0.4. ps1a-j171 correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki 1 , shigeo kinomura 1 , kazunori sato 1 , shinya uchida 1 , ryoi goto 1 , kentaro inoue 1 , ichiro tsuji 2 , hiroyuki arai 2 , ryuta kawashima 3 , hiroshi fukuda 1 1 department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; 2 tohoku univ. grad. school of med., sendai, japan; 3 niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of 111 community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h13-kenko-008), (h13-choju-007, h13-choju-023) ps1a-j172 effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the 8 weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook 1 , d.g. goodenowe 1 , y. yamazaki 1 , j. flax 2 1 phenomenome discoveries inc., saskatoon, canada; 2 precisionmed inc., san diego, ca, usa dementia affects about 10% of the population over the age of 65 and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an 8-metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the 8-metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin-2 (chr2). in this study, we quantitatively investigated the rapidness of the light-gated current of chr2 expressed in pc12 cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about 10-fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr2 is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori 1 , shigeki ohta 1 , kenji hamada 2 , naofumi yamada-okabe 2 , yonehiro kanemura 3 , hideyuki okano 4 , yutaka kawakami 5 , masahiro toda 1,6 1 neuroimmnology research group, keio univ., tokyo, japan; 2 chugai co. ltd., kanagawa, japan; 3 inst. cli. res., onh, japan; 4 physiology, keio univ., tokyo, japan; 5 cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; 6 neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa1110 gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa1110 homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa1110 protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day 15 were dissociated. after the cells were treated with 100 nm estradiol-17beta (e2) or ethanol for 8 days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis (2d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than 4000 spots were detected from 2d dige. compared with ethanol treatment, e2 increased the expression of 501 spots in the hypothalamic cells and 169 spots in the cortical cells (p < 0.2, difference >1.5). proteomics analysis showed different effects of e2 for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e14 days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp90 client protein family. 17-allylamino-17-demethoxygeldanamycin (17-aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. 17-aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of 17-aag in a mouse model of sbma. administration of 17-aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of 17-aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp90 function by 17-aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb9 promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately 5-20% of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps1a-k181 selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura 1 , yoshiyuki ishii 1 , kazuto kobayashi 2 , yasufumi sato 3 , keiichi itoi 1 1 lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; 2 dept. mol. genet., fukushima med. univ., japan; 3 inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a1, a2 and a6) and dopaminergic (a9, a10 and a12) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg4b has been shown to play an important role in the processing of lc3, a mammalian homologue of yeast atg8, but the tissue distribution of atg4b remains unknown. to understand the role of atg4b in rat tissue cells, we prepared an antibody to atg4b and pc12 cells in which atg4b expression was knocked down by rnai. in rnaitreated pc12 cells where atg4b expression was 10% of that in the wild-type pc12 cells, the expression of cytosolic lc3-i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc3 to lc3-i. moreover, the expression of atg4b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg4b was intense in neurons, especially in the cerebellum. these results suggest that atg4b plays a major role in the processing of lc3, while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps1a-k183 spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev 67n strain into the central nervous system was examined after its subcutaneous inoculation (10 5 pfu) in the rat hind footpad. on day 3 post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day 4 p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day 5 p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the 4th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki 1 , hiroshi kameda 1 , hisashi nakamura 1 , taro okunomiya 1 , koji ohira 1 , kouichi nakamura 1,2 , takahiro furuta 1 , takeshi kaneko 1,2 1 dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; 2 crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn1, rta1, mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap43 n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii ␤ receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣1 and gly␤ immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has 10 armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca1 hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos7 cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr2 increased the surface expression of glur2 in cos7 cells compared with grip/glur2 or ␦-catenin/glur2 transfection. this study points to ␦-catenin as a regulator of glur2 receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca1 area in wild type (wt) and gad65 knockout (ko) mice. this parameter was twice lower in gad65ko mice (wt: 2.0 ± 0.5 pa/pf, n = 7; gad65ko: 0.9 ± 0.3 pa/pf, n = 8; p = 0.038). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba (5 m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad65ko animals (wt: 12.4 ± 0.7 ms, n = 14; gad65ko: 10.8 ± 0.6 ms, n = 12; p = 0.047). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad65ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that 5 hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p5 to p8) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent 5 hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur1 but not by mglur5 because it was antagonized by the mglur1 antagonist ly367385 but not by the mglur5 antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur1 is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps1p-a004 involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita 1 , michiko ohtomi 2 1 cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; 2 department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d1 receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d1 antagonist sch23390 into pfc by using a microdialysis method impaired md-pfc ltp but the d2 antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by 110-120%). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer 2/3 pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya 464-8601, japan we showed in previous study that somatic inhibitory synapses of neocortical layer 5 pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer 5 pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer 5 pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer 6 pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate (200 nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr1 in the regulatory mechanisms for ltp in the amygdala, we utilized ntr1-knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr1-ko mice than in wild-type mice. the bla-ltp in ntr1-ko mice was attenuated by sulpiride, a d2 receptor antagonist. these results suggest that d2-ntr1 interaction regulates the extent of ltp in the mouse la-bla synapses. ps1p-a010 facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over 28-day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca 2+ sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca 2+ at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin-1, and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps1p-a012 inhibitory modulation of the hippocampal ca3 transmission and plasticity by glucagon-like peptide-2 jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode-2 (glp-2) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp-2 (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp-2 effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp-2 on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices (400 m) were prepared from 7 to 35 days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca3 in the whole-cell mode using glass microelectrodes (resistance: 4-8 m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp-2 (10 nm-1 m) inhibited spontaneous excitatory postsynaptic current. glp-2 (10 nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp-2 may play an inhibitory role in the dg-ca3 transmission. ps1p-b013 quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp-2 and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a1, a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, 100 hz for 1 s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, 1 hz for 900 s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of 50 m apv, 3 m bicuculline, but not 500 m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when 20 mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at −10 mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine (5-100 m) or capsaicin (0.1-1 m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin (1 m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after 5 min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv1), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji 1 , akira komatu 2 , akinao nose 1 1 dept. phys., univ. tokyo, tokyo, japan; 2 dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila 3rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca 2+ -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t287d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t287d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr1* flies did not show mg 2+ blockade, and dnr1* flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf-1, and notch. we examined how mg 2+ blockade of dnr links to these transcription factors. research funds: kakenhi ps1p-b020 response properties of wind-sensitive giant interneurons in the 4th-instar nymphs of the cricket tetsuya matsuura 1 , masamichi kanou 2 1 dept. of welfare eng., iwate univ., morioka, japan; 2 dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) 8-1, 9-1, 9-2 and 9-3 in the 4th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from 12 different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi 8-1 increased with stimulus velocity up to 300 mm/s regardless of the stimulus direction. the response magnitudes of gi 9-1 reached a plateau at a stimulus velocity of 30 mm/s in most stimulus directions. the response magnitudes of gis 9-2 and 9-3 increased with stimulus velocity up to 300 mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi 8-1, the ipsilateralfront and contralateral-rear in gi 9-1, the ipsilateral-rear in gi 9-2 and the ipsilateral-front in gi 9-3, designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca1. serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca 2+ , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca1 region. we found that ptp induced by the 100-hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca 2+ channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca 2+ channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma 1 , kunihiko kaneko 1,2 , makoto asashima 1,2 1 grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; 2 jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca 2+ concentration ([ca 2+ ] i ) may change because of nonsynaptic communication. we loaded pc12 cells with a ca 2+ indicator, indo-1 am, and the [ca 2+ ] i of more than 10,000 cells was measured using flowcytometry. the [ca 2+ ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca 2+ ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca 2+ -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss5 from human and mouse cns. in mouse cns, four isoforms (types 1-4) were expressed. subcellular localization analysis revealed that type 4 (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type1 variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc34 and nb2a derived from neuronal cell express only type 4, whereas os3 and kt-5 derived from astrocyte express both type 4 and type 1. interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os3 and kt-5. we analyzed promoter region of m-spinesin gene, and identified that 5 -flanking region from −224 to −188 bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme 3-phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca 2+ signals, such as intra-and intercellular ca 2+ waves; however, their physiological roles remain elusive. the overexpression of an ip 3 -hydrolyzing enzyme, ip 3 5-phosphatase, suppressed the spontaneous ca 2+ signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca 2+ -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca 2+ -signal-deficient astrocytes. these results indicate that spontaneous ca 2+ signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai 1 , noriko osumi 1,2 1 tohoku univ. sch. med., japan; 2 crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax6 transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax6 in postnatal mouse brain and found that pax6 was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax6 in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax6 heterozygote mutant (sey/+) mice at 8 weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, 6 and 24 h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax6 regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim21 to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., 2006) . we previously reported a unique expression of dim21 ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., 2005) . we show here that mab dim21 also recognizes adult neural stem cells and glial cells at postnatal period. dim21, brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim21 ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk2 (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l7 promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk2 cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad1/5/8 (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk2. lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in 1950s and 1960s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in 6-ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after 48 h (34 and 19%) and 72 h (56 and 33%) exposure to 10 −4 and 10 −5 m 6-ohda respectively. it was accompanied by morphological changes and induction of gfap, s-100 and ox42. 6-ohda (10 −6 m, 72 h) was found to cause a significant impairment in 3 h glutamic acid uptake (31%) and gsh levels (27%). further neurons (in coculture with 6-ohda pre exposed glial cells) on exposure to 6-ohda (10 −6 m), showed loss of th expression and significant neuronal cell death (34%). the results of the present study suggest that 6-ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at 1 day after injection, then started to repair at 3 days and returned to the normal levels at 6 weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at 3∼7 days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng2) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba1+ cells accumulated in stab wounds and ischemic lesions were found to express ng2, of which molecular weight of its core protein was higher by 10 kda than that of ng2 expressed in contralateral brain region. this was due to the lack of shedding of ng2 in the brain lesions. we found that iba1+ cells accumulated in stab wounds and ischemic core lesion, most of which were ng2+/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd163 and von willebrand factor. ng2+ mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng2+ mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy 1 all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from 20 cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from <1 to 40 m 2 . most of the fibers were myelinated and counts produced a mean of 16,891 (12,000-21,500). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from 0.29 to 3.16 m (from fetal age to 60 years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p5-10. costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca1, ca2 pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps1p-d042 impact of s100b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani 1 , akiko seto-ohshima 2 , shigeyoshi itohara 2 , hajime hirase 1 1 neuronal circuit mechanisms research group, japan; 2 lab. for behavioral genetics, bsi, riken, wako, japan s100b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s100b, we have recorded hippocampal spontaneous activities from urethane anesthetized s100b ko and wt mice. typical eeg patterns including theta (3-8 hz) and sharp wave associated fast ripple (120-180 hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca1 radiatum, where ictal event was characterized by hyper-synchronous gamma band (30-80 hz) activity. while both populations developed ictal event within 40 min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s100b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s100b related pathways could potentially be activated. yoshiko takagishi 1 , erina okabe 2 , xiaoyang sun 1 , sen-ichi oda 2 , yoshiharu murata 1 1 riem, nagoya univ, nagoya, japan; 2 grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps1p-d044 regulation of hippocampal neurocircuit activity by glutamate transporter glt-1 noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt-1, the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap5) and the glt-1 blocker (dhk) with the signal subtraction, we could visualize the activity of glt-1 as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt-1 by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt-1 function in the ca3 region was elicited by much lower concentration of dhk than that in the ca1 region. ps1p-d045 monoclonal antibody rip specifically recognizes 2 , 3 -cyclic nucleotide 3 -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg-4 cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was 2 ,3 -cyclic nucleotide 3phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg-4 cells. moreover, the same localization was also observed in rat cnp1 transfected hek293t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca 2+ signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca 2+ signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca 2+ signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki 1,2 , hisatsugu koshimizu 1 , megumi kashihara 1 , tomoko hara 1 , masami kojima 1,2 1 research institute for cell engineering; 2 sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that 3-day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p38) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p38 expressed in the neural progenitor cells at embryonic 14 days (e14) mice brain. to ascertain the function of p38 on these cells, we treated the cultured e14 cells with the selective chemical inhibitor for p38 (sb203580) for 7 days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p38 in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit 1 , takashi yamazaki 1,2 , takeshi oya 1 , yoko ishii 1 , masakiyo sasahara 1 1 pathology ii, university of toyama, toyama, japan; 2 oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on 3 to 28 days after injury, and activation of pdgfrs, mapks, akt and p38 were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from 3 to 21 days after injury. p-tyr was highly detected from 3 to 21 days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to 7 days after injury and then gradually decreased. activation of akt and p38 continuously increased from 3 to 28 day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln1 is a cerebellum-specific protein structurally related to the c1q and tumor necrosis factor families. recently, we have shown that cbln1 is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln1 was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln1 needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln1 proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln1 mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln1 released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to 3 -untranslated region (utr) of bdnf mrna to investigate the effect of the 3 utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf3 utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf3 utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer1a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps1p-e056 bmp4 expression in the adult rat brain bone morphogenetic protein-4 (bmp4) is a member of the transforming growth factor ␤ (tgf-␤) superfamily and plays important roles in multiple biological event. although bmp4 expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp4 expression in the adult rat cns using immunohistochemistry. bmp4 is intensely expressed in most neurons and their dendrites. in addition, intense bmp4 expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp4 protein. these data indicate that bmp4 is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp4 plays pivotal roles also in the adult brain. ps1p-e057 hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and 30 min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl-2 protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl-2 expression, decreasing the level of bax expression in c57bl/6 mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase 1. here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda 1 , yoko tohyama 1 , shinichi kohsaka 2 , tadashi kurihara 1 , kazuyuki nakajima 1,2 1 soka university, hachioji, tokyo, japan; 2 dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt-3, and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf␤2 among tgf␤ family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba 1 , chiaki nakagawa 1 , masako tsuzuki 1 , motoko matsuda 1 , satoshi ichisaka 2 , yoshio hata 3 1 inst. dev. res., aichi human ser. ctr., kasugai, japan; 2 div. neurobiol., tottori univ., sch. med., yonago, japan; 3 div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats 12 h after beginning an 8-h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin-3 (nt-3) in selected brain regions from 14-day-old male rats and compared to those from adults. the changes in levels of bdnf and nt-3 were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci (200 kdyne, ih injury device) at t8/9 and were assigned to three groups; a (placebo), b (mp treated; 30 mg/kg i.v. immediately after injury), c (g-csf treated; 15 g/kg i.v. for 5 days after injury). animals were assessed with the bbb locomotor rating scale for 6 w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi (16390427) ps1p-e064 glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons (12 div) exposed to glutamate for 15 min at 100 m. glutamate exposure led to a decrease in mrna of nd1 and nd6, which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least 2-12 h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk-801. these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj-1 is enhanced by oxidative stresses. although exact functional significance of dj-1 has still unknown, it is thus proposed that dj-1 is protective against neural damage under oxidative stresses. in this study, we tested expression of dj-1 in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj-1 was markedly increased in the molecular layer of the dentate gyrus on days 4 and 14 post tmt injection. on day 14 post tmt injection, enhanced expression of dj-1 was observed in the stratum lucidum of the ca3. in glutathione-depleted mice, tmt was more effective in enhancing expression of dj-1, compared with that in untreated mice. furthermore, double staining of dj-1 and gfap demonstrated that most of cells highly immunoreactive to dj-1 were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj-1 is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt (2.8 mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus 2 days later, with visually apparent recovery of the granule cell layer 14 days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone (0.5-5 mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of 2.0 mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb (1 g/ml) on the pc12 cells was elicited through apoptotic cell death. activation of caspase-3/7 was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin (1 nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase-3/7 activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc12 cells. ps1p-e068 kinesin superfamily protein 4 (kif4) regulates activity-dependent neuronal survival by suppressing parp-1 enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein 4 (kif4), a microtubule-based molecular motor. the c-terminal domain of kif4 is a module that suppresses the activity of poly (adp-ribose) polymerase-1 (parp-1), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif4 from parp-1, resulting in upregulation of parp-1 activity, which supports neuron survival. after dissociation from parp-1, kif4 enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif4 controls the activity-dependent survival of postmitotic neurons by regulating parp-1 activity in brain development. research funds: 1684304 ps1p-e069 expression of hsp105, apg-1, and apg-2 in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt (2.8 mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp105, apg-1, and apg-2 in the granule cell layer of the dentate gyrus, but not in ca1-ca3 pyramidal cell layer, 16 h to 2 days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp105 highly expressed in survival neurons in the culture, apg-1 and apg-2 highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day 18. thirty gray of x-irradiations were performed on the cultured cells at 7 or 21 days in vitro (div). then the cells were fixed at 24 h after the irradiation with dapi. at 7-div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at 21-div. this indicates that the radiosensitivity of 7-div immature neurons is higher than that of 21-div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦2 glutamate receptor (glur␦2), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦2 is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca 2+ , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin-1 and found a novel gene down-regulated in response to mutant ataxin-1 in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s6 as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca1 molecular pathology. ps1p-f080 permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca 2+ -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca 2+ -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi3 kinase (pi3k) pathways. interestingly the up-regulation of bcl-2, a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat3 probe. scat3 is an indicator protein for caspase-3 activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase-3 cleavage sequence. using this probe, we could monitor the activation of caspase-3 at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt (2.8 mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex 2 days later. in cortical neurons, tmt exposure for 48 h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases 3 and 8, but not caspase 9, were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride (2.5 mg/kg, i.p.) in 3-(3w) and 7-week-old (7w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in 3w mice. by contrast, slight, diffuse neuronal damage was found in the ca1 and ca3 subfields and dentate gyrus of 7w mice. the hippocampal putrescine level was elevated markedly in 3w mice on tmt administration, whereas a minor putrescine increase was detected in 7w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps1p-f086 establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai 1 , toshinori hongo 2 , kimisato naito 2 , shigeto sasaki 2 1 department of physiology, kyorin university, school of medicine, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for 0.3-1.0 s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of 0.1 s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in 17 volunteers. experiment was performed by three tasks, control task is a forearm rotation, task1 is simulation of tools-manipulation, task2 is execution of tools-manipulation. we found different brain regions by this experiment. task2 to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at 1 khz, to record simultaneously and independently the 12 • horizontal saccades of each eye for 12 subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at 1 day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr␤ is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi (17022042) ps1p-f094 dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼20 hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from 35 healthy subjects, high and low scorers (n = 12 each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band (18-24 hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a 3-t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = 2.7 s) with online monitoring of meps. the intensity of tms pulses was varied from 30% to 95% at a 5% step (frequency at ∼0.15 hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above 80% stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = 0.46, p = 0.001). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps1p-f097 shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda 1,2 , yukihiro gomi 1 , itsuki imai 3 , nobuaki shimoda 1,2 , hiroyuki kato 1,2 1 international university of health and welfare, ohtawara, japan; 2 crest, jst, kawaguchi, japan; 3 nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels (22 channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of 15 s task and 30 s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky 1,2 , rieko osu 2 , hiroshi imamizu 2 , mitsuo kawato 1,2 1 computational brain project, icorp, jst, japan; 2 atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed 1 hz isometric force pulses to seven targets separated by 30 • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a 2d strain gauge. twenty repetitions of each condition were performed in 12 s blocks (tr = 2 s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of 661 voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only 31.3% (chance: 14.3%), errors were distributed normally about the target such that 80.4% (chance: 59.2%) of the data was classified correctly to within 60 • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from 28 • c, fish showed repetition of short time swimming in the range 32.5-40 • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in 3-5 days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in 3 days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps1p-f100 to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu 1 , ken-ich morishige 2 , jun nakanishi 1,3 , hiroyuki miyamoto 2 , mitsuo kawato 1 1 atr computational neuroscience labs, kyoto, japan; 2 kyushu institute of technology, japan; 3 jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p1 pups of the surviving cases increased greatly between p4 (30% of the control value) and p7 (52%), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p1, dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p4 and p7 pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p4 to p7. we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully (180 • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative 1, 4, and 7 days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps1p-g110 role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa 1 , tatsumi nagahama 2 1 dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; 2 fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps1p-g112 neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto 1 , sayaka hihara 1 , kazuo okanoya 2 , atsushi iriki 1 1 lab for symbolic cognitive development, bsi, riken, japan; 2 lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about 38.5 • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps1p-g115 prostaglandin e 2 -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, 1-23-1 toyama, shinjuku,162-8636, japan unilateral microinjection of pge 2 into the region around the rostroventral wall of the third ventricle (av3v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of 20-1000 pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide (0.5 mm, 100 nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge 2 injection site was examined. this treatment alone had no effect on the o 2 consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge 2 -induced responses were blocked 10 min after the pretreatment with bicuculline, and recovered at ∼70 min. pretreatment with vehicle saline had no effect on the pge 2 -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge 2 -induced thermogenesis. tetsufumi ito 1 , hiroyuki hioki 2 , kouichi nakamura 2 , takeshi kaneko 2 , yoshiaki nojyo 1 1 dept. anat., univ. of fukui, fukui, japan; 2 dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad67-ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad67-positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad67. we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad67-ir. after cutting t1-t4 vrs and drs, almost all gad67-ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama 1 , ning ma 1 , reiji semba 2 , satoru naruse 3 1 dept. of anat. ii, mie univ. school of med., tsu, japan; 2 inst. for developmental research, kasugai, japan; 3 dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin 1 (aqp1) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp1 immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp1-immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp1-ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp1-ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp1-ir fibers were found both in the myenteric and submucosal plexi. many aqp1-ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp1-ir fibers penetrated into the muscle layers. these results suggest that aqp1-ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi (13137204) ps1p-g118 abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days 1-4. the increase in the volume and frequency setting of the artificial ventilator (fio2 = 50%, fico2 = 0%) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico2 was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad67-gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = 40) in medullary transverse slices. about 20% of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about 50% of gabaergic neurons recorded in the superficial rvm were co 2 inhibitory, and all of the remaining neurons were co 2 insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with 0 and 10 mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with 10 mv forehead spla, in comparison to subgroups 0 mv forehead spla. such law was common for both for sg and for cg. in two subgroups of 0 and 5 mv nasal zone spla in sg the greater rt gain after il was in 0 mv nasal spla subgroup, and for cg in 5 mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin (5ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via 5ht2 receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain 5ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c57bl/6n) were pretreated with pcpa (0.1 g/10 ml/kg body weight) or saline intraperitoneally for consecutive 4 days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o 2 inhalation was changed to stepwise 5, 7 and 9% co 2 in o 2 inhalation every 5 min. hypercapniainduced increases in tidal volume and minute ventilation during 9% co 2 inhalation were reduced by pcpa pretreatment; these results suggest that 5ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a 1.08% gosha-jinki-gan diet group. after 4 weeks, continuous cystometry with physiological saline or 0.1% acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with 0.1% acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps1p-g127 differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to 90 db white-noise for 10 min. 90 db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the 90 db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase 67 (gad67), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad67 mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were 2.0 on the small neurons, 3.1 on the medium-sized neurons, 1.2 on the large neurons, and 4.2 on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about 80% of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large (3.16 m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps1p-h131 neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c4 nerve and facial nerve activities. addition of 4 mm k + to the standard superfusate decreased burst rate of c4 activity. addition of 6 or 8 mm k + caused initial inhibition of c4 burst and subsequent high frequency c4 burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi (16500208) ps1p-h132 regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp (1 m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at −60 mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin 1 receptor (nk1r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk1r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps1p-h133 effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for 1 h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min-6 cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm-3000, shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda 2300, usa) at the right middle finger from 12 volunteers (22.8 ± 2.1 years). mild thermal stimuli (17 ± 1 • or 40 ± 1 • ) were administered every 2 min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm-3000. we examined the effects of color environments on cognitive function in 12 healthy subjects and 12 patients after traumatic head injury using p300 components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p300 latency in the red environment was significantly shorter in controls than in patients. the p300 amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area 10 in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about 800 ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps1p-h139 neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi 1,2 , mikinobu takeuchi 1,2 , takahiro omote 3 , naoyuki yosimura 3 , etsuro hori 1,2 , kazuo sasaki 3 , taketoshi ono 2,4 , hisao nishijo 1,2 1 system emotional science, univ. toyama, toyama, japan; 2 crest, japan science and technology agency, japan; 3 bio-information engineering, univ. toyama, toyama, japan; 4 molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems (103 channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate (1) human facial stimuli with different gaze directions and (2) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the 3d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi 1 , sachiko koyama 2 , ayumu matani 3 , andrzej cichocki 1 1 riken, wako, japan; 2 hokkaido university, sapporo, japan; 3 university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at 40 hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: (1) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. (2) the functions were averaged across subjects. (3) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. (4) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi (17022004) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps1p-h142 the reflection of category perception of sound in the auditory evoked n1m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for 2 different fundamental frequencies (f0) and 12 different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n1m source strength for nonlinear sounds was significantly larger than that for linear sounds. n1m peak latency only for vocal sounds was not affected by f0. these results suggest that perceptual categorization was reflected in n1m source strength and location (linear or nonlinear), and in n1m latency (vocal or nonvocal). sunao iwaki 1 , hiroko kou 1 , kouichi sutani 1 , mitsuo tonoike 2 1 national institute of advanced industrial science and technology, osaka, japan; 2 chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p3, related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p3 components, suggested the involvement of frontal and anterior cingulate cortex in the early p3 component (p3a) and the contribution of the parietal and temporal regions to the later component (p3b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l1 and l2, respectively). we compared the brain activity of 11 chinese learners of japanese as l2 with that of 28 japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm2. while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l1 and l2. neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around 100 ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around 200 ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around 500 ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a 1.5 t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for 6 times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya 1 , mie matsui 2,3 , tsuneyuki kobayashi 2,4 , hisao nishijo 2,4 , michio suzuki 2,5 , yasuhiro kawasaki 2,5 , masayoshi kurachi 2,5 , jun nakazawa 6 , kyo noguchi 7 , hikaru seto 7 1 neuropsychiatry, univ. toyama, toyama, japan; 2 crest, japan science and technology corporation, japan; 3 psychology, univ. toyama, toyama, japan; 4 system emotional science, univ. toyama, toyama, japan; 5 neuropsychiatry, univ. toyama, toyama, japan; 6 developmental psychology, univ. chiba, chiba, japan; 7 radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura 1 , atsushi sekiguchi 2 , keisuke wakusawa 2,3 , yuko sassa 2,4 , hyeonjeong jeong 2,5 , kaoru horie 5,6 , shigeru sato 5,6 , ryuta kawashima 2,6 1 miyagi university of education, sendai, japan; 2 niche, tohoku univ., japan; 3 dep. pediatrics, tohoku univ. school of medicine, japan; 4 ristex, jst, japan; 5 gsics, tohoku univ., japan; 6 lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain 10, 50, 100 points or lose 100, 50, 10 points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps1p-h152 brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara 1 , masato taira 2,3 , toshio iijima 1 , ken-ichiro tsutsui 1 1 div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; 2 arish, nihon university, tokyo, japan; 3 appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either 0 yen or a gain or loss of 200, 400, or 800 yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps1p-i153 neural activation during experience-based reasoning chisato suzuki 1,2 , takashi tsukiura 1 , hiroko mochizuki-kawai 1 , yayoi shigemune 1,2 , toshio iijima 2 1 neurosci. res. inst., aist, japan; 2 div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in 16 healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps1p-i156 the difference of brain activity elicited by different styles of art hiromi yamamura 1 , yasuyuki kowatari 1,2 , shigeru yamane 2 , miyuki yamamoto 1,2 1 comprehensive human sciences, university of tsukuba, tsukuba, japan; 2 system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm2. while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba9, ba46). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j-3) ps1p-i157 effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were 20-30 aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi 17,6577 ps1p-i158 the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo 1 , michio nomura 2 , jun kawaguchi 3 1 ntt commun. sci. labs., ntt corp., atsugi, japan; 2 dept. psychol., tokai women's univ., kakamigahara, japan; 3 dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo 1,2,3 , yulri nonaka 1 , katsumi mizuno 4 , kazuo okanoya 1,5 1 riken, bsi, biolinguistics, saitama, japan; 2 chiba university, chiba, japan; 3 jsps, japan; 4 department of pediatrics, showa university, tokyo, japan; 5 presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for 18 neonates within 3 days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three (3) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented 1 s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for 12 different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around 700-900 ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism (9:4-14:11) and 18 agematched typically developing children (9:5-15:2) were instructed to name 50 pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = 1.2 ± 0.2, loudness = 1.4 ± 0.3) than in the children with autism (duration = 1.1 ± 0.2, loudness = 1.2 ± 0.3) (p < 0.001). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at 0.5/s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser (532 nm, 180 mw) to the muscle surface (28.3 mm 2 ) significantly delayed its attenuation (p < 0.05). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for 2 min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < 0.05). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps1p-i165 nedl1, a novel e3 ubiquitin ligase for dishevelled-1, targets mutant superoxide dismutase-1 and interacts with p53 yuanyuan li 1,2,3 , kou miyazaki 1 , toshinori ozaki 1 , akira nakagawara 1 1 division of biochemistry, chiba cancer center research institute, chiba, japan; 2 production technology development center, the furukawa electric co., ltd., ichihara, japan; 3 hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e3 ubiquitin ligase gene termed nedl1. previous study has shown that nedl1 is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled-1, a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl1 for uniquitination and proteasome-mediated degradation. on the other hand, nedl1 bound and ubiquitinated mutant (but not wild-type) sod1 in a mutant sod1 type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl1 physically bound p53, and induced apoptosis in a p53-dpendent manner. taken together, our results suggest that nedl1 may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod1 and p53. spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc70interacting protein), u-box type e3 ubiquitin ligase, has been shown to interact with hsp90 or hsp70 and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc12 cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca 2+ current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox-2 inhibitor, rofecoxib in pentylenetetrazol (ptz, 40 mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily 45 min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the 16th day of ptz treatment. chronic treatment with rofecoxib (2.0 and 5.0 mg/kg, p.o.) for 15 days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced (80 mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered 45 min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin (10 and 20 mg/kg, p.o.), naproxen (7 and 14 mg/kg, p.o.), nimesulide (1-5 mg/kg, p.o.) or rofecoxib (1-4 mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib (1 mg/kg) or nimesulide (1 mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox-2 inhibitors. cox-2 inhibitors also antagonized the effect of flumazenil (4 mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps1p-j171 cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca3 subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps1p-j172 a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus (4 mg/4 min). after 20 min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that 8-oxoguanine (8-oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca3 subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that 8-oxog accumulated greatly in mtdna of the ca3 microglia. wild-type and mth1-null mice, the latter lacking an ability to hydrolyze 8-oxo-dgtp and 8-oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca3 neuron loss after kainate administration, however, levels of 8-oxog accumulated in mtdna and cellular rna in the ca3 microglia were significantly increased in mth1null mice in comparison to wild-type mice. we thus demonstrated that mth1 efficiently suppresses the accumulation of 8-oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps1p-j176 transcription factor nrf2 regulates brain response to kainate-induced excitotoxicity yukihiko dan 1 , kosuke kajitani 1 , noriko yutsudo 1 , ken itoh 2 , masayuki yamamoto 2 , yusaku nakabeppu 1 1 kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; 2 univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e2 related factor 2 (nrf2) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf2 has been reported to increase neuronal death under increased oxidative stress, nrf2 seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca3 subregion of hippocampus. to unveil the functional significance of nrf2 in the brain, we compared seizure responses between wild-type and nrf2-null mice after systemic kainate administration. we found that nrf2-null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps1p-j177 synaptic plasticity and 4-aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine (4-ap) at the concentration below 0.1 mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the 4-ap induced discharges in ca3 region was studied. ten m 4-ap induced the epileptic discharges with the frequency of 0.33 ± 0.04 hz (mean ± s.e.m.; n = 3) and the amplitude of 1.23 ± 0.86 mv. when ap-5, an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap-5 was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of 4-ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. 7ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps2a-a001 influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno 1 , hugh p.c. robinson 2 1 engineering science, osaka university, osaka, japan; 2 university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣1a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the 3-day-old rat pup spinal subarachnoid space at the rate of 1 l/2.5 min. the rats were sacrificed 48 h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣1a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps2a-a003 on the membrane potential profile of ca1 pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga 1,2 , yoko tominaga 1 1 dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; 2 lab. for dynamics of emergent intelligence, riken bsi, hirosawa 2-1, wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps2a-a004 overexpression of inwardly rectifying k + channel 2.1 in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) 2.1 have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca1 pyramidal neurons. then, egfp and kir2.1 expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer 5 pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer 5 pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer 2/3 neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer 5 pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types 1 and 2 of ryanodine receptors (ryr1 and ryr2) in the cerebellum of a ca 2+ channel ␣ 1a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr1 but not ryr2 in the rmn cerebellum: a less ryr1 mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr1 immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr2 immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr1 expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr1-mediated ca 2+ release, contributing altered ca 2+ homeostasis in the rmn purkinje cells. ps2a-a007 dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than 5 s. d1 receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi 1,2 , hiroyoshi miyakawa 3 , masashi inoue 3 , masato okada 2,4 1 tokyo institute of technology, japan; 2 brain science institute, riken, japan; 3 tokyo university of pharmacy and life science, japan; 4 the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai 1 , maya yamazaki 1,2 , mika tsujita 1 , manabu abe 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, niigata, japan; 2 sorst/jst, saitama, japan we previously reported that stargazin family (␥2, ␥3, ␥4, and ␥8) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., 2004) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥4 subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥4 subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day 11, while the reduction was not so significant in adult brain. these results suggested that ␥4 might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph 6.8) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop1). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop1-transfected cells. mvsop1induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn 2+ sensitivity. neutralization of a positive charge in the s4-like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca1 pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh414). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca1 region. in the pyramidal layer, the amplitudes of the responses to the 1-hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the 3-10-hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels 2 (asic2) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic2-expressing cells in rat central nervous system (e17-p14) using immunohistochemical techniques. asic2positive fiber first appeared in brain stem and spinal cord at e17-18 stage. asic2-expresseing cells appeared in white matter of brain stem and spinal cord at e20 stage. in early postnatal stages asic2expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p4 stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic2 mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval (1st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long 1st isi by whole-cell recordings in rat slices. application of 100 m apamin, an sk-type ca 2+ -activated k + (kca) channel blocker, shortened the 1st isi and decreased the amplitude of the slow ahp. the shortening of the 1st isi was observed when membrane potentials were depolarized. moreover, application of 3 m mibefradil, a t-type ca 2+ channel blocker, shortened the 1st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca 2+ influx through t-type ca 2+ channels. research funds: kakenhi (c) (17500270) jafar vatanparast 1,2 , mahyar janahmadi 1 , houri sepehri 2 , ali haeri-rohani 2 , ali reza asgari 1 1 neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; 2 dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px (0.6 m, within 5 min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within 20 min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca 2+ spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca 2+ dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca1 slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca1 pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm8). repeated menthol application induced trpm8 desensitization. this desensitization was depended on extracellular ca 2+ , indicating that involvement with ca 2+ -dependent kinase. pkc activator (pma) desensitized trpm8 and go6976 (pkc inhibitor) abolished pma-induced trpm8 desensitization. pma similarly desensitized wild type trpm8 and mutant trpm8, in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm8. as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm8 causes pkc to desensitize trpm8 itself. yosuke sawada 1 , hiroshi hosokawa 1 , kiyoshi matsumura 2 , shigeo kobayashi 1 1 dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; 2 dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below 17 • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa1 is activated by pungent compounds stimuli. if trpa1 responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa1 is activated by cooling or not is still controversial. here, we investigated that trpa1-expressing hek293 cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was 17.5 ± 2.7 • c. in inside-out singlechannel recording, cooling activated trpa1 directly. single channel conductance was 74.1 ± 18.8 ps. single channel currents showed inword rectification. in conclusion, trpa1 is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn1-4) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn1-4 demonstrated that samples from the nerve and the heart showed an hcn2 band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn1, hcn3 and hcn4 could not be found in those samples. when an hcn channel blocker, zd7288 was applied on the stimulus portion of sciatic nerve and the nerve was elicited at 0.5/s by a duration of 10 ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < 0.01). it is suggested that hcn2 channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ 1 adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir3) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir1.1 and kir2.1 channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps2a-b022 proliferation of rat c6 glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c6), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control (140 mm) to 190 or 240 mm. an increase in [na + ] i was revealed by an image processor in c6 cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr-1, which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr-1 level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than 1 khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma (20-50 m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma (5 m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi (17021021) il-sung jang 1 , in-sun choi 1 , eun-ju park 1 , jin-wha cho 1 , man-gee lee 2 , byung-ju choi 1 1 kyungpook national university, school of dentistry, south korea; 2 kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca3 pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (<10 m), but decreased those induced by gaba at higher concentrations (>100 m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav1.1-deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav1.1-deficient mice as negative controls to examine nav1.1 distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav1.1 expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav1.1 distribution, in the present study, higher magnification analysis revealed that nav1.1 is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav1.1 antibodies used in these previous studies, none of which utilized nav1.1-deficient mice. based on our findings, we propose that nav1.1 might be involved in propagating action potential to presynapses. keiji miura 1,2 , masato okada 2,3,4 , shun-ichi amari 4 1 department of physics, kyoto university, kyoto, japan; 2 "intelligent cooperation and control", presto, jst, japan; 3 department of complexity science and engineering, university of tokyo, chiba, japan; 4 brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. 14084212 and 16500093) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [1-13 c] glucose intravenously (1 g/kg) 24 or 48 h after the intraventricular injection of colchicine (75 g/10 l) and the amino acid fractions were extracted from the brains at 10, 20 or 40 min after the glucose injection. the amount of [1-13 c] glucose in the cerebra was increased, however, the 13 c incorporation into glutamine, glutamate, gaba and aspartate from [1-13 c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a ␤ alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes1 or hes5 gene, phes1-and phes5-egfp transgenic (tg) mice were generated (ohtsuka et al., 2006) . in each transgenic mouse, a promoter of hes1 or hes5 gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes1 or hes5 regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day 10 (e10) to postnatal day 60 (p60). in the phes1-egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e10 to p60. in the phes5-egfp tg mouse inner ear, gfp-ir cells were observed from e16.5 to p15. gfp-ir cells in phes1-gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., 2006. mol. cell neurosci. ps2a-c033 expression of zfh-5 in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine 1 , kenji nakamura 2 , motoya katsuki 1 , tetsuo yamamori 1 1 national institute for basic biology, okazaki, japan; 2 mitsubishi kagaku institute of life science, machida, japan zfh-5 is a transcription factor containing three homeodomains and 18 zn fingers and expressed in differentiating neurons. we have reported that the level of zfh-5 mrna is negatively regulated by antisense transcripts of the zfh-5 gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh-5 antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh-5 mrna starts to be expressed. recently, we have raised an antibody against mouse zfh-5 and examined the expression profile of the zfh-5 protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh-5 protein was not detected even when the zfh-5 mrna was already expressed. this observation together with other data suggested that the zfh-5 protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue 1 , maya ota 1 , katsuhiko mikoshiba 2 , jun aruga 1 1 laboratory for comparative neurogenesis, riken bsi, saitama, japan; 2 laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic1-5) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic1/zic3 compound mutant mice. mice carrying homozygous zic1 mutant allele together with zic3 null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic1-or zic3-single mutant. these observations indicate that zic1, in cooperation with zic3, have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp58, a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp58 gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp58 has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp58 gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp58 transcripts are first detected at embryonic day 10 in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day 12-13 embryos, rp58 transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e15, rp58 transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp58 is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c2hc-type zinc finger motifs. using p19 cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf-2 and nzf-3 respectively. nzf-2 null mice are born alive, but die within 10 min after birth with cyanosis. on the other hand, nzf-3 null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf-2 and nzf-3 by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , 2006) . in the present study, to examine the role of runx3 in the development of drg in more detail, we examined the development of drg neurons in runx3-deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx3−/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days 14.5 and 15.5. shin hisahara 1,2 , susumu chiba 2 , hiroyuki matsumoto 2 , yoshiyuki horio 1 1 department of pharmacology, sapporo medical university, sapporo, japan; 2 department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt1 is still unclear. recent studies indicated that sirt1 interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes1 promoter. we performed overexpression of sirt1 and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt1 and n-cor in the sirt1 with n-cor. sirt1 and n-cor suppressed hes1 transcription by notch-icd in the luciferase assay. hes1 transcription was suppressed in overexpression of sirt1 and n-cor, suggesting that interaction between sirt1 and n-cor represses hes1 transcription. consistent with this, chip assays revealed that not only n-cor but also sirt1 bind to the promoter of hes1 gene. taken together, these results indicate that sirt1 and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes1 promoter site and repressing hes1 transcription. yasushi maruyama 1 , mitsuhiko kurusu 2 , masataka okabe 3 , katsuo furukubo-tokunaga 1 1 grad. school life and envir. sci., univ. tsukuba, japan; 2 natl. inst. genetics, mishima, japan; 3 inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf-␤ and bmp signaling. we reported previously that runx3 mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx3 regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., 2002) . furthermore, we announced previously that runx3 mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx3 on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx3 knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx3 knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi 1 , hirohide takebayashi 2 , takayuki manabe 1 , kazuhiro ikenaka 2 , akio wanaka 1 1 dept. anatomy, nara med. univ., kashihara, nara, japan; 2 division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng2proteoglycan and olig2 at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig2 promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig2 is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig2 lineage cells in the forebrain is largely unknown. here we examined fates of olig2 expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig2-creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) 12.5 or 14.5, when most of olig2+ cells are observed in the basal forebrain. the olig2+ cells at e12.5 gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig2 lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig2+ cells at e14.5 generated more glial cells than neurons. these results indicate that olig2 lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi 1,2 , masato yano 1,2 , satoru hayashi 1,2 , hirotaka j. okano 1,2 , hideyuki okano 1,2 1 dept. physiol., keio univ. sch. med, tokyo, japan; 2 sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf90 family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p21, p27 and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf90 and nf45, binding partner of nf90, respectively. in mouse embryonic brains, we found that nf45/90 expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf45/90 formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er81 and brn2) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi (16027202) ps2a-d047 protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes 2 days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-␤iii tubulin immunoreactivity increased through the re-cultivation for 1-4 weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-␤iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng2 + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng2 is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng2 + cells in the neocortex are the opcs. we investigated about subtypes of ng2 + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng2 + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng2 + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math1 + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp4 and wnt3a, a significant proportion of these neural cells become math1 + . the induced math1 + cells mitotically active and express markers characteristic of granule cells precursors (pax6, zic1, and zipro 1). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math1 + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l7 + /calbindin-d28k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf8 rather than by wnt3a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p21 cip1 , p27 kip1 and p57 kip2 mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p27 kip1 mrna was shown. transfection of p27 kip1 into npcs induced activation of neurod promoter and increase of number of ␤tublin iii-positive cells. treatment with deferoxamine to npcs from e12.5 rat midbrain and hb1.f3 cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p27 kip1 elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g1/s phase did not activate mapk and pi3-k signaling. yuichi tanaka 1 , yusuke tozuka 1 , dai muramatsu 1 , kin-ichi nakashima 2 , tatsuhiro hisatsune 1 1 departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; 2 graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng2+ and olig2+ cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj-1+ or map-2+ neuronal cells by adding retinoic acid or bdnf. more than 90% of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod1 significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi 1 , michiko imanishi 2 , teiko kuroda 2 , masao tachibana 1 , masahiko takada 2 1 department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv3 subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv3 channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv3.1b and kv3.3 were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv3.3 in the frog retina and by kv3.1b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv3.1b. thus, the differential expression of kv3 channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou 1,2 , kazunori yamamoto 1 , masayuki hara 1 , akinori homma 1 , akimichi kaneko 3 , masahiro yamada 1 1 tokyo metropolitan univ., hino, tokyo, japan; 2 astellas pharma. inc., osaka, japan; 3 sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko (2003) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by 5-hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as 0.21 ± 0.05 ph unit by 100 mm k + . acidification of pho was suppressed by 0.4 m bafilomycin a1, a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps2a-e055 analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar 64-channel field potential recording system in slice preparations obtained from 16 to 24 days old mice. stimulation at ssc induced negative field potential with short latency and short duration (100-200 ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing 10 m bicuculline, the same stimulus induced a large negative field response with long duration (200-400 ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of 50 m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya 1 , ken tsutsui 2 , kimiko tsutsui 1 1 dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; 2 dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap-2 binding, and the c-terminal sh3 domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with 3 -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe-19, also expresses amph ir. arpe-19 thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe-19 cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura 1 , satoshi ichisaka 2 , chihiro hayashi 2 , hirotoshi maki 2 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v1) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v1 and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v1 receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi (176959) ps2a-e058 rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut)1 and vglut2, respectively. a complementary distribution of vglut1 and vglut2 was established by postnatal day (p)18 and both signal intensities increased further by p29-30 (peak cp). the immunoreactivity for vglut2 decreased around layer iv after brief monocular deprivation (4dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to 4dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area 17, with a small projection to layer vi of areas 17 and 18a. cells in layer vi of area 17 projected to dlgn. in addition, cells in layer v of area 17 projected to dlgn, which is not observed in normal rats. in area 18a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of 18a. cells in layers v and vi of area 18a projected to lp. smaller number of cells in layer v of area 17 also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps2a-e060 activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida 1 , takemasa satoh 2 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v1) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v1 to the lgn. we focused on the density of type 1 metabotropic glutamate receptor ␣ (mglur1␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur1␣ in the lgn decreased after elimination of v1, showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur1␣ signal in the lgn of young rats. yu morishima 1 , hiroshi sakamoto 2 , takahumi akasaki 1 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, (1) how rapidly the reverse od shift proceeds and (2) whether the shift is induced only in young animals. we infused the cortex with muscimol in 4-week-old kittens and in adults. the reverse od shift was observed after 6 days md, but not significant after 3 days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after 6 days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps2a-e062 experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai 1 , nafiseh atapour 1 , john renger 1 , john roder 2 , peter seeburg 3 , takao hensch 1 1 neuronal circuit dev., riken, bsi, wako, japan; 2 mount sinai hospital, toronto, canada; 3 max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur1, 2) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation (1d md) rapidly alters both the phosphorylation state and surface expression of glur1 in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur1 (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after 4d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as 20d md eventually led to full od shifts. in glur2 ko mice, even 4d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur1 may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx2 promotor and dlx5/6 enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca1 and revealed a nonpyramidal neuron projecting to ca3 and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita 1 , takao oishi 2 , motoharu hayashi 2 1 div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; 2 dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than 60% of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area (5.3%) than in other areas, such as the prefrontal (10.8%), primary motor (9.8%), somatosensory (10.0%) and secondary visual areas (8.5%), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than 80% of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe 1 , guy n. elston 2,3 , tomofumi oga 1 , hiroshi tamura 1,3 , ichiro fujita 1,3 1 osaka univ., japan; 2 univ. queensland, australia; 3 crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v1). pyramidal cells in prefrontal cortex, such as area 12, have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v1 (n = 52), area te (n = 46), and area 12 (n = 43) of a 3-week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area 12, followed by area te, and v1. thus, considerable heterogeneity in pyramidal cell structure already exists 3-weeks after birth. hiroaki matsushita 1 , mahito ohkuma 1 , masami watanabe 2 , ei-ichi miyachi 1 1 department of physiology, fujita health university, aichi, japan; 2 department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura-2 based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the 50% of axotomized rgcs. bath application of 100 m carbachol, an ach receptor agonist, rose [ca 2+ ] i in 22% of normal rgcs. although the 85% of rgcs responded to carbachol at 3 days after axotomy, no responsive rgcs appeared during 7-15 days. ach responsiveness recovered in axonal regenerated rgcs (17%). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi (17500217 to em, 16591780 to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., 2004) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor (75 hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at 30 deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames (107 ms) or only one frame (13 ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps2a-e069 motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima 1,3 , tohru kiryu 2 , kazuhiko ukai 3 , takeshiko bando 1 1 div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; 2 div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; 3 dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. 20 subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura 1 , kenichiro miura 1 , masakatsu taki 1 , hiromitsu tabata 1 , naoko inaba 1 , kenji kawano 1 , frederick a. miles 2 1 grad. schl. med., kyoto univ., kyoto, japan; 2 lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn 2005) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency 3f and 5f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps (100 hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the 5f forwards and the 3f backwards. the contrast of the 5f was fixed at 4, 8, or 16%, while the contrast of the 3f was varied from one-fourth to four times the contrast of the 5f. when the contrast of the 3f (5f) was less than about half that of the 5f (3f), the 5f (3f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < 0.05). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito 1 , yaoxing chen 2 , yukiko tanada 1 1 dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; 2 china agricul. univ., beijing, china twenty white-leghorn chicks (p0-8) were perfused with 1% paraformaldehyde through the heart under deep anesthesia of nembutal (32 mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of 225 rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, 2004). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to 32 in 121 tec-rgcs, 28 in 77 tha-rgcs, and 16 in 38 hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers 1-3). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie 1 , kenji yuda 3 , eiichi okawa 4 , katsuyoshi maruyama 4 , hiroshi uozato 5 , hiroko horie 1 , satomi nakajima 1 , kenkichi tanioka 6 , yuji ohkawa 6 , tomoki matsubara 6 , wolfram tetzlaff 7 1 advanced res. centr. biological sci., waseda univ., tokyo, japan; 2 technomaster co. ltd., yokohama, japan; 3 kikuna yuda eye clinic, yokohama, japan; 4 healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; 5 dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; 6 nhk sci. technical res. labo., tokyo, japan; 7 icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between 25 and 70 cm toward and away in a high speed (top speed: 1000 mm/s) from the subjects. the device significantly improved visual performances in over 60% of the school-aged children with myopia and 80% of adults with presbyopia. hiroyuki miyamoto 1 , toral s. surti 2 , takao k hensch 1 1 laboratory for neuronal circuit development, riken brain science institute, wako, japan; 2 san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v1) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v1. here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad65 ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with 4 day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa 1 , miquel bosch 2 , shuzo sakata 3 , yoshio sakurai 4 , tetsuo yamamori 1 1 division of brain biology, national institute for basic biology, okazaki, japan; 2 mit, ma, usa; 3 rutgers university, nj, usa; 4 department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., 2004) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v2lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v2lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki 1 , youichi miyawaki 2 , tsunehiro takeda 1 1 department of complexity science and engineering, university of tokyo, chiba, japan; 2 mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly (10 ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = 0-990 ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa (10 and 20 ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p1, n170 and area under curve of a late positive potential expanding from 300 to 500 ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse 1 , ayumu matani 1,2 , tomoe hayakawa 2,3 , norio fujimaki 2 1 university of tokyo, kashiwa, japan; 2 nict, kobe, japan; 3 teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around 500 ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of 1000 flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p100 amplitude becomes. this suggested that a factor of the variability of the p100 amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. 16300083) hitoshi sasaki 1 , takuya ishida 1 , masayoshi todorokihara 2 , junichiro miyachi 1 , tahei kitamura 3 , ryozo aoki 1 1 dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; 2 dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; 3 dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama 1 , nobuo usui 1 , izuru nose 1,2 , masato taira 1,3 1 department of applied system neuroscience, nihon university school of medical science, tokyo; 2 faculty of human science, bunkyo university, saitama, japan; 3 arish, nihon university, tokyo, japan when an object is moving, perception of its 3d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps2a-f083 local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a1) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = 830 nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., 1998) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., 1993) . moreover, aaf has dense synaptic connections within the area (budinger et al., 2000) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were 50-ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima 1 , yasuo takeda 2 , yasushi shimoda 1 , kazutada watanabe 1 1 department of bioengineering, nagaoka university of technology, nagaoka, japan; 2 department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb-2 that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb-2 expression is prominent in the auditory system. nb-2 knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb-2 is indispensable for the function of auditory system. here we report the detailed analysis of the nb-2 localization using anti-nb-2 monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb-2 was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb-2 immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb-2 regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation (4 khz octave band, 125 db, 5 h), and then subjected to electrophoretic mobility shift assay to determine activator protein-1 (ap-1) dna binding. ap-1 binding was increased 2-12 h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea 2 h later. taken together, ap-1 may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki 1 department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c57bl/6 mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli (5-26 khz for 500 ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by 5 khz were different from those elicited by 20 or 25 khz. however, the areas activated by 5 khz were also activated by the mixture of 20 plus 25 khz but not by that of 19 plus 26 khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps2a-f092 functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh795 were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by 55-90%, whereas the inhibition of dc resulted in reduction only by 20-50%. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh-795. a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato 1,2 , kazuo okanoya 1 1 laboratory for biolinguistics, riken bsi, wako, saitama; 2 graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, 2000) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented 100-500 times at a fixed interval, through a speaker at 50-70 db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (<10 hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (>100 hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki 1,2,3,4,5,6 1 hokkaido university, sapporo, japan; 2 hokkaido university, sapporo, japan; 3 sakushin gakuin university, utsunomiya, japan; 4 kobe shoin women's university, kobe, japan; 5 riken, wako, japan; 6 sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children (8-12 years old, n = 8) and adults (19-23 years old, n = 12). there were six gap conditions with durations of 8, 16, 32, 64, 128 and 256 ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was 500 hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n1 and p2 complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps2a-f098 an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua 1 , tamaki miyamoto 2 , atsushi terao 2 , sachiko koyama 2 , takashi omori 2 , harumitsu murohashi 2 , shinya kuriki 2 1 jst, saitama, japan; 2 hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba9), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation (12 subjects, p < 0.05 corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi 1 , yuko toyosawa 2 , fumiya takeuchi 3 , michinao matsui 4 , shinya kuriki 1 1 research institute for elecronic science, hokkaido university, sappro, japan; 2 jst, saitama, japan; 3 hokkaido university, school of medicine, japan; 4 kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = 11). four synthesized vowels were used as stimuli (stimulus duration 600 ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used (1200/4800 ms). for the native vowels, an early sf (250-300 ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf (400-600 ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi (16500166) ps2a-f100 spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto 1,2 , junsei horikawa 1 1 department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; 2 riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh 795) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli (5-20 khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for 40 min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe 1 , go ashida 1,2 , kazuo funabiki 2 1 graduate school of informatics, kyoto university, kyoto, japan; 2 hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi 1 , shigenori takebayashi 2 , masahiko bundo 2 , masazumi fujii 3 , toshihiko wakabayashi 3 , jun yoshida 3 , hiroyuki fujisawa 1 , kazunori ando 1 , kazumasa hayasaka 1 1 department of radiology, national hospital for geriatric medicine, obu, japan; 2 department of neurosurgery, national hospital for geriatric medicine, obu, japan; 3 department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using 1.5 tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in 11 out of 12 patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = 8) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, 1987) . surprisingly, subjects tended to misidentify the second toe as the third (47.2%), and the third toe as the fourth (37.2%), while the reverse misidentification rarely occurred (third as second, 4.5%; fourth as third, 7.1%). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps2a-g105 effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi 1,2 , shunjiro moizumi 1 , ayami okuzumi 1 , humine saito 1 , shigeru kitazawa 1,2 1 department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; 2 crest, jst, saitama, japan morrone et al. (2005) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade (24 • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of 100 ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within 300 ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps2a-g106 function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda 1,3 , miki taoka 2,3 1 department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; 2 secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; 3 department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas 3b and 1 of four conscious macaque monkeys was studied by recording single-neuron activities. a total of 115 electrode penetrations were made in areas 3b and 1. in 44 penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining 71 penetrations, however, 14% of adjacent pairs (311/2153) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas 3b and 1 might be responsible for forming composite rfs of area 2 neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi (12771111, 14771029) miki taoka 1 , michio tanaka 1 , hisayuki ojima 1 , atsushi iriki 2 1 secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; 2 laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded 168 neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them (44% contra-, 55% bi-and 1% ipsilateral hand movements), we could determine receptive fields for only 43 neurons (25%). most of activities (138 neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta 1,2 , kouichi nakamura 1 , takeshi kaneko 1 1 department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; 2 crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata 1,2 , shanlin chen 2 , hiroki kitaura 1 , masaharu kudoh 1 , minoru shibata 2 , katsuei shibuki 1 1 department of neurophysiology, brain research institute, niigata university; 2 department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane (1.7 g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation (50 hz for 1 s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi 1 , nanae fukushima 1 , tetsuhiro fukuyama 2 , akira kakegawa 1 , tetsuji moriizumi 1 1 department of anatomy, shinshu university school of medicine, matsumoto, japan; 2 department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima 1 , takeshi kawano 1 , hidekuni takao 1,2,4 , kazuaki sawada 1,2,4 , hidekazu kaneko 3,4 , makoto ishida 1,2,4 1 department electrical & electronic engineering, toyohashi university of technology, aichi, japan; 2 issrc, toyohashi university of technology, aichi, japan; 3 aist, hsbe, ibaraki, japan; 4 jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, 2 m in diameter, 60 m in length; array: 40-m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = 0.91). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi (17206038), the 21st century coe program "intelligent human sensing" ps2a-g113 a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko 1 , hiroshi tamura 2 , shinya s. suzuki 1 , takahiro kawashima 3 1 aist, hsbe, ibaraki, japan; 2 laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; 3 department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over 2 h. seiji matsuda 1 , takehiro terashita 1 , tetsuya shimokawa 1 , kyoujy miyawaki 1 , yuji miguchi 1 , takuya doihara 1 , jie chen 1 , shuang-yan gao 1 , chun-yu li 1 , min wang 1 , zhong wang 1 , bing xue 1 , naoto kobayashi 1,2 , kazuhiro shigemoto 3 1 department anatomy, ehime university of medicine, ehime, japan; 2 medical education c, ehime university of medicine, ehime, japan; 3 department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than 1.2 m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat3, whereas other genes known to be regulated by stat3 were not detected. stat3 is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat3 in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat3 in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat3 and stat3phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat3 are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto 1 , susumu hyodo 2 , makoto kawasaki 1 , minori shibata 1 , takeshi saito 1 , hiroaki fujihara 1 , takashi higuchi 3 , yoshio takei 2 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; 3 department of integrative physiology, university of fukui, japan adrenomedullin 2 (am2) (identical to intermedin) belongs to the super family of am. centrally administered am and am2 activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am2 on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am2 was stronger than those of am and the effects of am2 on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am2 would have unknown receptor except cgrp and am receptor. arata oh-nishi 1 , makoto saji 1 , taku uchida 1 , sen-ichi furudate 2 , nobuyuki suzuki 1 1 division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; 2 division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr1 expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, 2004) . here we report that the density and morphology of spines of pyramidal neurons in ca1 region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for 2 h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca1, both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day 8-15, the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day 15. the staining of somatostain mrna in individual neurons was diminished when examined on day 35 or 70, albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps2a-g121 short chain sugar acid, 2-buten-4-olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki 1 , tatsushi onaka 2 , hirofumi hashimoto 1 , hiroaki fujihara 1 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 department of physiology, jichi medical school, tochigi, japan 2-buten-4-olide (2-b4o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of 2-b4o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at 15-60 min after intraperitoneal (i.p.) administration of 2-b4o (100 mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei 120 min after i.p. administration of 2-b4o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of 2-b4o. these results suggest that peripherally administered high dose of 2-b4o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps2a-g122 estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within 4 h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto 1,2 , shinpei higo 1,2 , yasushi hojo 1,2 , kouhei nakajima 1,2 , hironori nakanishi 1,2 , hirotaka ishii 1,2 , suguru kawato 1,2 1 department of biophysics & life science, university of tokyo, tokyo, japan; 2 crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps2a-g125 gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from −40 to -10 mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than -50 mv and most of them were ∼−80 mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr1, galr2 and galr3, in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr1 selective agonist m617 induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr1. hiroaki fujihara 1 , tomoki fujio 1 , david murphy 2 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking 2% saline for 5 days in 3, 6, 12 and 24 months old tg rats. in 3 and 6 months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats (12 and 24 months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps2a-h128 prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera 1 , hiroaki fujihara 2 , hirofumi hashimoto 2 , makoto kawasaki 2 , tatsushi onaka 3 , takakazu oka 1 , sadatoshi tsuji 1 , yoichi ueta 2 1 department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; 2 department of physiology, school of medicine, university of occupational and environmental health, japan; 3 department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin 1 , afsaneh eliassi 1 , nasser naghdi 2 , ali ghanbari 1 1 ghsemi; 2 pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d1 receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above (0.5 mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from 30 min up to the end of experiments (90 min). at the peak acid response, the vmh microinjection skf38393 (0.1,1) significantly reduced the amount of gas (p < 0.001). there was no any effect by microinjected sch23390 (0.1) into the vmh. injecting skf38393 into the vmh, 5 min after sch23390, had no effect on gas in compare with control. but, the acid suppressant effect of skf38393 was completely removed by peripheral injection of sch23390 (p < 0.01). our results show that the vmh d1 dopamine receptors have regulatory mechanisms of gas by interaction with h2 receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p 2x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p 2y purinoceptors (p 2y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p 2y r agonists increased [ca ++ ] i in son, with p 2y1 r agonist being the most effective. the possibility that p 2y1 r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p 2y1 r antagonist, mrs2179. atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs2179 (100 m) in 2 mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs2179. furthermore, combined treatment with mrs2179 and ppads (10 m, a p 2x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p 2y1 r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps2a-h131 analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii 1,2 , yasuhiro sonoki 1,2 , aizo furukawa 2,3 , yasushi hojo 2 , tetsuya kimoto 1,2 , suguru kawato 1,2 1 department of biophysics and life science, university of tokyo, tokyo, japan; 2 crest, jst, japan; 3 kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day 1(pd1) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd1 to pd14 with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai 1,2 , gen murakami 1 , shirou kominami 3 , john h morrison 4 , william g.m janssen 4 , tetsuya kimoto 1,2 , suguru kawato 1,2 1 department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo 153-8902, japan; 2 crest project, jst, japan; 3 faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima 739, japan; 4 kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc-19, mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca1, ca3, and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud 1 , song t. yao 1 , jing qiu 1 , julian fr paton 2 , david murphy 1 1 university of bristol, hw-line, uk; 2 department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic (2d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from 3 days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase 1, has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt1-7. delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt1-7 cells cultured in dmem supplemented with 10% fbs for 3 days. bk current was increased by addition of 17␤-estradiol (e2) in culture medium in a physiological concentration range. this action of e2 was blocked by ici-182,780, a potent estrogen receptor (er) antagonist. we further examined whether e2 acted through er ␣ or er ␤ by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e2 but ppt had no effect. e2 had no effect on the i k . these results indicate that e2 increases the bk current by activating er␤ without affecting the i k . research funds: kakenhi 16590180, 16086210 ps2a-h136 myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi 1 , haruko kumanogoh 2 , shun nakamura 2 , seiji miyata 3 , shohei maekawa 1 1 department of biosystems science, kobe-university, kobe, japan; 2 division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; 3 department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, 25 kda protein, was identified as myelin protein zero (p0) from mass analysis and this result was confirmed by western blotting that a specific antibody to 25 kda band reacted to an authentic p0 prepared from rat sciatic nerve myelin. p0 is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p0 has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p0 expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan 17␤-estradiol (e2) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e2 (hojo et al., 2004) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e2 and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca1, ca3 and dg with a custom made multi-electrode measuring system (med64). ltd was enhanced by e2 dose-dependently in ca1, ca3 and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito 1 , takushi x. watanabe 2 , tomoko urabe 2 , hirofumi hashimoto 1 , hiroaki fujihara 1 , yukio hirata 3 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 peptide institute, inc., osaka, japan; 3 department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin-␤ was newly discovered as a bioactive endogenous peptide. low concentration of salusin-␤ stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin-␤ coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin-␤ in body fluid homeostasis, we examined the effects of salt loding for 5 days on salusin-␤-like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin-␤-li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin-␤ may play a role of body fluid balance by regulating avp release. naoyuki yamamoto 1 , hao-gang xue 1 , yuji ishikawa 2 , yoshitaka oka 3 , hitoshi ozawa 1 1 department of anatomy and neurobiology, nippon medical school, tokyo, japan; 2 national institute of radiological sciences, chiba, japan; 3 department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki 1 , eiji munetsuna 1 , asuka kamogawa 1 , suguru kawato 2 , shiro kominami 1 1 graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; 2 graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p450arom in the hippocampus. method: the cultured hippocampus slices from 10 days male rat were treated with 100-1000 nm of tbt for 48 h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p450arom was determined by quantification of [3h]estradiol from [3h]testosterone. result: forty-eight hours treatment of hippocampal slices with 100 nm tbt induced increases in mrna contents of p450arom, and with 1000 nm tbt induced that of 3␤-hsd. estradiol content was increased by the treatment with 100 nm tbt, but not affected by 1000 nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p450arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for 4 h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of 30, 60, 90 and 120 ng in 200 nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent 4 h. the 30 ng dose of bicuculline methoiodide had minimum effect whereas 60 and 90 ng produced arousal. maximal wakefulness was observed at dose of 90 ng with no further increase in wakefulness at higher dose of 120 ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe 1 , hiroyuki tsuda 2 1 department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; 2 department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee1). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., 200 mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in 24 h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, (1 antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at 1 h after from hibernation onset compared with before hibernation. in addition, a localized ␤-endorphin-like immunoreactivity (␤-end ir) was observed in neuronal perikarya in arc at 1 h after from hibernation onset. although ␤-end ir in arc got weak, the ␤-end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the ␤-endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via 1-opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d 2 is a potent somnogenic substance, and isomerized from pgh 2 through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl 4 into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl 4 decreased rapid eye movement (rem) and non-rem sleep for 5 h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl 4 administrations. these findings indicate that pgd 2 plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd 2 involved in sleep-wake regulation. under baseline conditions, h 1 r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (<16 s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h 1 r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h 1 r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., 2006) . ps2a-h147 dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = 500 nm). the scn neurons responded to the light in intensities above 10 11 photons/cm 2 /s and were activated maximally at around 10 15 photons/cm 2 /s, they were about 1.6 log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca 2+ in scn neurons. since cytosolic ca 2+ regulates diverse cellular systems, we have hypothesized that the cytosolic ca 2+ rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca 2+ rhythms, mouse bmal1 and its dominant negative sequence (mbmf1r5) are transfected into the organotypic culture of scn with a yellow cameleon ca 2+ sensor by the gene gun. the results demonstrated that over-expression of bmal1 or mbmf1r5 significantly inhibited the circadian ca 2+ rhythms and thus we concluded that the native bmal1 rhythm is essential for cellular output processes of the murine clock system. ps2a-h149 the activation of ␣2 adrenergic receptor increases the frequency of carbachol-induced ␤ oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses ␤ rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce ␤ oscillation in rat hippocampal slices like ␤ rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced ␤ oscillation in ca3 region of rat hippocampal slices. carbachol (30 m) induced ␤ oscillation with the frequency and the amplitude of 14.8 ± 0.1 hz, and 0.7 ± 0.1 mv, respectively (mean ± s.e.m.; n = 3). epinephrine (50 m) significantly increased the frequency of 16.2 ± 0.1 hz (**p < 0.01), not change the amplitude. clonidine (50 m), an ␣2 receptor agonist, alone significantly increased the frequency at the concentration of 50 m (*p < 0.05). yohimbine, an ␣2 receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal ␤ rhythm via ␣2 receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart 0.4 mm from each other in the hippocampal ca3 region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency 100-300 ms), oscillatory (2-4 hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties (8/9 slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca3 region. ryozo aoki 1 , hiroshi wake 2 , hitoshi sasaki 1 , kiyokazu agata 3 1 dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; 2 dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; 3 dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as 1/f, ranging from 10 − 1 to 10 + 2 hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond 40 min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to 60 min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over 30 min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida 1 , hiroki sato 1,2 , naoki tanaka 2 , atsushi maki 1,2 1 japan science and technology agency, crest, saitama, japan; 2 advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from 8 to 12 hz) decreases and the sleep spindle power (from 12 to 16 hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from 1 to 8 s in (i), and from 8 to 15 s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim 1 , kazuki nakajima 1 , yutaka oomura 2 , kazuo sasaki 1 1 div. of bio-information eng., univ. of toyama, toyama, japan; 2 dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca 2+ exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng 1 , wei zhang 1 , akira nakamura 2 , masashi yanagisawa 3 , yasuichiro fukuda 2 , tomoyuki kuwaki 1,2 1 dept. molec. integ. physiol., chiba univ., japan; 2 dept. autonom. physiol., chiba univ., japan; 3 dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb-334867 (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb-334867. icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co2-sensitivity at least during waking periods. research funds: kakenhi 16590162,17590183 junko hara 1 , taizo matsuki 2 , katsutoshi goto 1 , masashi yanagisawa 3 , takeshi sakurai 1,2 1 department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; 2 yanagisawa orphan receptor project, erato, jst, tokyo, japan; 3 howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps2a-i157 prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto 1 , kazuhiko kubo 2 , shuji aou 1 1 dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; 2 dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. 2006) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. 16209006, s.a.) shuji aou 1 , tetsuya fujimoto 1 , yumi ichihara 1 , kimiya narikiyo 1 , toru ishidao 2 , hajime hori 2 , yukiko fueta 2 1 dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; 2 dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan 1-bromopropane (1-bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to 1-bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to 700 ppm of 1-bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. 1-bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that 1-bp is the potential candidate of endocrine disruptors which affect brain development. (16651027) ps2a-i159 changes in hippocampal excitability of rats prenatally exposed to 1-bromopropane yukiko fueta 1 , toru ishidao 1 , susumu ueno 2 , yasuhiro yoshida 3 , hajime hori 1 1 department of environmental management, school of health sciences; 2 department of pharmacology; 3 department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to 1-bromopropane (1-bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to 1-bp (700 ppm) during gestational day 1-20 (6 h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca1 at the pnd 12-15. conversely, s/r curves of fepsp as well as ps in the ca1 were inhibited at the age of 6-8 weeks. our results suggest that 1-bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca1 of the offspring during development. research funds: grant-in-aid for exploratory research (16651027) ps2a-i160 effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura 1,2 , kyoko itoh 1 , takeshi yaoi 1 , tohru sugimoto 2 , shinji fushiki 1 1 dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; 2 dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously 20 g/kg of bpa daily from embryonic day 0 (e0). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e14.5 and e16.5, whereas those cells increased in the cortical plate at e14.5, as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant 15390334 keiko ikemoto 1 , teruko uwano 2 , hisao nishijo 2 , taketoshi ono 2 , masayuki ito 3 , ikuko nagatsu 4 , katsuji nishi 5 , shin-ichi niwa 1 1 dept. neuropsychiat., fukushima med. univ. sch. med.; 2 toyama med. pharm. univ.; 3 faculty med., mie univ., mie, japan; 4 fujita health univ. sch. med., toyoake, japan; 5 dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day 9 and 19 after fertilization. pups were perfused at postnatal day 8. in the frontal cortex, the number of largesized (more than 7 m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c1/a1 catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps2a-i162 developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi 1,2,3 , kaori morohoshi 3,4 , rie yanagisawa 3 , erina saita 5 , gen watanabe 6,7 , masatoshi morita 3 , kazuyoshi taya 6,7 , hirohisa takano 3,4 , toshiyuki himi 1,2 , hideki imai 3,8 1 dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; 2 res. inst. pharmaceut. sci., musashino univ., tokyo, japan; 3 nation. inst. for environ. stud., ibaraki, japan; 4 grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; 5 wildlife rescue veterinarian associ., tokyo, japan; 6 facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; 7 the united grad. sch. veterinary sci., gifu univ., gifu, japan; 8 div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor ␤, which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps2a-i163 the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic (5-ht) systems in stressinduced anxiety, we measured brain da and 5-ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of 5-ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for 60 min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and 5-ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although 5-ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl1-4) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl3 and nl4 in autistic disorders. nls have a relative identical structure (∼70%); nl1 and nl2 localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl3 seems express in the olfactory glia, but nl4 distribution is unknown. here we have generated antibody against human nl4, and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl4 was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl4 was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl4 is important for neuroendocrine function. nl4 cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl4 were rough and thicker than those of nl1 or nl3 transformants. it suggested the unique activity of nl4 for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca1 pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting 1-3% of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below 50. here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura 1 , daisuke horiuchi 1 , tomoyuki miyashita 2 , minoru saitoe 2 , hitoshi okazawa 1 1 department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein-1 (pqbp-1) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp-1 disease that pqbp-1 mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp-1 in xlmr pathology, we produced two kinds of flies, human pqbp-1 overexpression flies (hpqbp1 flies) and drosophila pqbp-1 knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp1 flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp-1 in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun 1 , sentaro takahashi 1 , yoshihisa kubota 1 , rui zhang 2 , chun cui 2 , yoshihiro fukui 2 1 natl. inst. radiol. sci., chiba, japan; 2 sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr1 subunits paired with two nr2 subunits. nr1 and nr2 (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr1/nr2a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr1/nr2b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca1 pyramidal neurons from mice (at 12 weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr2b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca1 region after ischemia. in blood, corticosterone levels increased biphasically at 30 min and 12 h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca1 region. by 3 days after ischemia, mr and gr were not significantly altered in the ca1 region. from 4 days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca1 region, and at 7 days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase-3 and midkine in the mca area. stroke was induced by a 90-min mca occlusion using an intraluminal filament. rats were exercised for 20 min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase-3 expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps2a-j174 reversed astrocytic glutamate transporter glt-1 crucial to the ca 2+ paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca 2+ paradox" is the phenomenon whereby the intracellular concentration of ca 2+ paradoxically increases during reperfusion with normal ca 2+ -containing media after brief exposure to a low ca 2+ solution. the present study aims to characterize the ca 2+ paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca 2+ solution for 60 min significantly injured only neurons after reperfusion with a normal ca 2+ medium for 24 h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt-1. the present findings suggested that ca 2+ paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt-1 during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch3 concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch3 were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c577t, which results in pro167ser. the second patient carried the missense mutation c302g, which results in arg75pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch3 receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps2a-j177 mci -186, a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3kinase madinyet niyaz 1 , tadahiro numakawa 2 , yoshinori matsuki 1 , emi kumamaru 2 , yuki yagazaki 2 , harumi kitazawa 1 , hiroshi kunugi 2 , motoshige kudo 1 1 pathology department of tokyo medical university, tokyo, japan; 2 department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci -186, a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci -186 prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci -186 exposure induced the activation of both the map kinase (mapk) and pi3 kinase (pi3k) pathways and that the mci -186-dependent survival effect was blocked by the inhibitors, u0126 (an mapk pathway inhibitor) or ly294002 (a pi3k pathway inhibitor). these results suggested that mci -186 exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru 1 , noriko yoshimura 1 , tetsuro sakamoto 1 , takaaki kitano 2 , naoko nisimaru 1 , isao yokoi 1 1 dept. physiol., sch. med., oita univ., japan; 2 med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by 31 p-nuclear magnetic resonance (25 • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with 2 mm ep (ep-1), acsf replaced by acsf with 2 mm ep after ischemia (ep-2), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for 1 h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep-1 than in control (p < 0.01). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) #17591639 from mext to t.k. hideaki tamai 1 , kuniko shimazaki 2 , norimasa seo 1 1 department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; 2 department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc6), yintang (ex-hn3) and sanyinjiao (sp6), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through 5-bromo-2,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc6, ex-hn3 and sp6. the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago 1 , keiko takahashi 1 , shigeo nakamura 1 , akemichi baba 2 , toshio matsuda 1 1 laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; 2 laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug ␤-cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara 1 , tatsuya mishima 1 , takefumi kofuji 2 , kimio akagawa 1 1 department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; 2 radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc-1/syntaxin 1a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc-1/syntaxin1a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc-1/syntaxin1a knock-out mouse will be discussed. research funds: kakenhi (15700292) ps2a-k182 effects of local administration of the gaba agonists into the hippocampus ca1 area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka 1 , hiroko miyakubo 1 , junichi tanaka 1 , yasushi hayashi 2 , yukio hattori 2 , masahiko nomura 3 1 department of curriculum, teaching and memory, naruto university of education, tokushima, japan; 2 department of human nutrition, notre dame seishin university, okayama, japan; 3 department of physiology, saitama medical school, saitama, japan in fischer 344 male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca1 area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite 5-hydroxytryptamine (5-hiaa) concentration in the ca1 area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca1 area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad-67. fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka 1 , sho koyasu 1 , akinori nishi 2 , tomoyuki furuyashiki 1 , toshiyuki matsuoka 1 , shuh narumiya 1 1 department of pharmacology, university of kyoto, kyoto, japan; 2 department of physiology, university of kurume, kurume, japan prostaglandins e2 (pge 2 ) exert their actions in various organs through specific receptor, ep1 to 4. the previous study suggests that ep1 modulates da system. to investigate the roles of ep1 in da system, we examined ep1ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼300% in both wild-type and ep1ko mice. however, increase of locomotor activity in ep1ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d1 receptor (d1r) signaling, we tested the density of d1r and d1r signaling with phosphorylation of darpp-32. there were no differences in d1r binding. d1r signaling was significantly attenuated in the striatal slices from ep1ko mice. the effect of d1r agonist on locomotor activity was also attenuated in ep1ko mice. these results indicate that pge 2 has enhancing effects on locomotor activity via ep1 by potentiating the d1r signaling. central serotonin (5-ht) function has been implicated in impulsivity. the present study examined rats with 5-ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of 5-ht and its metabolites (5-hiaa) in the brain. for both tasks, significant negative correlations between learning performance and 5-ht and 5-hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and 5-ht and 5-hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of 5-ht roles on selective indices of impulsivity. yuki sato 1,2,3 , tatsushi onaka 2 , norimasa seo 1 , eiji kobayashi 3 1 dept. anesthesiol., jichi med. univ., tochigi, japan; 2 dept. physiol., jichi med. univ., tochigi, japan; 3 div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p1-21; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p4 in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p7; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io␤) by p11. within the vestibular nucleus of p7-9 rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p21 and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p1 to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p3 in the vestibular nucleus as fuzziness about neuronal cell bodies. from p9 onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p12. during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p21. with ul, the pn network on the lesioned side remained compact until 5 days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by 11 days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by 14 days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca1 region of rat hippocampal slices, 100 hz/20 pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at 100 hz. ikuko yao 1 , hiroshi takagi 1 , hiroshi ageta 1 , tomoaki kahyo 1 , ken hatanaka 1 , kaoru inokuchi 1 , mitsutoshi setou 1,2 1 mitsubishi kagaku institute of life sciences, tokyo, japan; 2 university of tokyo, tokyo, japan; 3 okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps2p-a003 presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., 10 mv, 1 s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca 2+ currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca 2+ channel knockout mice. we conclude that the p/q-type specific ca 2+ channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi 1 , koji ikegami 1 , ken hatanaka 1,2,3 , yoko fujiwara-tsukamoto 1 , mineo matsumoto 1 , ikuko yao 1 , mitsutoshi setou 1,2,4 1 mitsubishi kagaku institute of life sciences, japan; 2 presto, japan; 3 school of pharmaceutical sciences, the university of tokyo, japan; 4 okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa22(−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa22 mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki 1 , shingo kimura 1 , reiko fujita 2 , shuji watanabe 1 , kazuhiko sasaki 1 1 dept. of physiol., sch. of med., iwate medical univ., morioka, japan; 2 dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c3, a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p50rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦2 glutamate receptor (␦2r) plays a crucial role in cerebellar functions. although ␦2r has a putative channel pore domain, and ␦2r displayed ca 2+ -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦2r functions as a channel. here we introduced a ␦2r transgene, which had a mutation (gln618arg) in the putative channel pore conserved in ca 2+ -permeable glutamate receptors, into ␦2 −/− mice. surprisingly, a mutant ␦2r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦2 −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca 2+ influx through ␦2r is not required for its function in the cerebellum in vivo, and that wild-type ␦2r may not function as a ca 2+ -permeable ion channel. research funds: kakenhi (17700316) and takeda science foundation ps2p-a007 distribution of tarp -8 on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya 1 , mika tsujita 2 , maya yamazaki 2 , etsuko kushiya 2 , manabu abe 2 , kaori akashi 2 , masanobu kano 3 , haruyuki kamiya 4 , kenji sakimura 2 , masahiko watanabe 1 1 department of anatomy, hokkaido university school of medicine, sapporo, japan; 2 department of cellular neurobiology, brain research institute, niigata, japan; 3 department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; 4 department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the -8 is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized -8 on excitatory synaptic and extrasynaptic membrane. in -8-ko mice, ampars were reduced in hippocampal homogenates (46% of control) and psd fraction (35%). immunogold labeling also exhibited reduction of extrasynpatic (47%) and synaptic (35%) ampars in ca1 pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites (36%) and spines (38%). ampar-mediated responses were reduced at ca1 synapses (52%). therefore, -8 is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa 1 , yugo fukazawa 1 , elek molnar 2 , masahiko watanabe 3 , ryuichi shigemoto 1,4 1 div. cerebral structure, nips, okazaki, japan; 2 mrc, univ. of bristol, bristol, uk; 3 hokkaido univ., sapporo, japan; 4 sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed 1.7 to 3 times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed 1.6 to 3 times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda 1,2,3 , hitoshi kita 1 1 dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; 2 japan society for promotion of science, tokyo, japan; 3 dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp55845 abolished the sipsp. an mglur1 antagonist, but not an mglur5 antagonist, partially blocked the sdepo. in addition, cgp55845 enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur1-mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps2p-a010 essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto 1 , yoshikazu isomura 1,2 , michiko imanishi 1 , tomoki fukai 2 , masahiko takada 1 1 system neurosci., tokyo met. inst. neurosci., tokyo, japan; 2 neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for >20 s after the induction. gaba amediated cl-influx lasted for 1.5 s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura 1 , yuko sekino 1,2 , toshiya manabe 1,2 1 division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; 2 crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < 30), using electrophysiological methods and optical imaging. in immature animals (p10), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate (<p5) with a significant change in the paired-pulse ratio. the presynaptic effect of baclofen was gradually decreased with development (p2-18). in situ hybridization and immunohistochemistry experiment revealed gaba b r expression in mntb neurons was also gradually decreased. the property of mntb-lso synapses in gaba b r knock out mouse remained immature at p14-16 compared with control mouse. thus, gaba b receptor seems to play an important role in development of synaptic property. pyramidal cells in the hippocampal ca1 area receive gabaergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (ais). here, we used the sdsdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣1, ␣2, and ␤2/3 subunits quantitatively on distinct subcellular compartments of pyramidal cells. labelings for these subunits were accumulated over clusters of intramembrane particles (imp) on the protoplasmic face (p-face) of the plasma membrane, indicating that many imp clusters of a certain size represent gabaergic synapses. the synaptic labelling densities for gaba-a receptor subunits were not significantly different on the tested subcellular compartments. double labelling experiments showed that the majority of synapses contains ␣1, ␣2, and ␤2/3 subunits. in the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. therefore, we investigated the effects of the ␣ 1 -or ␣ 2 -adrenoceptor agonist on firing rates of presynaptic interneurons (ins). presynaptic cell-attached recordings showed that spontaneous activity of gabaergic ins was enhanced by the ␣ 1 -adrenoceptor agonist phenylephrine, while reduced by the ␣ 2 -adrenoceptor agonist clonidine. immunohistochemical studies showed that ␣ 1 -adrenoceptors were expressed in the molecular layer and the purkinje cell (pc) layer, while ␣ 2 -adrenoceptors were observed in cell bodies of pcs and ins. these results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ 1 -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of ins via ␣ 2 -adrenoceptors, which were located in the presynaptic in soma. thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of gabaergic inputs from ins to pcs. research funds: kakenhi (16700344) ps2p-b015 involvement of basal pkc and erk 1/2 activities in constitutiveinternalization of ampa receptors in cerebellar purkinje cells tetsuya tatsukawa 1 , takahiko chimura 2 , azumi matsumoto 2 , kazuhiko yamaguchi 2 1 lab. for motor learning control, riken, bsi, japan; 2 lab. for memory & learning, japan ampa receptor (ampar) internalization provides a mechanism for cerebellar ltd, which is the underlying basis of motor learning and motor coordination. however, the relationship between cerebellar ltd and constitutive ampar internalization in parallel fiber (pf)-purkinje cell (pc) synapses remains unclarified. in the present study, we demonstrated that tetanus toxin (tetx, a blocker of exocytosis mediated by vamp) infusion into pcs caused the rundown of pf-epsc amplitude through the constitutive ampar internalization, and then addressed question whether intracellular signals involved in cerebellar ltd induction modify the constitutive ampar internalization at pf-pc synapses using electrophysiological and immunocytochemical techniques. our results suggest that the regulation of actin polymerization is involved in the surface expression of ampars and the surface expressing ampars are constitutively internalized depending on both basal pkc and mek-erk1/2 activities at pf-pc synapses. taisuke miyazaki 1 , kohichi tanaka 2 , masahiko watanabe 1 1 department of anatomy, school of medicine, hokkaido university, sapporo, japan; 2 department of molecular neuroscience, tokyo medical and dental university, tokyo, japan glutamate released to the synaptic cleft has to be removed by glutamate transporter. in the cerebellum, glutamate transporter glast is richly expressed in bergmann glial cells (bg) that enwrap synapses, dendrites and somata of purkinje cells (pcs). in this study, anatomical analysis was applied to glast-deficient mice. in the mutant mice, lamellate processes from bg fibers became atrophic, resulting in incomplete sealing of synaptic cleft. furthermore, the distribution of climbing fiber (cf) terminals was decreased proximally. by anterogradely labeling, multiple cf innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. these results suggest that glast is essential for complete ensheathment of pc synapses and for the establishment of cf mono-innervation by suppressing local ectopic innervation between nearby pc dendrites. miwako yamasaki 1,2 , kouichi hashimoto 2 , taisuke miyazaki 1 , masahiko watanabe 1 , masanobu kano 2 1 dept. of anatomy, grad. sch. of med., hokkaido univ., sapporo, japan; 2 dept. of cellular neuroscience, grad. sch. of med., osaka univ., suita, japan the ␥ isoform of protein kinase c (pkc␥) is highly expressed in cerebellar purkinje cells (pcs), and its deficiency impairs the late phase of climbing fiber (cf) synapse elimination. in the present study, we analyzed multiple cf innervation in pkc␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of cfs. in general, cf innervation regressed proximally in the pkc␥-deficient mouse. in some mutant pcs, a considerable part of the proximal dendrites lost association with cf terminals. in the majority of multiply innervated pcs, single main cf innervated broad region of proximal dendrites, whereas surplus cfs innervated the somata and basal portion of the proximal dendrites. moreover, immunoelectron microscopy revealed synaptic contact of cf terminals to somatic spines. these results suggest that pkc␥ is crucial for strengthening innervation by a main cf and elimination of surplus cfs from proximal somatodendritc compartments of pcs. keiji imoto 1,2 , takashi kodama 1,2 , ryuichi shigemoto 2,3,4 , yugo fukazawa 2,3 , yasuo mori 5 1 division of neural signaling, nips, okazaki, japan; 2 school of life science, graduate university for advanced studies (sokendai), okazaki, japan; 3 division of cerebral structure, nips, okazaki, japan; 4 sorst, japan science and technology agency, kawaguchi, japan; 5 department of synthetic chemistry and biological chemistry, kyoto university, kyoto, japan a murine ataxic mutant, rocker, has a point mutation in the ca v 2.1 (p/q-type) ␣ subunit gene and shows the mildest phenotype among the series of ca v 2.1 mutants. although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-purkinje cell synapses was considerably impaired. we demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of ampa receptors in psd, whereas the presynaptic function remained normal. we also found the dendrites of purkinje cells showed poor arborization in rocker mice. these lines of evidence indicate that ca v 2.1 exerts its strong influence on postsynaptic maturation and dendritic structure. bai-chuang shyu, chia-ming lee, wei-chih chang institute of biomedical sciences, academia sinica, taipei, taiwan roc the aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the anterior cingulate cortex (acc) using a novel mouse brain slice preparation protocol. the mono-and poly synaptic responses in the acc were excited by direct thalamic activation. the synaptic transmission involves both ampa and nmda glutamate receptors. several thalamus-evoked responses were identified: the early negative component (n1) emerged in layer vi near cingulum. subsequently a positive component (p) appeared in layer vi. the second negative component (n2) became apparent in layers ii/iii and v. then the activities spread downward to layer vi and developed as n3 component in a medial-to-lateral direction. we confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. the thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the acc and this process was modulated extensively by glutamatergic synapses. ps2p-b020 target-dependent extracellular ca-dependence of the short-term plasticity of the solitary complex synapses in the rat lab. neurophysiol., dept. neurosci., jikei univ. sch. med., tokyo, japan the discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (sc) forms the primary filter of the visceral information. such frequency filtering of the synaptic transmissions from the solitary tract (ts) to neurons in the sc depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (ppr). here we analyzed the mechanism underlying distinct pprs among distinct types of synapses. in synapses from ts to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, ppr significantly increased from 0.4 ± 0.2 to 1.1 ± 0.2 (n = 6; mean ± s.d.) by reducing [ca 2+ ] o from 2 to 0.5 mm, whereas that in low-pass synapses from ts to the neurons in dorsal motor nucleus of the vagus was affected only slightly. these results suggest that the synaptic frequency filtering in the sc is under distinct control depending on the function of the postsynaptic target systems. ps2p-b021 response of spinal monosynaptic reflex to high frequency stimulation in newborn rat the susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. in an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (msrs) evoked by dorsal root stimulation, were mediated by both nmda and non-nmda glutamate receptors. msrs were constant in amplitude at 1/15 s, and were depressed completely by cnqx. when stimulus rate was increased to 1/s, msr amplitudes were greatly reduced initially, and recovered later. this recovery of msr was eliminated by apv. non-nmda component of msrs was eliminated completely at 1/s, but appeared in constant amplitudes in the presence of cyclothiazide. from these results, non-nmda glutamate receptor mediated almost most of msrs at 1/15 s, but not at 1/s due to desensitization. in contrast, nmda glutamate receptor does not mediate msrs, but activated at 1/sec, in place of non-nmda receptor. in conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission. kenichi ono 1 , keisuke shiba 2 , ken nakazawa 3 , ichiro shimoyama 4 1 department of otolaryngology, chiba university, chiba, japan; 2 department of otolaryngology, kimitsu central hospital, chiba, japan; 3 department of integrative neurophysiology, chiba university, chiba, japan; 4 section for human neurophysiology, research center for frontier medical engineering, chiba university, chiba, japan we determined synaptic source of the respiratory-related activity of laryngeal motoneurons (lms) using spike-triggered averaging of lm membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. in inspiratory (i) lms, monosynaptic epsps were evoked by spikes of i neurons with augmenting firing patterns, and monosynaptic ipsps were evoked by spikes of expiratory (e) neurons with decrementing firing patterns and of i neurons with decrementing firing patterns. in elms, monosynaptic ipsps were evoked by spikes of i neurons with decrementing firing patterns and of e neurons with augmenting firing patterns. we conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of lm membrane potentials. ps2p-b023 in-vivo gene transfer of exogenous protein to the primary afferent neurons chiaki yamada 1,2 , eiji shigetomi 1,3 , fusao kato 1 1 lab. neurophysiol., jikei univ. sch. med., tokyo, japan; 2 dept. basic biol. sci., kyoritsu univ. of pharm., tokyo, japan; 3 jsps, japan in order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. in the anesthetized young wistar rats, we injected pcaggs-egfp plasmid vector (through courtesy of drs. j. miyazaki and k. nakajima) into the nodose ganglion (ng) at the neck and transferred it by electroporation. two days after transfection, the ng was extirpated and fixed to observe egfp signals. a large portion of somata in the ng as well as the centrally and peripherally projecting afferent fibers expressed high levels of egfp with few damaged cells. this technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain. research funds: kakenhi (17650116, 176865) ps2p-b024 laminar sources of synaptic input to layer 1 neurons in rat visual cortex takuma mori, edward m. callaway salk institute, usa layer 1 of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. very little is known about connectivity of these cells with cells in other cortical layers. we examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer 1 neurons of rat visual cortex, using scanning laser photostimulation. we find that axon descending cells get excitatory synaptic inputs primarily from layer 2/3. neurogliaform cells receive more excitatory inputs from layers 4 and 5. the dominant inhibitory synaptic inputs onto both types of cells originate from layer 2/3. these neurons also get inhibitory inputs from layers 1 and 4-6. the presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer 1 are highly integrative. takako nishi 1 , tsukasa gotow 2 , shiro nakagawa 2 1 ins. nat. sci., senshu univ., kawasaki, japan; 2 dept. neurol., kagoshima univ. grad. sch. med.-den. sci., kagoshima, japan four extra-ocular photoreceptors, the photoresponsive neurons, a-p-1, es-1, ip-1 and ip-2 which respond directly to light, exist in the abdominal ganglion of the mollusc onchidium. a-p-1/es-1 of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other ip-1/ip-2. the present study was conducted to examine the functional significance of the above ip-1/ip-2, having a peak sensitivity at 510 nm light. the body wall of the animal transmitted enough 510 nm to hyperpolarize ip-1/ip-2 in the ganglion covered by the body wall. ip-1/ip-2 were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. ip-1/ip-2 were not coupled to a-p-1/es-1 one another. finally, ip-1/ip-2, the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera. ps2p-c026 cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken tatsuo sato, iwao fukui, harunori ohmori department of physiology, faculty of medicine, kyoto university interaural level difference (ild) and timing difference (itd) are the major two cues to localize the sound source, and are processed separately. although the details of itd processing are well investigated, not much is known about ild. in the barn owl, it is proposed that ild is calculated in lldp (posterior part of the dorsal lateral lemniscal nucleus). we confirmed already that in vivo lldp calculates ild in the chicken as well. in this work using the patch clamp recording with the brainstem slice preparation, we found that lldp neurons fire tonically under the regulation of two k + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. we are intending to clarify the cellular mechanism of ild calculation as the interaction of both excitatory and inhibitory synaptic inputs. to understand the functional organization of the cns, it is essential to know how sensory information is processed within the cns. we have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. in this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. stimulation applied to the olfactory nerve elicited epsp-related optical signals in the olfactory bulb from the 7-day old embryonic stage. the epsp was mediated by glutamate, and nmda-and non-nmda-receptor components were identified. in more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. the results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. yoko momose-sato, katsushige sato dept. physiol., tokyo med. dent. univ. sch. med., tokyo, japan in an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around e7. it is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. we found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. at early stages of development (e7-e8), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. as development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. the oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. we examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. akiyoshi shimada 1 , nyberg tobias 1 , nahoko kasai 1 , yuriko furukawa 2 , keiichi torimitsu 1,2 , kunihiko obata 3 , yuchio yanagawa 2,4 , tadaharu tsumoto 2,3 1 ntt basic research laboratories, ntt corporation, kanagawa, japan; 2 sorst, jst, saitama, japan; 3 neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; 4 department of genetic and behavioral neuroscience, graduate school of medicine, gunma university, gunma, japan in the immature stage of cerebral cortex, gaba a receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. cortical neurons of glutamic acid decarboxylase 67-green fluorescence protein (gad67-gfp) knock-in mice at postnatal day 0-1 were cultured on a microelectrode array. spontaneous firing of cortical neurons appeared within the first 5 days in vitro (div). when a gaba a receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least 14 div: increase, decrease, disappearance and no change. these results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of gaba a receptors. ps2p-c030 pathway specific signal modulation by purinergic receptors in the parallel processing system makoto kaneda 1 , toshiyuki ishii 2,4 , yasuhide shigematsu 3 , toshihiko hosoya 2 , yukio shimoda 3 1 dept. physiol., keio univ. sch. med., tokyo, japan; 2 bsi, riken, wako, japan; 3 mri, tokyo womens' medical univ., tokyo, japan; 4 dept. biomolecular science, univ. toho, funabashi, japan the retina divides visual information into on-and off signals, and processes them with independent subsets of neurons. so far no difference between these pathways has been found for neurotransmitter functions. we have previously found that p2x2-purinoceptors are selectively present on the off-type, but not on the on-type cholinergic amacrine cells of the mouse retina. we therefore asked if atp has different functions in the two pathways. as expected, only the off-type, but not the on-type, cholinergic amacrine cells responded to atp. this response was mediated by the p2x2-purinoceptors. furthermore, a p2x-purinergic receptor antagonist activated the tonic phase firing of the off ganglion cells, but not of the on cells. thus atp has off-pathway specific modulatory functions mediated by p2x-purinergic receptors. bhupesh mehta, gulnaz begum, preeti g. joshi department of biophysics, national institute of mental health and neuro science, bangalore, india cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic ca 2+ . these oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. the synchronized ca 2+ oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. these spontaneous oscillations are inhibited by activation of purinergic receptors. we observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists atp and utp, but an enhanced oscillatory pattern was observed when the action of 2mesatp was observed. the inhibition of 2mesatp response by p2y1 receptor specific antagonist mrs 2179 indicates that different subtypes of purinergic receptors play different role in the regulation of synchronized ca 2+ oscillations. data will be presented on the p2 receptor mediated regulation of synchronized ca 2+ oscillations in cultured hippocampal neurons. mahomi kuroiwa, akinori nishi department of pharmacology, kurume university school of medicine, kurume, japan presence of additional d1-like dopamine receptors that selectively couple to gq/phospholipase c (plc) has been proposed. in recent studies, skf83959 was shown to selectively stimulate plc-linked d1-like dopamine receptors. darpp-32 is a phosphoprotein that regulates the efficacy of d1 receptor signaling. activation of d1 receptors that couple to golf/pka signaling leads to darpp-32 phosphorylation at thr34. we investigated the effect of skf83959 on darpp-32 phosphorylation at thr34 by using mouse neostriatal slices. treatment of neostriatal slices with skf83959 increased thr34 phosphorylation. the effect of skf83959 was enhanced by a plc inhibitor, u73122. in contrast, the effect of skf83959 was partially blocked by a d1 receptor antagonist, sch23390, and the sch23390-insensitive increase in thr34 phosphorylation was completely abolished by an adenosine a 2a receptor antagonist, zm241385. thus, analysis of darpp-32 phosphorylation revealed that skf83959 activates at least three signaling cascades in the neostriatum: (1) plc, (2) d1 receptor/golf/pka, (3) adenosine a 2a receptors. ps2p-c033 thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus naoshi fujiwara division of medical technology, niigata university school of health sciences, japan effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. cortical and hippocampal slices of the c57bl6 mouse were dyed with a voltage-sensitive dye rh414. in cortical slices, electrical single-pulse stimulation to the layer v evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers ii-iii and widely expanded in these layers. the excitation propagation in the layers ii-iii was significantly inhibited by thiamylal (200 mol/l). in hippocampal slices, excitation propagation elicited by the same stimulation to the ca1 stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. on the other hand, an ampa/kainate receptor antagonist cnqx (20 mol/l) inhibited excitation propagation in both cortical and hippocampal slices. the results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus. research funds: grant-in-aid from jsps no.15390471 ps2p-c034 brain-derived neurotrophic factor regulating ampa receptor translocation to postsynaptic density through ip3r and trpc calcium signaling hiroko nakata, shun nakamura national institute of neuroscience, tokyo, japan the change in the number of postsynaptic ampa-type glutamatergic receptors (ampars) by neuronal activity is implicated in the excitatory synaptic plasticity. here, we showed that bdnf induced ampar translocation to postsynaptic site of the dissociated cortical pyramidal neurons. using calcium imaging, we located the spines that were activated by bdnf. with thus identified spines, we analyzed the ampar subunit glur1 localization to postsynaptic density (psd) by immunostaining. we found that bdnf increased the ratio of surface glur1 at psd within 20 min. bdnf selectively translocated ampar, but not nmdar. the essential signaling was ca 2+ transients evoked by the activation of trkb/plc␥ pathway. both the intracellular ca 2+ rise, that is, ca 2+ release from ip3r and ca 2+ influx through storeoperated cation channel trpc contributed to the increase of the surface expression of glur1 at psd. these results suggest an important role of bdnf in the regulation of ampar trafficking by spatial and temporal ca 2+ signaling regulation. research funds: health sciences research grant of nano-1 hiroyuki konishi 1 , kazuhiko namikawa 1,2 , keiji shikata 3 , yuji kobatake 1 , taro tachibana 3 , hiroshi kiyama 1 1 dept. anat. & neurobiol., osaka city univ., grad. sch. med., osaka, japan; 2 dept. anat., asahikawa med. col., hokkaido, japan; 3 dept. bioeng., osaka city univ., grad. sch. eng., osaka, japan activation of akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. in this study, we attempted to identify novel substrates for akt by using an antibody, which recognized phosphorylated consensus motif by akt. using pc12 cells, which overexpressed constitutively active akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. we then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. these results suggest that peripherin is a novel substrate for akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. the truncated trkb receptor, t1, is involved in the control of cell morphology via the regulation of rho proteins via rho gdi1. however, it is unclear whether t1 regulates the downstream of rho signaling and the actin cytoskeleton. in this study, we investigated this question using c6 rat glioma cells, which express t1 endogenously. rho gdi1 was dissociated from t1 in a bdnf-dependent manner, which also causes decreases in the activities of rho-signaling molecules such as rhoa, rock, pak and erk. moreover, bdnf resulted in the disappearance of stress fibers in the cells treated with lpa, an activator of rhoa, and in morphological changes in cells. furthermore, a competitive assay with cfp fusion proteins of t1-specific sequences reduced the effects of bdnf. these results suggest that t1 regulates the rho signaling pathways and the actin cytoskeleton. anjana kalita eukaryotic gene expression laboratory, national institute of immunology, new delhi, india jak and stat are activated in response to many cytokines and growth factors. this pathway has been primarily studied in non-neuronal origin cells. it has been shown that stat activated cellular transformation, occurs through transcription regulation of specific genes like c-myc, cyclin d1, bcl2. the present study was undertaken to identify target genes of jak/stat pathway in response to tnf-alpha & igf-1 in neuronal origin cells. we also looked at the mechanism of activation of these target genes. we saw that jak/stat pathway was involved in upregulation of cyclind1 in response to igf-1 both at protein & rna levels but no change was observed in c-myc, bcl2 levels. interestingly, mapk-erk, which is also known to activate cd1 in other systems, had no effect on the cd1 level in neuronal cells. on the other hand, pi3kinase not only activated jak/stat pathway but also affected on cyclind1 level. thus it appears that jak/stat is activated by pi3k, which in turn upregulates cyclind1 level. pi3k also activated erk without changing cd1 level, thus we show that jak/stat pathway is important for survival of neurons, activated by pi3k. cd1 is a one of the major downstream targets of jak/stat. hiroko inoue 1 , haruhisa kawasaki 2 , ritsuko fujii 2 , tohru yoshioka 2 , kazunori sango 3 , toshihiko kadoya 4 , hidenori horie 2 1 sch. of sci. and eng., waseda univ., tokyo, japan; 2 advanced res. center for biol. sci., waseda univ., tokyo, japan; 3 tokyo metropolitan inst. for neurosci., tokyo, japan; 4 pharmaceut. res. lab. kirin brewery co., ltd., gumma, japan oxidized galectin-1 (gal-1/ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. but, the mechanism how gal-1/ox stimulates macrophages remains to be clarified. in the present study with rt-pcr and western blotting, we searched for the molecules contributing to the mechanism. rt-pcr analysis with cultured rat peritoneal macrophages revealed that the application of gal-1/ox enhanced mrna expression of nerve regeneration-related factors such as il-6, il-1 ␤. however, those of gdnf, ngf, bdnf, nt-3, nt-4, igf-i, and igf-ii scarcely detected in control condition were not changed by the application of gal-1/ox. since il -6, il-1, and lif can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with gal-1/ox may enhance nerve regeneration after nerve injury. kennichi katou, taku iwamoto, satoshi kida dep. of bioscience, tokyo univ. of agriculture, japan ␣camkii has been known to play essential roles in synaptic plasticity. in response to increase in ca 2+ concentration, ␣camkii is activated by the interaction with ca 2+ /cam. prolonged increase in ca 2+ level leads to further activation of ␣camkii through autophosphorylation at t286. to understand the molecular dynamics of ␣camkii activation in vivo, we have tried to visualize and monitor the ␣camkii activity in various types of cells using fret. in hela cells, increase in ca 2+ level induces continuous interaction between ␣camkii and cam and conformational change of ␣camkii, indicating that the conformational change of ␣camkii mainly reflects the interaction with cam. in sh-sy5y cells, the increase in ca 2+ level induces only transient interaction between ␣camkii and cam but the conformational change of ␣camkii is maintained following the termination of the interaction with cam, suggesting that conformational change of ␣camkii is induced by the interaction with cam and then maintained by autophosphorylation. thus, mechanisms for regulation of ␣camkii activation is cell-type dependent. akifumi kamata, yusuke takeuchi, kohji fukunaga dept. pharmacol., grad. sch. pharmaceu. sci., tohoku univ., sendai, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is highly expressed in brain including dopaminergic neurons, however, the role of camkii in the dopaminergic neurons remains to be unclear. camkii consist of four isoforms, ␣, ␤, ␥ and ␦, and differential isoforms are implicated in the different neural functions. we have examined the isoforms of camkii expressed in rat substantia nigra (sn). rt-pcr and immunoblot analyses revealed that the ␥ and ␦ isoform mrnas including ␦3 isoform, a nuclear isoform of camkii, were predominantly expressed in sn. the immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the sn dopaminergic neurons, and immunoreactivity against anti-camkii␦1-4 antibody was detected in both nucleus and cytoplasm. furthermore, we defined expression of bdnf mrnas having the exon ii and iv in sn. taken together with our previous observation, the camkii␦3 isoform was involved in the expression of bdnf in sn dopaminergic neurons. ps2p-d041 the role of ca 2+ /calmodulin-dependent protein kinase ii in neuronal functions revealed by inactivated camkii␣ knock-in mouse yoko yamagata 1,2 , hiroyuki sakagami 3 , keiji imoto 1,2 , kunihiko obata 4 , yuchio yanagawa 5 1 okazaki, japan; 2 sokendai, okazaki, japan; 3 tohoku univ., sendai, japan; 4 riken, wako, japan; 5 gunma univ., maebashi, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. we engineered knock-in mice with the inactivated ␣ subunit of camkii by replacing k42 with r42 to study functional significance of camkii activity in intact animals. as expected, camkii activity was selectively reduced, while camkii subunit protein levels were comparable to those of wild type controls in homozygous mutants. in situ hybridization revealed normal expression patterns of camkii subunit mrnas, including dendritic localization of camkii␣ mrna. further analyses of these mice will be presented in the meeting. research funds: a grant-in-aid for scientific research from japan society for the promotion of science (kakenhi , #17500218) ps2p-d042 characterization of ca 2+ -dependent membrane binding proteins of rat brain shohei maekawa 1 , katsutoshi taguchi 1 , haruko kumanogoh 2 , shun nakamura 2 1 division of biosystems science, grad. sch. of sci/tech, kobe-u kobe 657-8501, japan; 2 division of biochemistry and cellular biology, national inst. neurosci. kodaira, tokyo 187-0031, japan ca 2+ -dependent membrane binding proteins were purified through a ca 2+ -dependent membrane binding and egta-induced membrane dissociation protocol from a crude triton solubilized membrane fraction. molecular characterization of these ca 2+ -dependent membrane binding proteins through lc-ms/ms identified annexin vii, annexin xi, and copin isoforms, in addition to annexin vi and neurocalcin␣. since the membrane fraction contains phosphatidylethanolamine (pe), cholesterol, and phosphatidylcholine (pc), the membrane binding activity of annexin vi, a major protein in this fraction, was studied using artificial liposomes. annexin vi showed a pe-dependent, but not cholesterol-dependent liposome binding activity. immunostaining of annexin vi in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. these results suggest the participation of annexin vi in the membrane cycling at the presynaptic region. ps2p-d043 generation of dominant negative mutants of transcription factor crest takuzou simo, kenichi kato, hiroshi hosoda, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan transcription factor calcium-responsive transactivator (crest) has been shown to be required for dendritic growth of neuron. previous studies have shown that crest contains two functional domains; multifunctional domain (mfd; aa251∼322) required for transactivation, nuclear localization and homodimerization and c-terminal transactivation domain (ctd; aa238∼402). to further understand the function of crest on brain function, we tried to generate the dominant negative mutant of crest to use mouse genetics study. indeed, we generated fusion proteins of gal4-dbd with mutants of crest; crest c ( 238∼402) lacking c-terminal transactivation domain, mfd and mfd-nls that is a fusion protein of mfd with nuclear localization signal (nls). from the experiments using gal4 one hybrid system, we found that mfd-nls shows the strong dominant negative effects on transcriptional activation by crest. ps2p-d044 characterization of iq-arfgef, a novel exchange factor for arf6, in mouse brain hiroyuki sakagami, hisatake kondo department of cell biology, tohoku university graduate school of medicine, sendai, japan adp-ribosylation factor 6 (arf6) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. to understand its neuronal functions, we have focused on guanine nucleotide exchange factors (gef) for arf6. in this study, we determined the entire sequence of a partially identified cdna (mkiaa0522) encoding a novel sec 7 domain. the deduced protein contained coiled coil, iq, sec7, plekstrin homology domains. interestingly, it contained a type i pdz domain-binding motif at its cterminal tail. arf pull-down assay demonstrated its ability to active arf6 more selectively than arf1. thus, we named this protein as iq-arfgef. in situ hybridization analysis demonstrated the preferential expression of iq-arfgef in the forebrain and cerebellar granule cells. furthermore, iq-arfgef mrna was localized not only in the hippocampal neuronal layers but also in their dendritic fields. our present findings suggested that iq-arfgef may play important roles in dendrites through activation of arf6 and interaction with various pdz proteins. eriko miura 1 , masahiro fukaya 1 , tokiharu sato 2 , kazushi sugihara 3 , masahide asano 3 , katsuji yoshioka 2 , masahiko watanabe 1 1 dept. anat., hokkaido univ. sch. med., sapporo, japan; 2 dept. mol. cell. biol., cancer res. inst., kanazawa univ., kanazawa, japan; 3 adv. sci. res. center, kanazawa univ., kanazawa, japan the c-jun n-terminal kinase (jnk) is one of the major mitogenactivated protein kinases (mapks), and jsap1 (or jip3) is a jnkassociated scaffold that controls the specificity and efficiency of jnk signaling cascades. here we studied its expression and distribution in mouse brains. jsap1 mrna was expressed in developing and adult brains, showing spatial patterns similar to jnk1∼3 mrnas. in embryos, jsap1 immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. in adults, jsap1 was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with ser and cell membrane. this wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to jnk mapk pathway and other intracellular machineries. yasukazu hozumi, kaoru goto department of anatomy and cell biology, yamagata university school of medicine, japan diacylglycerol kinase (dgk) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. to address the functional implications of dgk isozymes in the brain, we have raised specific antibodies against the isozymes. by immunoblot analysis, we found that the immunoreactive bands for dgk␤, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. immunohistochemical examination on rat brain revealed that dgk␤ was detected as dot-like structure in the cell membrane, and dgk␥ as round-shaped structure in the juxtanuclear region of neurons. dgk was detected in the perinuclear region and in the dendrites of purkinje cells. these data show that dgk isozymes localize differentially to the internal membrane system in neurons, suggesting that dgk isozymes play unique roles in distinct internal membrane system. kotaro hattori 1 , shigeo uchino 2 , tomoko isosaka 1,3 , shinichi kohsaka 2 , takeshi yagi 3 , shigeki yuasa 1 1 dept. ultrastractural res., nat. inst. neurosci., ncnp, kodaira, japan; 2 dep. neurochem., nat. inst. neurosci., ncnp, kodaira, japan; 3 kokoro biology group, fbs, osaka univ., suita, japan recently, we have found that fyn-mediated tyrosine-phosphorylation of nmda-r subunits is required for haloperidol-induced catalepsy. haloperidol induced catalepsy in the control mice, but such response was significantly reduced in fyn-deficient mice. fyn activation and enhanced tyrosine-phosphorylation of the nmda-r nr2b subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in fyn-deficient mice. both nr2b phosphorylation and the nmda-induced calcium responses increased after dopamine d 2 -r blockade in cultured striatal neurons of the control, but not in fyn-deficient neurons. accordingly, d 2 -r antagonist activates striatal fyn and the subsequent tyrosine phosphorylation of nr2b alters striatal neuronal activity, thereby inducing catalepsy. we also report further proteomics analysis on this molecular pathway. hiroaki okuda, shingo miyata, tsuya taneda, atsushi yamaguchi, shinsuke matsuzaki, natsuko kumamoto, masaya tohyama department of anatomy & neuroscience, graduate school of medicine, osaka university, osaka, japan the schizophrenia is a well-known debilitating psychiatric disorder. recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including dysbindin (dtnbp1: dystrobrevin binding protein 1) at 6p22.3. however it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. since, there is an important another factor, an environmental factor, for the schizophrenia. although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the n-terminal region of dys-bindin as a bait. as a result, we detected several transcriptional factors. thus, we further examine that these interactions regulate the expression level of particular genes in the neurons. ryota adachi 1 , naoko oda 2 , ryuzo shingai 1,2 1 21st coe program, iwate university, morioka, japan; 2 department of welfare engineering, faculty of engineering, iwate university, morioka, japan the response to environmental stimuli is important to live for organisms. the soil nematode caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. c. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. the sensory neurons detected these stimuli are in the head region. the neural network and genes required in each neuron are well known. however, it is under investigation about the integration of chemical and thermal stimuli. to understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. wild type worms cultivated at 10 • c showed decreased chemotaxis response to water-soluble chemicals and alcohol. alteration of assay temperature did not affect response to cl − but that to na + . to discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay. takashi takekawa 1 , masaki nomura 2,3 , toshio aoyagi 2 , tomoki fukai 1 1 riken brain science institute, saitama, japan; 2 graduate school of informatics, kyoto university, kyoto, japan; 3 crest, japan science and technology agency, kawaguchi, japan the neuron model proposed by izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. however, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. in fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. here, we study the phase responses of the izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks. honghai cui, hirotaka sakamoto, mitsuhiro kawata department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan the amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. in the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (sps) paradigm as ptsd (post-traumatic stress disorder) model. rats were perfused 7 days after sps, and intracellular injections of lucifer yellow were carried out in neurons of central (cea) and basolateral amygdala (bla). at the cellular level, we observed a significant increase of dendritic arborization in bla pyramidal neurons but no effect on the cea neurons. these results suggest that sps-induced structural plasticity of the bla provides a cellular substrate for affective disorders that are triggered in ptsd. takahiko kawasaki 1,2 , tatsumi hirata 1 1 division of brain function, national institute of genetics, sok-endai, mishima, japan; 2 prest, kawaguchi, japan in macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (mob) and the accessory olfactory bulb (aob), respectively. the secondary projections from the mob and aob first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. therefore, we investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of mob and aob neurons. ps2p-d053 an essential role for click-iii/camki␥ in regulation of neurite extension natsumi ishihara, sayaka takemoto-kimura, hiroyuki okuno, haruhiko bito dept. of neurochem., univ. of tokyo, tokyo, japan click-iii/camki␥, a membrane-anchored camk, is a novel member of the camki family that acts downstream of camkk. in order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged click-iii was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. phenotypic studies in pc-12 cells expressing various mutants of camkk and click-iii revealed that depolarization-induced activation of camkk-click-iii signaling is sufficient to robustly induce neuritogenesis. interestingly, click-iii localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. furthermore, lipidification mutants of click-iii were impaired in their ability to facilitate activity-dependent neuritogenesis. our results thus indicate a novel role of click-iii in activity-dependent neurite formation and extension. takuro maruyama 1 , masahiro matsuura 2 , nobuhiko yamamoto 1 1 grad. sch. of frontier biosci., osaka univ., suita japan; 2 riso kagaku corp., tsukuba japan during development, thalamocortical (tc) axons invade into the cortex and stop growing in layer 4. to study tc axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. the dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered fc-tagged extracellular domains of several candidate molecules. after four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-a5, sema7a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. a tendency for tc axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. these results suggest that ephrin-a5, sema7a and kit ligand may contribute to regulating tc axonal growth in the developing cortex. research funds: kakenhi 17023030, kakenhi 15300107 ps2p-e055 expression pattern of the guidance molecules and their receptors during gnrh neuron migration from the nose to the ventral forebrain shizuko murakami 1 , katsuhiko ono 2 1 dept. anat., juntendo univ. sch. of med., tokyo, japan; 2 div. of neurobiol. and bioinfo., nat. ins. for physiol. sci., okazaki, japan using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of gnrh neurons of chick embryos. netrin-1 expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. a few gnrh neurons expressed netrin-1 receptor neogenin. slit-1 was expressed in the forebrain, but its receptor robo-1 was not expressed in gnrh neurons. semaphorin (sema) 3a expression was detected in the olfactory-forebrain region. within the forebrain, sema3a was absent in the restricted region of the medial forebrain where gnrh neurons migrated in association with a subset of olfactory fibers. at the end of this pathway, sema3a expression was observed in the caudal forebrain, and then gnrh neurons changed their course ventrally. many gnrh neurons expressed sema3a receptor neuropilin-1, suggesting that sema3a may act as a repellent during the migration of gnrh neurons. kazuto fujishima 1,2 , qing-hua jin 1 , junko kurisu 1 , tomoo hirano 2 , mineko kengaku 1 1 riken, bsi, lab. for neural cell polarity, saitama, japan; 2 department of biophysics, graduate school of science, kyoto university, kyoto, japan during development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. dendrite morphology is also regulated by cell-cell contacts with neighboring cells. recent studies have implicated notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. we previously showed that delta/notch-like egf-related receptor (dner), neuron-specific transmembrane protein, acts as a notch ligand and mediates neuron-glia interaction during cerebellar development (eiraku et al., 2005) . dner is expressed in the dendrite of developing cortical pyramidal neurons. here, we analyzed the role of dner in the development of pyramidal neuron dendrites. we quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and dner knock out mice. reelin signal controls neuronal migration in the developing brain. because the binding of reelin to apoer2/vldlr induces dab1 phosphorylation, dab1 is a critical component of reelin signal. recent studies suggested that reelin signal is involved not only in neuronal migration but in other aspects of neural development. we investigated a possible function of the reelin signal in the filopodia and neurite formation. administration of reelin protein increased number of filopodia in a lesser extent than bdnf treatment. pp2 suppressed effects of reelin and bdnf on filopodia formation, suggesting that both signal pathways involve src kinase function. in spite of these similarities of reelin and bdnf functions in the filopodia formation, bdnf did not phosphorylate dab1. reelin treatment of the neurons derived from dab1 deficient mice did not induce significant increase in the number of filopodia. these results suggest that the filopodia and neurite formation involves the reelin-dab1 signal pathways. hidenobu mizuno 1,2 , tomoo hirano 1,2 , yoshiaki tagawa 1,2 1 dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan; 2 crest, jst, kawaguchi, japan the left and right hemispheres are interconnected via the corpus callosum. in mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area 17/18 border of the other hemisphere, where they terminate in layers 2/3 and 5. we used in utero electroporation-mediated gene transfer of gfp to assess distribution of callosal projections during development. gfp-labeled callosal axons arrived at the 17/18 border and started to invade the cortical plate around p5. they generated exuberant axonal arborizations by p13 then underwent remodeling to form the adult pattern by p15. neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. exogenous expression of a potassium channel kir2.1 in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers 2/3. area-specific targeting to the 17/18 border was not affected. results suggest that certain stages of callosal axon development require neuronal activity in cortex. ps2p-e060 identification of a novel tetraspan membrane protein, nplp, that is up-regulated after the facial nerve axotomy chihiro akazawa, yoh sasaki, masato hoshi, yasuko nakamura, shinichi kohsaka department of neurochemistry, national institute of neuroscience, ncnp, japan facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. to identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cdnas expressed in the injured facial nucleus. we identified a novel putative tetraspan protein designated as neuronal pmp22like protein (nplp) that has a distant homology to pmp22/claudin family proteins. the mrna expression of nplp was detected on the cell bodies of neurons meanwhile mrna of pmp22 resided on the cell bodies of schwann cells. the nplp mrna level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. nplp has a large extracellular domain that may interact with other membrane molecules. overexpression of nplp showed the microspike formation in pc12 cells. our results suggest that nplp plays an important role in the regeneration of injured motor neurons. shinsuke shibata 1 , shin-ichi sakakibara 2 , hirotaka j okano 1 , hideyuki okano 1,3 1 dept. physiol., keio univ., sch. med., japan; 2 dept. histol. neurobiol., dokkyo med. univ., japan ; 3 crest-jst, japan musashi (msi) is an evolutionarily conserved rna-binding protein. drosophila-msi regulates asymmetric cell division in neural development. in mammalian cns we identified two members, msi1 and msi2, which are co-expressed in neural precursor cells including neural stem cells, suggesting msi proteins regulate the immaturity of cns stem cells. to reveal the roles of msi family in vivo, we generated msi1 and msi2 deficient mice. msi1−/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. msi2−/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of pns. msi1−/− msi2−/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. in vitro and in vivo analysis show msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian cns and pns development. youhei koshimizu 1 , satoshi yamagoe 2 , kazuo suzuki 2 , michiko ohtomi 1 1 department of biomolecular science, graduate school of science, toho university, chiba, japan; 2 department of bioactive molecules, national institute of infections diseases, tokyo, japan we have previously suggested that lect2 (leukocyte cell-derived chemotaxin 2) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. however, the mechanisms of these regulations have not yet been clarified. in this study, we carried out multi-immunostaining against lect2 and tau or map2 in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of lect2 in neurons. at the early stages in cultured neurons, lect2 was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. in tissues of adult mouse brain, lect2 was localized to soma, axons and dendrites in neurons. these results suggest the possibility that lect2 influences the extension of dendrites and axons from the early stage of neuritic development. furthermore, it is thought that lect2 may regulate the dendritic and axonal morphology in mature neurons. mitsuru noda 1,2 , takahiro moriya 1 , hideyuki terazono 1 , suguru kudo 5 , masahiro yamaguchi 3 , kenji yasuda 4 , izumi nagata 2 , kazuyuki shinohara 1 1 div. neurobiol. & behav., nagasaki univ. grad. sch. biomed. sci., nagasaki, japan; 2 dpt. neurosurg. radiol. nagasaki univ. nagasaki, japan; 3 dpt. life sci., grad. sch. arts & sci., univ. tokyo, tokyo, japan; 4 dpt. physiol., grad. sch. med., univ. tokyo, tokyo, japan; 5 special division for human life technology, national institute of advanced industrial science and technology (aist), japan neural stem cells (nscs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of bdnf. the individual nscs from e12.5 nestin-promoter gfp transgenic mice were placed into 32 pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of bdnf. we observed that a part of nscs exhibited neuron-like morphology and extended some neurites. the application of bdnf increased the rate of neurite outgrowth. thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of bdnf. ayumi kyuka, ryoichi yoshimura, yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan in order to investigate the relationship between neurite outgrowth and target cells, ng108 cells were co-cultured with vascular smooth muscle cells (sm-3). sm-3 cells promoted the neurite outgrowth, but repelled the contact of their growth cones. the dna microarray analysis indicated that neuropilin-1 mrna was specifically up regulated in ng108 cells. but the western blotting analysis did not show the significant increase of neuropilin-1. sema3a, a candidate of ligands for neuropilin-1, was found in ng108 cells rather than in sm-3 cells. the immunocytochemical analysis revealed that neuropilin-1 was increased in the surface of ng108 cells co-cultured with sm-3 cells or cultured with the conditioned medium of sm-3 cells. these results suggest that neuropilin-1 may be transferred from cytoplasmic pool to surface of ng108 cells by some soluble factors released by sm-3 cells. we will discuss about kinetics of neuropilin and chemorepellents. yoshikuni edagawa 1 , j. nakanishi 2 , h. hamada 1 , y. yokomachi 1 , k. yamaguchi 3 , n. takeda 1 1 inst. biomed. eng., waseda univ., tokyo, japan; 2 riken, japan; 3 kanagawa univ., japan we show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of uv light. this molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. for the neurite outgrowth, pc12 cell was recruited as a model of neuron. by irradiating uv light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ecm. to draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. ngf stimulated each pc12 cell to extend a neurite along the narrow path: the elongation required both photo-activated path and ngf. the successive irradiation in front of developing neurite achieved bending or branching of the neurite. these results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system. tetsuhiro kakimoto, hironori katoh, manabu negishi lab. of molecular neurobiology, grad. sch. of biostudies, kyoto univ., kyoto, japan n-wasp is important for actin polymerization and is strongly expressed in brain. toca-1 was shown to be required for cdc42 to activate n-wasp in vitro, although its physiological role is unknown. here we studied neural function of toca-1. toca-1 is strongly expressed in developing brain. knockdown of toca-1 in pc12 cells significantly enhances neurite elongation. consistently, overexpression of toca-1 suppresses neurite elongation through its amino terminus including the f-bar/efc domain, which induces plasma membrane invagination. in addition, knockdown of n-wasp also enhances neurite elongation in pc12 cells, in clear contrast to the previous report using dominant negative mutants. on the other hand, knockdown of toca-1 in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of n-wasp enhances both axon elongation and branching. these results suggest that a vesicle trafficking regulating protein toca-1 regulates different aspects of neuronal morphology from n-wasp. research funds: kakenhi (17079003) ps2p-e067 pragmin, a novel effector of rnd2 gtpase, stimulates rhoa activity and regulates neurite outgrowth hironori katoh, hiroko tanaka, manabu negishi kyoto university, japan the rho family small gtpase rnd2 is specifically expressed in brain. although rnd1 and rnd3 display an antagonistic action for rhoa, signaling pathways of rnd2 are not well known. here we have performed a yeast two-hybrid screen using rnd2 as bait and identified a novel rnd2-effector protein, pragmin (pragma of rnd2). pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. in in vitro and in vivo binding assays, pragmin specifically bound to rnd2 among the rho family gtpases in a gtp-dependent manner. rnd2-bound pragmin significantly stimulated rhoa activity and induced cell contraction through rhoa-and rho-kinase-dependent pathway in hela cells. in pc12 cells, expression of pragmin inhibited the ngf-induced neurite outgrowth in response to rnd2, and knockdown of pragmin by a pragmin-specific small interfering rna enhanced the neurite elongation. therefore, these results suggest that rnd2 controls neurite outgrowth by regulating rhoa activity through pragmin. ps2p-e068 regulation of growth cone motility by collapsin response mediator protein-2 (crmp-2) masumi iketani 1 , yanai shigeki 1 , fumio nakamura 1 , yoshio goshima 1,2 , kohtaro takei 1,2 1 dept. of mol. pharmacol. & neurobiol., yokohama city univ. grad. sch. of med., yokohama, japan; 2 crest, japan sci. & tech. agency, kawaguchi, japan nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. collapsin response mediator protein-2 (crmp-2) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. the local functions of crmp-2 within growth cones, however, remains unclear. chromophoreassisted laser inactivation (cali) that can inactivate selectively a target protein with high spatial and temporal resolutions. acute localized loss of function of crmp-2 in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. asymmetric inactivation of crmp-2 in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. these findings suggest that a spatial distribution of crmp-2 activity within the growth cone can regulate growth cone motility and direct neurite outgrowth. research funds: crest, jst ps2p-e069 eph receptors are negatively controlled by protein tyrosine phosphatase receptor type o takafumi shintani 1,2 , masaru ihara 1,2 , hiraki sakuta 1,2 , hiroo takahashi 1,2 , ikuko watakabe 1 , masaharu noda 1,2 1 division of molecular neurobiology, national institute for basic biology, okazaki, japan; 2 crest, jst, kawaguchi, japan eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (ptps) responsible for the negative regulation of eph receptors have not been elucidated. here, we identified protein tyrosine phosphatase receptor type o (ptpro) as a specific ptp that efficiently dephosphorylates both epha and ephb receptors as substrates. using the retinotectal projection system, we show that ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections. ps2p-e070 neogenin acts as a displacement factor that releases rho from rho-gdi katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan repulsive gudiance molecule (rgma) is a potent inhibitor of axon regeneration in the adult central nervous system. rgma inhibits mammalian cns neurite outgrowth by a mechanism dependent on activation of the rho/rho-kinase pathway. here we show that direct interaction of the rho gdp dissociation inhibitor (rho-gdi) with neogenin which is a receptor of rgma initiates the activation of rhoa, and this interaction between neogenin and rho-gdi is strengthened by rgma. we also found that neogenin facilitates the release of prenylated rhoa from rho-gdi. thus, understanding of the molecular mechanisms of rgma may be useful for developing methods to overcome the inhibitory signals. nobuyuki fukushima, masumi nakashima, ryou tokunaga, ryutaro moriyama department of life science, kinki university, higashiosaka, japan lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through g protein-coupled lpa receptors, including lpa 1 and lpa 2 . here we examine how lpa 3 , third lpa receptor subtype, is involved in neuronal development by using pheochromocytoma 12 (pc12) cells. pc12 cells expressed low levels of endogenous lpa 3 mrna. when pc12 cells were infected with retrovirus expressing lpa 3 and treated with nerve growth factor under serum-free conditions for 3 days, neurite formation was observed in 55% of total infected cells, which was similar to that in control virus-infected pc12 cells. however, the mean length of neurites was increased in pc12 cells expressing lpa3, compared with that in control pc12 cells. interestingly, multiple neurite sprouts were frequently observed in lpa-treated lpa 3 -expressing cells. indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. these results indicated that lpa 3 was involved in neuritogenesis and branch formation. masao sakurai 1 , tomoko aoki 1 , kyoko ishikawa 1 , shingo yoshikawa 2 , john b. thomas 2 , chihiro hama 1 1 cdb, riken, hyogo, japan; 2 salk institute, usa in drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (al). each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (orns) and projection neurons (pns). in an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified wnt5 as a regulatory factor for glomerular patterning. in the wnt5 mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by wnt5 expression in orns. previous studies in embryos have shown that wnt5 repels the axons expressing derailed (drl). in the als of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the wnt5 mutant. this and other genetic data suggest that drl is an antagonist for wnt5 signaling. we propose that wnt5 signal transmitted from orns to pns provide one of mechanisms for glomerular patterning in drosophila. ps2p-e073 a homeodomain transcription factor, homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the drosophila brain mai ando, yoko totani, katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan the precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. we are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. marcm mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. currently, we are looking for genes that are either downstream or work cooperatively with hth. based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons. ps2p-e074 differential microarray analysis of mushroom body transcripts using chemical ablation ayako ino 1 , masatomo kobayasi 1 , taiki nakajima 1 , lydia michaut 2 , indrayani ghangrekar 1 , kuniaki takahashi 3 , ryu ueda 3 , kaoru saigo 4 , walter j. gehring 2 , katsuo furukubo-tokunaga 1 1 grad. sch. life envir. sci., univ. tsukuba, japan; 2 biozentrum, univ. of basel, switzerland; 3 genet. strains res. ctr., natl. inst. genet.; 4 dept. biophys. biochem. grad. sch. sci., univ. tokyo, japan mushroom bodies (mbs) are the centers for olfactory associative learning in the drosophila brain. we have surveyed mb transcripts using a drosophila whole-genome microarray and identified 1,465 genes that exhibited reduced expression levels with pharmacological mb ablation. among the identified genes, we have further selected 102 genes that exhibited a most consistent reduction of expression levels in the ablated brains. in situ hybridization analyses confirmed preferential mb expression patterns for the majority of the identified genes. moreover, we also demonstrate that rnai of many of the identified genes causes mild to strong mb defects. these results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the mb neurons in brain development and plasticity. mikiko inaki 1 , yoshie suzuki 1 , hiroyuki aburatani 2 , akinao nose 1 1 dept. phys., univ. tokyo, tokyo, japan; 2 rcast, univ. tokyo, tokyo, japan in drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. to understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. we focused on two neighboring muscles, 12 and 13, which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. by comparing gene expression data from the two muscles, we identified ∼170 genes with more than two-fold difference in expression level. for 26 candidate genes out of 33 (78%), the differential expression was confirmed by in situ hybridization and/or quantitative pcr. we focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. ectopic expression and/or knockdown of some of them altered the target specificity of muscles 12 or 13, implicating them in neuronal target recognition. makoto aoki, hiroshi segawa, mayumi naito, hitoshi okamoto laboratory for developmental gene regulation, brain science institute, riken, saitama, japan the sensory neurons have two axons, the central and peripheral axon. previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the lim domains of islet-2. to find the molecules which regulate the formation of peripheral axons of sensory neurons in the islet-2 signaling cascade, we have created cdna library from trigeminal sensory neurons of zebrafish larvae. most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. most of them show cns or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the lim domains of islet-2. loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry. hiroshi yamamoto, kiyokazu agata development of biophysics, graduate school of science, kyoto university, kyoto, japan highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. they act through receptors of the deleted in colorecal cancer (dcc) and unc5 receptor families. freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. the planarian central nerve system (cns) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. we have identified eight netrin related genes from planarians, dugesiajaponica, including four different netrin homologues and three unc5 homologuesand one dcc homologue. we analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted rnai experiments to investigating their functions in the process of regeneration of visual neurons. here we will summarize these results and speculate their molecular actions during eye regeneration in planarians. ps2p-e078 analysis of the significance and mechanism of neurite elimination using c. elegans as a model yu hayashi 1 , takaaki hirotsu 2 , eriko kage 1 , hideaki takeuchi 1 , hirofumi kunitomo 3 , yuichi iino 3 , takeo kubo 1 1 dept. biol. sci., grad. sch. sci., univ. tokyo, tokyo, japan; 2 dept. biol., fac. sci., kyushu univ. grad. sch., japan; 3 mol. genet. res. lab., univ. tokyo, japan during brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. we previously showed that elimination of neurites occurs in the nematode c. elegans and that a novel transcription factor mbr-1 is involved in this process. in the present study, we attempted to clarify the significance of this phenomenon. the mbr-1 mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. by executing cell-specific rescue experiments, we suggest that mbr-1 functions in a set of interneurons involved in olfactory processing. we are now examining whether neurite elimination occurs in these neurons using gfp reporters. we expect that our research provides a useful model for genetic dissection of neurite elimination. taro asakura 1 , ken-ichi ogura 1 , yoshio goshima 1,2 1 dept. of mol. pharmacol. and neurobiol., yokohama city univ., grad. sch. of med., yokohama, japan; 2 crest, japan sci. and tech. agency, kawaguchi, japan netrin is an evolutionary conserved axon guidance molecule. in c. elegans, netrin/unc-6 is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. one of the neurons guided by netrin/unc-6 is the hsn neuron. the hsn neuron of c. elegans has interesting axon guidance. netrin/unc-6 is required for the first ventral growth of the hsn neurons. however, what molecules participate in the axon guidance is largely unknown. in this study, we found that the vulval precursor cells strongly expressed netrin/unc-6 during the axonal guidance of the hsn neurons. silencing of netrin/unc-6 only at the vulval precursor cells resulted in abnormal axon guidance of hsn. in addition, expression of netrin/unc-6 only at the vulval precursor cells in unc-6 null mutants partially restored the hsn guidance defects. these results suggest that netrin/unc-6 expressed in vulval precursor cells plays essential roles on axon guidance of the hsn neuron. tomomi sato, takanori hamaoka, hidenori aizawa, hitoshi okamoto brain science institute, riken, saitama, japan the optic tectum is a visual center in vertebrates. it receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. here, we established a stable transgenic line tg(brn3a-hsp70:gfp) in zebrafish to visualize the retinotectal and the tectobulbar projections. to explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxp and gal4/uas systems. using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. in addition, we showed that ephrinb2a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere 2. these results suggest a neuroanatomical and a molecular basis for the motor map in the tectum. research funds: grant-in-aid for young scientists (b) 16700296 masahiko taniguchi 1 , yoshinori mikami 2 , tomoyuki masuda 3 , tomoyuki yoshida 2 , naoto matsuda 2 , masayoshi mishina 2 , takao shimizu 4 1 department of biochemistry, cancer research institute, sapporo medical university, sapporo, japan; 2 department of molecular neurobiology and pharmacology, graduate school of medicine, university of tokyo, japan; 3 department of anatomy, fukushima medical university, japan; 4 dapartment of biochemistry and molecular biology, faculty of medicine, university of tokyo, japan semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. here we report the identification of zebrafish semaphorin 6d (sema6d). the orf of sema6d is 3054 bp (1018 aa). zebrafish sema6d protein showed a 70.8% identity with mouse sema6d protein. to clarify the expression pattern of sema6d, in situ hybridization was performed. in the embryos, sema6d is expressed in the lens and hindbrain. we also found that sema6d has the repulsive activity. these results indicate that sema6d repels specific types of the axons and functions in the nervous system development. research funds: kakenhi on priority areas from the mext (17023013) mayumi yamada 1 , shohei maekawa 2 , seiji miyata 1 1 department of applied biology, kyoto institute of technology, kyoto, japan; 2 division of bioscience, graduate school of science and technology, kobe university, kobe, japan obcam belongs to the immunoglobulin superfamily cell adhesion molecule (cam) and was synapse-specific cam in cortical and hippocampal neurons in vivo. however, it is uncertain how obcam is involved in synaptic functions. in this study, we showed that obcam was highly concentrated on spines of cultured mature neurons. the inhibition of obcam function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. the suppression of obcam mrna expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. in contrast, overexpression of obcam augmented the formation of synapses. moreover, obcam was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. these results suggest that obcam, a spine-specific cam, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity. ps2p-f083 semaphorin 4d inhibits ␤1 integrin activity through the r-rasgap activity of plexin-b1 izumi oinuma, hironori katoh, manabu negishi lab. mol. neurobiol., grad. sch. biost., kyoto univ., kyoto, japan plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. we have recently reported that the semaphorin 4d (sema4d) receptor, plexin-b1 functions as a gap for r-ras, a member of ras family gtpases implicated in regulation of integrin activity and cell migration. here we characterized the role of r-ras downstream of sema4d/plexin-b1 in cell migration. activation of plexin-b1 by sema4d suppressed the ecm-dependent r-ras activation, r-ras-mediated pi3-k activation, and ␤1 integrin activation through its r-ras gap domain, leading to inhibition of cell migration. in addition, inactivation of r-ras by overexpression of the r-ras-specific gap or knockdown of r-ras by rna interference was sufficient for suppressing ␤1 integrin activation and cell migration in response to the ecm stimulation. thus, we conclude that r-ras activity is critical for ecm-mediated ␤1 integrin activation and cell migration, and that inactivation of r-ras by sema4d/plexin-b1-mediated r-ras gap activity control cell migration by modulating the activity of ␤1 integrins. shun hamada 1 , emi fukuda 2,3 , shota katori 2,3 , takeshi yagi 2,3 1 dept. nutrition health sci., fukuoka women's univ., fukuoka, japan; 2 grad. sch. frontier biosci., osaka univ., osaka japan, 3 crest, jst, japan cnr/protocadherin (pcdh)␣ is a subfamily of the clustered pcdhs that comprise >50 cadherin-related molecules and are encoded by three gene clusters (␣, ␤ and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking 34 or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga1 is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga1 is expressed by layer 2/3 neurons throughout the neocortex at p7 mice, but is absent in adults. between e15.5 and late p0, stages that span the generation and radial migration of layer 2/3 neurons, mdga is expressed in patterns consistent with its expression by migrating layer 2/3 neurons, suggesting a role for mdga1 in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e15.5 to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga1 protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga1 acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca1 pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on 7 days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on 14 div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day 2, but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps2p-f088 analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono 1 , yoshiyuki kuroda 1 , fumiko matsui 1 , yoshihito tokita 1 , keiko nakanishi 1 , michiru ida 1 , masahito ikawa 2 , masaru okabe 2 , katsuhiko ono 3 , atsuhiko oohira 1 1 institute for developmental research, aichi human service ctr., kasugai, japan; 2 research institute for microbial diseases, osaka university, suita, japan; 3 national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi (17500246) ps2p-f089 phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata 1 , tatsuhisa watanabe 2 , ikuko suzuki 1 , junichi kitagawa 1 , akiko ogawa 3 , kenro kanda 4 , kazunao kuramoto 5 1 dept. of physiol., sch. of dent., nihon univ., tokyo, japan; 2 dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; 3 dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; 4 shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; 5 division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats (30-34 months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura 1 , koichi obata 1 , masafumi sakagami 2 , koichi noguchi 1 1 department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; 2 department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk5 (also known as big mapk 1 or bmk1) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk5, mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days 1 and 3 after cfa injection. furthermore, time course of phosphorylated-erk5 level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk5 in drg neurons may contribute to the development of inflammatory pain. ps2p-f091 activation of erk5 in drg neurons contributes to acute pain toshiyuki mizushima 1,2 , koichi obata 1 , takashi mashimo 2 , koichi noguchi 1 1 department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; 2 department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p38 in dorsal root ganglion (drg) neurons. however, there has been no study examining erk5 (also known as big mapk 1 or bmk1) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk5 phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk5 in small-to-medium diameter sensory neurons with a peak at 2 min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk5 in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk5 (also known as big mapk 1 or bmk1) is activated in the drg and spinal cord and participate in pain-related behaviors in the l5 spinal nerve ligation (snl) model. l5 snl induced an increase in the phosphorylation of erk5 not only in the injured l5 drg, but also in the spared l4 drg at day 7 after surgery. furthermore, l5 snl induced a striking increase in erk5 phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk5 in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai 1 , minoru asada 1 , naoki seno 2 , hidenori suzuki 1 1 department of pharmacology, nippon medical school, tokyo, japan; 2 pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣-1, which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps2p-f094 interleukin-1␤ enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda 1 , jun kadoi 1 , msanori nasu 2 , masayuki takahashi 1 , shigeji matsumoto 1 1 dep. physiol and 2 res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il-1␤ paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il-1␤-immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il-1␤ induced depolarization in inflamed rats was larger than that in control rats. il-1␤ application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il-1␤ paracrine mechanism following peripheral inflammation. junichi kitagawa 1 , mamoru takeda 2 , jun kadoi 2 , yoshiyuki tsuboi 1 , shigeji matsumoto 2 , koichi iwata 1 1 dept. of physiol., sch. of dent., nihon univ., tokyo, japan; 2 dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day 3 after ion treatment and the threshold decrement was lasting more than day 14. single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day 3-14 after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for 90 min. at 6, 12, 24, 48 and 120 h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at 6 h, peaked at 12 h and then decreased. intense gfap immunoreactivities were also detected at 6 and 12 h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for 90 min at 25 m/min. after the running, rats were applied the weight on the gastrocnemius muscles for 30 min, and then spinal cord and soleus muscles were removed from the rats. productions of il-1beta, il-6 and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at 6 h post-running, number of fos-positive neurons was increased, peaked at 12 h and then decreased to control level at 24 h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at 2 days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo 1 , yusuke sakai 2 , aya masawaki 3 , takashi shimoda 2 , masayuki moritani 1 , motohide takemura 1 1 dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; 2 dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; 3 dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip 2 weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for 90 min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep3 receptor (ep3r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca 2+ ] i ) in a ptx-sensitive manner in cho cells expressing canine ep3r and mouse bk b2 receptor (b2r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk (1 nm) was applied twice with a 6-min interval to the cells expressing b2r, the second [ca 2+ ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h-89 (1 m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk (10 pm) treatment for 1 h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca 2+ ] i was at least in part mediated by pka. ps2p-f100 contribution of peripheral 5ht2a or 5ht3 receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral 5ht2a or 5ht3/r in male rats with tmj inflammation. here we tested if these two 5ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c2. fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c2 transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for 7 days compared to naive rats. we tested if local 5ht2a or 5ht3/r antagonist affects fos expression in both groups. the number of fos-li in the vc/c2 but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral 5ht2a and 5ht3/rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of 5-ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼60% of sg neurons examined, 5-ht induced an outward current. the outward current was mimicked and suppressed by a 5-ht 1a agonist and 5-ht 1a antagonist, respectively. in ∼10% of sg neurons, 5-ht evoked an inward current which was mimicked by a 5-ht 3 agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that 5-ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of 5-ht 1a and 5-ht 3 receptors, respectively. the reciprocal postsynaptic actions of 5-ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by 5-ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a␤ fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic 50 of bupivacaine and r-bupivacaine were almost equal on a␤, a␦ and c neurons. however, ic 50 of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a␤ neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps2p-g103 nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than 2 h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi (17700306) and km (16500262) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla 2 activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about 90% of neurons examined, melittin (1 m) superfused for 3 min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at 0 mv in the presence of bicuculline. this action was visible about 2 min after the beginning of its superfusion and subsided within 8 min after washout. these melittin actions were reduced in extent by a pla 2 inhibitor 4-bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla 2 activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi (17500275) ps2p-g105 presynaptic p2y 1 receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p2y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied 2-methylthio adp (2mesadp, 30 m), a p2y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about 20% of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by 2mesadp with no change in the amplitude. the 2mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs2179 (30 m), a selective p2y 1 receptor antagonist. these results indicate that the activation of presynaptic p2y 1 receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p2y 1 receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi (17700370), the japanese health sciences foundation (kh21006) ps2p-g106 presynaptic enhancement by proteinaseactivated receptor-1 agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)-1 but not par-2 agonist (each 1 m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par-1 mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par-1 agonist (sfllrn, 1 m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at -70 mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par-1 antagonist (yfllrnp, 1 m). these results indicate that the activation of par-1s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par-1 activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi (16700340) ps2p-g107 effect of tramadol metabolite m1 on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m1, which is one of its metabolites, at 1 mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m1 reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at −70 mv. this action was not seen in the presence of a -opioid receptor antagonist ctap (1 m). m1 also reduced the peak amplitudes of epscs which were monosynaptically evoked at −70 mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m1 inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m1 would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk-801 or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk-801 injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk-801. these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps2p-g109 studies on pain control system (rept. 57): changes in phosphorylation of nr2b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for 14 weeks. under these conditions, the immunoreactivities of phosphorylated-ser-1303 nr2b (p-ser-1303 nr2b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr-1472 nr2b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr2b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr2b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps2p-g110 prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, 100 hz, 20 a, 30 s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by 6-ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps2p-g111 the nav1.3 sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav1.3, a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav1.3. four weeks post-sci, nav1.3 protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav1.3 caused a significant reduction in nav1.3 expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda 1 , hiroyuki ichikawa 2 , ryuji terayama 2 , tomosada sugimoto 2 1 department of oral maxillofacial rehabilitation, okayama university, okayama, japan; 2 department of oral function and anatomy, okayama university, okayama, japan ib4-sap is a neurotoxin designed for targeting primary nociceptors with ib4 binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib4-sap (2.5 l of 0.067% solution for each ganglion) directly into the 4th and 5th lumbar (l4 and 5) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib4-binding. after counterstain, the cell body size of neurons were measured. ib4-sap reduced the total number of drg neurons in l4 and 5 ganglia combined by 21% (11652 ± 2213 on untreated side versus 9208 ± 1609 on treated side). small neurons (<1200 m 2 ) were reduced by 28% whereas large ones (≥1200 m 2 ) were not affected. ib4-binding neurons were mostly small (≥96%) and were reduced by 46%. the number of small neurons, that were not stained for ib4-binding, increased by 35% (1994 ± 286 versus 2700 ± 600). schuichi koizumi 1 , kaoru nasu-tada 1 , makoto tsuda 2 , emiko kunifusa 1,2 , kazuhide inoue 2 1 div. pharmacol., natl. inst. hlth. sci., tokyo, japan; 2 dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p2x4 receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p2x4 receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p2x4 receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p2x4 receptor in microglia, leading to neuropathic pain. research funds: mf16, kakenhi (170230570) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. 2004). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at 24 h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a␤-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi 17109015 ps2p-g115 lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. 2004). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at 1 mm decreased the protein levels of myelin basic protein (mbp) at 24 h. this action was significantly inhibited by botulinum neurotoxin/c3 (bont/c3). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp22, mag, p0 in cultured fibers. the maximal decrease was observed all at as early as 3 h after the addition of lpa. bont/c3 and y27632 abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at 0 and 3, 7, 14, 21, 28 days after operation. pre-emptive co-administration of mk-801 and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk-801 decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama 1,2 , naomi akiyama 1 , mikie hirata 1 , takayuki nakagawa 2 , shuji kaneko 2 , masabumi minami 1 1 dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; 2 dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a ␤-adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for 3 h per day between postnatal day 1 and 14. visceral sensitivities were assessed in stomach and colon at 10 weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research 16500216 ps2p-g119 change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered 1 to 3 days after exercise in rats (8 w old). c-fos expression in the superficial dorsal horn increased in l4 spinal segment when the edl muscle was compressed 2 days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats 8, 80 (81-84) and 130 (131-133) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in 80 w old rats, but not significant. after lec, the threshold started to decrease 1 day after lec in all three age groups. it returned to the pre-lec level 4 days after lec in 8 and 80 w old rats only. recovery of 130 w old rats delayed up to 7 days after lec. increased c-fos expression in the superficial dorsal horn was observed in l4 as well as in l5 in 130 w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal4 males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal4 males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama 1 , masaharu akita 2 1 kanagawa life-science res., japan; 2 kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day 11 to 13 of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days 25-39 of age, they did not show the first estrus before 52 days of age with mean age of 54.7 ± 2.0 day. in contrast, most of normal mouse igg-treated controls showed the first estrus by 43 days of age with mean age of 42.8 ± 2.2 day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss-1 gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days 29 of age (d29), and first estrus on around d34. brain tissues were obtained on d21, 26, 31, 36 and 41. kiss-1 mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d21 to 26 and was kept at a high level thereafter. gpr54 mrna expression in the medial preoptic area increased significantly from d21 to 31. metastin-immunoreactive cells were not found on d21 but were apparent in the arc-me on d26 onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito 1 , sei-etsu fujiwara 1 , kenjiro konno 2 , takashi yamaguchi 3 , tetsu nemoto 4 , etsuko kasuya 1 , ryosuke sakumoto 1 1 anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; 2 inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; 3 grad. sch. sci. & eng., yamagata univ., yonezawa, japan; 4 sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps2p-g126 hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi 1 , teruko uwano 1,3 , anh tran 2,3 , eturou hori 1,3 , taketoshi ono 2,3 , hisao nishijo 1 1 system emotional science, univ. of toyama, japan; 2 integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; 3 crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a 2 s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of 85 hf neurons, 26 responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue 1 , atsu aiba 2 , kaoru inokuchi 1 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 grad. sch. med., kobe univ., kobe, japan vesl-1s/homer-1a and vesl-1l/homer-1c are splicing isoforms encoded by the vesl-1 gene. vesl proteins bind and regulate mglur1/5, ip3 receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl-1s is upregulated by tetanic stimulation that elicits l-ltp. vesl-1s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl-1s gene-targeting mice (ko) and examined whether vesl-1s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl-1s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta 1 , r. migishima 1 , s. kida 2 , k. inokuchi 1,3 1 mitsubishi kagaku inst. life sci (mitils), japan, 2 tokyo univ. agricul., japan; 3 crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin ␤a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within 3 d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps2p-h129 sex differences in the effects of chronic estrogen treatment on fear conditioning in c57bl/6j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c57bl/6j mice were implanted with a silastic capsule containing 0, 5, 10 or 20 g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps2p-h130 analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested 48 h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for 2 weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps2p-h132 analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps2p-h133 analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor 1 (cb1) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb1 and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb1 or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb1 and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb1 and lvgccs, we also found that cb1 is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda 1 , takahiro furuta 1 , kouichi nakamura 1,2 , takeshi kaneko 1,2 ps2p-h136 difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama 1 , tomo unzai 1 , kouichi nakamura 1,3 , sakashi nomura 2 , takeshi kaneko 1,3 1 department of morphological brain science, kyoto university, kyoto, japan; 2 department of physical therapy, kyoto university, kyoto, japan; 3 crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut)2, thalamostriatal inputs, were less dense in patch region, whereas vglut1-positive corticostriatal inputs were evenly distributed. quantitative analysis revealed 84% of vglut2positive synapses in patch region were formed with spines, whereas 70% in matrix region were made with dendritic shafts. in contrast, the targets of vglut1-positive inputs were mainly spines in both regions. moreover, vglut2-positive axospinous synapses in patch region were larger than vglut1-positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi (16500217, 17022024,16200025, 17022020 (0131),17650100) ps2p-h137 single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai 1 , fumino fujiyama 1 , takeshi kaneko 1,2 1 department of morphological brain science, university of kyoto, kyoto, japan; 2 crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi (16500217, 17022024, 16200025, 17022020(0131) , 17650100) ps2p-h138 lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui 1 , kayo nishizawa 1 , nobuyuki kai 2 , kazuto kobayashi 2,3 1 dept. of psychology, fukushima univ., japan; 2 dept. mol. genet., fukushima medi. univ., japan; 3 crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of 6-ohda injections into the nucleus accumbens (acb) in c57bl/6j mice. all mice were trained to press the lever for a food pellet using a fixed ratio 5 (fr5) schedule. the mice were injected with ascorbate vehicle or 6-ohda into the acb, and then tested post-surgically using the fr5 schedule again. the 6-ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area 29 is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area 29, it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area 29 in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area 29d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area 29c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas 29a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area 29, suggesting different functional roles played by each subdivision of area 29 in spatial memory and behavior. eiichi jodo 1 , yoshiaki suzuki 2 , tadahiro katayama 1 , ken-yo hoshino 2 , yukihiko kayama 1 1 dep. of physiol., fukushima med. univ., fukushima, japan; 2 dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, 30%) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about 0.1 s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in 19% out of 438 vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only 6% out of 254 it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., 1994) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., 2005) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., 2005) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of 2d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of 75 sessions in phase 3, although they learned easily the four problems in phases 1 and 2. our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto 1 , noriyuki higo 1 , munetaka shidara 2 1 neuroscience research inst., aist, tsukuba, japan; 2 grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d2 receptor study using antisense cdna showed that d2 receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d2mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d2 receptor antipeptide antiserum. d2 receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area 36 of the perirhinal cortex than in area teav. the differential localization between area 36 and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano 1 , natsuko yoshida 2 , kiyohisa natsume 3 1 kyushu kyoritsu university, fukuoka, japan; 2 kyushu institute of technology, fukuoka, japan; 3 kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f3 and f4 was the highest and suggests that the theta rhythm at f3 and f4 may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after 6-month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi (1565081) ps2p-h146 neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe 1 , sho yagishita 1 , hideyuki kikyo 1,2 1 department of physiology, the university of tokyo school of medicine, tokyo, japan; 2 department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a 1.5 t scanner system was used (te = 50 ms; tr = 14 s; acquisition time = 3.0 s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong 1 , motoaki sugiura 3 , yuko sassa 2 , keisuke wakusawa 2,4 , kaoru horie 1,5 , shigeru sato 1,5 , ryuta kawashima 2,5 1 gsics, tohoku university, sendai, japan; 2 niche, tohoku university, japan; 3 miyagi university of education, sendai, japan; 4 department of pediatrics, school of medicine, tohoku university, japan; 5 the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz 1 , christie l. sahley 2 , kenneth j. muller 1 1 physio. & biophys., univ. of miami, miami, fl, usa; 2 biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps2p-h150 sensory/motor modules regulating the development of peer social relationship mamiko koshiba 1,2 , shun nakamura 2 1 jst, crest, japan; 2 ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata 1 , minoru hattori 2 1 department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; 2 graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) 30-s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi 30-s procedure over 30 days. the 3-s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the 30-s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi 30-s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps2p-i152 the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka 1,2 , ritz oshio 1,3 , norihiro sadato 1,4 , manabu honda 5,6 1 nips, okazaki, japan; 2 jsps, tokyo, japan; 3 nagoya univ., nagoya, japan; 4 ristex, jst, tokyo, japan; 5 sorst, jst, tokyo, japan; 6 ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps2p-i153 sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi3k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi3k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi (17500429) ps2p-i154 selective manipulation of working memory through d1 and d2 receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d1 and d2 receptors. the computer simulation with this model shows that d1 receptor activation mainly affect working memory activity itself, while d2 receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d1 and d2 receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or 25 mg/kg mam on the 15th day of gestation. the activity of male and female offspring was measured for each 1 h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every 1 min interval for 1 h under 5 min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca1 pyramidal layer and ectopic neuron mass). ps2p-i156 long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur1. hamid gholamipour 1 , shirin babri 2 , khameneh saied 3 1 department of physiology, university of tabriz, tabriz, iran; 2 university of tabriz, iran; 3 university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca1region of hippocampus on memory formation. fifty male rats were divided into five groups. (1) control (2) sham operation (3) test (4) diabetic/saline (5) diabetic/insulin. groups 4 and 5 were made diabetic by treatment with stz (50 mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca1 region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < 0.05). compared to sham group diabetic/insulin group showed increased latency (p < 0.05) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca 2+ /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk␤ is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk␤ null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk␤, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of 19 per 100,000 in india. we studied the park 4 polymorphism in north indian population and parkin expression in early parkinson's disease (n = 30) and sporadic parkinson's disease (n = 30). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon-1, 3 and 6 in early pd and exon-1 and 12 in sporadic pd, heterozygous mutations in exon 4 and 9 in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position 167 of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin 4 mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul 1 , kittisak sripanichkulchai 2 , david finkelstein 3 1 faculty of medicine, srinakharinwirot university, thailand; 2 faculty of medicine, khon kaen university, thailand; 3 university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c57bl/6 mice were pretreated with 17beta-estradiol (e2), injected with mptp on the day 6 and brains were collected on day 11. e2 pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e2 was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e2 neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps2p-i163 effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki 1 , fumihiko yoshino 2 , masaichi chang-il lee 2 1 dept. of food sci. and nutrition, showa women's univ., tokyo, japan; 2 clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi (17700577) ps2p-i164 recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi 1 , kaori sawada 1 , haruo okado 1 , atsushi nambu 2 , masahiko takada 1 1 tokyo met. inst. neurosci., fuchu, tokyo, japan; 2 natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype-2 raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type-1) of raav (raav1) carrying αs cdna (raav1-␣s), and a cocktail of raav1-␣s and raav1 carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav1-␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser129 residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with ␤-band local field potentials (␤-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged 70-80s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of 0.5-1 (i), 1.5-2 (ii), 2.5-3 (iii), and 3.5-4 (iv). the ␤waves were rated with total periods (%) of 13-27 hz wavelets in 3-s sample records. rigidity was re-scored after the surgeries. the vl ␤-waves were rated 65-70% in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about 55% in group iv. the pvp ␤-waves were 60-80%, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp ␤-waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana 1,2 , hirokazu iwamuro 1,3 , masahiko takada 4 , atsushi nambu 1,2 1 div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; 2 sokendai; 3 dept. neurosurg., univ. tokyo, tokyo, japan; 4 dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd44 expression on microglia and also enhanced the mt1-mmp expression to shed off cd44 at the cell surface and degrade surrounding ecm to open the migratory way. a53t mutant ␣-synuclein showed greater level of cd44 shedding and cell migration. extracellular treated ␣-synuclein also increased cd44 and mt1-mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant (2004 grant ( -2006 ps2p-j169 serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai 1 , hiromasa yamada 1 , yoshinari aimi 1 , ikuko nagatsu 2 1 department of anatomy, shiga university of medical science, otsu, japan; 2 fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of 6hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba 1-and tb4distribution in microglia were immunohistochemically investigated at 0 h and 7 days after twice mptp-treatments (one treatment comprised of 4 intraperitoneal injections of 20 mg/kg mptp at 2 h interval) to c57bl/6 and balb/c at 6 months (mo) interval. the recognizable change of iba 1-and tb4-distibution in microglia of both mice strains was observed even 6 mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps2p-j171 activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada 1 , ryohei hishida 2 , yoko hirata 3 , kenji ono 4 , hiromi suzuki 4 , shin-ichi muramatsu 2 , imaharu nakano 2 , kunihiro tsuchida 1 , toshiharu nagatsu 1,4 , makoto sawada 4 1 school of medicine, fujita health university, aichi, japan; 2 division of neurology, jichi medical university, japan; 3 department of biomolecular science, gifu university, japan; 4 research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the 60 week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps2p-j173 phosphorylation of erp57 in adult rat brain with neonatal 6-ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of 6-ohda at 3(p3) and 6 days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p56, tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the 6-ohda lesioned rats at p56, and it was identified as disulfide-isomerase a3 precursor (erp57) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp57 may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi (20 or 25 nm, 48 h) was applied to cultures at 7 days in vitro and succeeding changes of da neurons were investigated up to 35 days. more than 95% of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately 2 weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps2p-j175 specific expression of proapoptotic factor pag608 on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p53-activated gene 608 (pag608), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p53 expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag608 in the internal capsule of l-dopatreated and constitutive expression in the smi-32-immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag608 in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag608 expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag608 in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps2p-j176 an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang 1,2 , yuzuru imai 2 , haruhisa inoue 1,2 , ayane kataoka 2 , sachiko iita 2 , nobuyuki nukina 2 , ryosuke takahashi 1,2 1 neurology, university of kyoto, kyoto, japan; 2 bsi, riken, saitama, japan since loss of parkin e3 activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., 2004) have found that application of an antisense oligodeoxynucleotide specific for nr1 have successfully knockdown the expression of nr1 gene expression in the striatum of 6-hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr1 was re-addressed using a small interfering rna (sirna) specific for nr1. in pc12 cells, reductions of nr1 proteins after a single application of nr1 sirna were found by western blot experiments. and after one single application of nr1 sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr1 immunofluorescence were found in the striatum after the sirna treatments. lai et al., 2004. neurochem. int. 45, 11-22. research funds: faculty research grant, frg/04-05/ii-27, hong kong baptist university ps2p-j178 homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy (0.25-1 mol in 1 l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on 19th day. animals with 1 mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps2p-j179 ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca14 takahiro seki 1 , takayuki shimahara 1 , naoko adachi 2 , naoaki saito 2 , norio sakai 1 1 dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; 2 lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type 14 (sca14), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca14. however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca 2+ homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f9 cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately 300 compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki 1 , yo sasaki 2 1 department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; 2 washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase1 (nmnat1) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least 6 different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at 10 min, but next increased at 2 h and returned to basal at 6 h after cocaine challenge. actin showed a similar pattern but was maintained at high level at 6 h. both psd-95 and glur1 were increased between 2 h and 6 h like actin. by contrast, some proteins such as drebrin were decreased after the peak at 2 h. interestingly, the 20 s proteasome subunit demonstrated a dramatic upregulation at 2 h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps2p-k184 effects of mdma and 5-meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, 3,4methylenedioxymethamphetamine (mdma) and 5-methoxy-n,ndiisopropyltryptamine (5-meo-dipt), on sert and dat were examined. as previously reported, mdma (0.3-10 m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, 5-meo-dipt (0.3-300 nm) evoked an outward current response in sertexpressing cells similarly to that of selective 5-ht reuptake inhibitors. no current response was observed when 5-meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that 5-meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka 1 , nobue kitanaka 1 , tomohiro tatsuta 1,2 , yoshio morita 2 , motohiko takemura 1 1 department of pharmacology, hyogo college of medicine, nishinomiya, japan; 2 department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine (30 mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of 0.1 mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of 1 and 10 mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth (0.5 mg/kg, i.p.), pretreatment with subchronic clorgyline (0.1-10 mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura 1 , nobue kitanaka 1 , tomohiro tatsuta 1,2 , yoshio morita 2 , junichi kitanaka 1 1 department of pharmacology, hyogo college of medicine, nishinomiya, japan; 2 department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline (3.0-30 mg/kg, i.p.) 15 min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except 30 mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum 20 min after drug challenge. these results suggested that the inhibitory effect of lobeline (3.0-10 mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with 1 mg/kg meth and locomotor activity measured daily for 14 days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, 5 nmol ufp-101 (a nop receptor antagonist) and 1 mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp-101 strongly suppressed locomotor activity. thus, it was unclear if ufp-101 suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp-101 co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d2dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine (1-10 mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine (0.5-5 mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine (1, 2.5 and 5 mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine (5 mg/kg, i.p.) caused more than 80% mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine (20 mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps2p-d191 regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: (1) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis 3(ca3) were increased after fs. the rule of this kind of changing was of region-and time-specific. (2) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi 1,2 , tomoaki shirao 1 1 dept of neurobiol & behav.; 2 ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only 22.7 ± 3.0% of drebrin in the spine is stable, with a turnover time of 5.8 ± 0.4 min. blockade of ampar by 20 m cnqx reduces the population of stable drebrin (8.9 ± 3.6%), and has no effect on a turnover time. on the other hand, blockade of nmdar by 100 m ap5 has no effect on the population of stable drebrin, whereas shortens a turnover time (4.0 ± 0.3 min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu 1 , yoshito saitoh 1 , kaoru inokuchi 1,2 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at 1 h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki 1,2 , daisuke okada 1 , kaoru inokuchi 1,2,3 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 yokohama natl. univ. environment information sci., kanagawa, japan; 3 crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk2 cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto 1 , mitsutoshi setou 1,2 , kaoru inokuchi 1 1 laboratory for molecular gerontology, mitils, japan; 2 laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps3a-a005 an activity that traps vesl-1s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl-1s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at 1 m, but not 50 nm. because 50 nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps3a-a006 acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu 1 , ling chen 2 , masahiro sokabe 1,3,4 1 dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; 2 dept. physiol., nanjing med. univ., nanjing, china; 3 sorst cell mechanosening, jst, nagoya, japan; 4 dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca1 region in rat hippocampal slices. an application of 0.1 dheas for 10 min to the slice augmented instantly the epsp, which was terminated within 30 min. however, even 1 h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for 10 min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai 1 , ling chen 2 , masahiro sokabe 1,3,4 1 dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; 2 dept. physiol., nanjing med. univ., nanjing, china; 3 sorst cell mechanosensing, jst, nagoya, japan; 4 dept. mol. physiol., nips, okazaki, japan to know whether 17␤-estradiol (e2) can protect ca1 neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion (4vo) for 10 min, and the effect of e2 against this ischemic injury was examined. the electrophysiological properties of ca3-ca1 synapses were examined by a real-time optical recording method 7 days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e2 (1 mg/kg) 3 h before 4vo was able to protect ca1 neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt (2 mg/kg) produced a similar protective effect, but the estrogen receptor-␤ agonist dpn (8 mg/kg) did not. above results suggest that e2 can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps3a-a008 non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima 1 , mari ogiue-ikeda 1 , yuki oishi 2 , suguru kawato 1,2 1 department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; 2 department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er␤ hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca1, ca3, and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er␤ hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps3a-a010 morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki 1 , gen murakami 2,3 , tetsuya kimoto 2,3 , suguru kawato 2,3 1 college of humanities and sciences, nihon university, tokyo, japan; 2 department of biophysics and life sciences, university of tokyo, tokyo, japan; 3 crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of 100 nm dex induced rapid modulation of the density and morphology of dendritic spines in ca1 pyramidal neurons within 1 h. the total spine density increased from 0.88 spines/m to 1.36 spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of 10 m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura 1 , yuji ikegaya 2 , sadao shiosaka 1 1 division of structural cell biology, naist, nara, japan; 2 laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: 21st century coe research ps3a-a012 actin organizations within single dendritic spines in ca1 pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with ␤-actin in rat ca1 pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of 1.2 min and 17 min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds (0.2-1.2 um/min) and lengths (0.2-0.7 um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori 1 , katsushige sato 2 , kohichi tanaka 1 , hiroko hamazaki 1 1 mol. neurosci., tmdu, tokyo, japan; 2 physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at 1 or 4 days post-hatching (p1 or p4), and at p7. this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p4, but they lose this ability at p7. in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps3a-b015 effect of estrogen on hippocampus in male and female mice takanori sugawara 1 , shinji hayashi 1 , victoria luine 2 1 graduated school of integrated sience, yokohama city university, yokohama, japan; 2 department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received 17 ␤-estradiol (e2) or oil-vehicle injections at adult before golgi impregnation. spine densities 10 m of apical dendrites of the pyramidal neurons in the hippocampus ca1 region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e2 treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto 1,2 , takafumi inoue 2,3 , akinori kuruma 1 , katsuhiko mikoshiba 1,2,3 1 lab. developmental neurobiology, brain science inst., riken, saitama, japan; 2 div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; 3 calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca 2+ reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p38 mapk and c-jun nh2-terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u0126, a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb203580 and sp600125, antagonists for p38 mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps3a-b018 regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey 1 , chou-chi li 2 , yumiko motoyama 1 , junko yamaoka 1 , masao ito 1 1 riken brain sci. inst., japan; 2 national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur2 co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur2 c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur2. glur2 phospho-ser880 promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep2m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur2-vcp interaction. yuichi kitagawa 1,2 , shin-ya kawaguchi 1,2 , tomoo hirano 1,2 1 dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp-1 and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps3a-b020 long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr2b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from 35 to 42 days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over 60 days old, when song learning is nearly complete, but ltd remained possible in birds over 60 days old who had been isolated from song tutors, and thus retained the capacity for learning. ps3a-b021 involvement of ca 2+ -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that 3 exposures to glu of cultured rat hippocampal slices at 24 h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur1 and glur2, showed that the number of glur1-positive puncta increased transiently after the repeated glu exposures, whereas the number of glur2-positive puncta increased gradually and persistently. jstx (a ca 2+ -permeable ampar blocker) suppressed fepsp amplitude recorded at ca3-ca1 synapses by 20-40% in the period corresponding to the transient increase of glur1-positive puncta. this transient increase should represent the delivery of ca 2+ -permeable (glur2-lacking/glur1-including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca 2+ -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka 560-0043, japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after 3 inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; 3 inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd 3 times (24 h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of 5-ht (10 m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the 5-ht 1b receptor agonist cp93129 mimicked the presynaptic inhibitory effect of 5-ht. 5-ht 1b receptor antagonist nas-181 reversed the 5-ht inhibitory effect, indicating that the 5-ht induced inhibitory effect occurs by mediating 5-ht 1b receptors. the presynaptic inhibitory effect of 5-ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, 5-ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this 5-ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that 5-ht, upon activating 5-ht 1b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku 1 , masato ano 1 , yuki hashimotodani 2 , tadasato nagano 3 , masanobu kano 3 1 dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; 2 dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; 3 dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, 5-ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a1, a2a, a2b and a3). adenosine a1 and a3 receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a1 and a3 receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a1 agonist (cpa) and a3 agonist (2-cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and 2-cl-ib-meca as reported previously. although ca currents were decreased by 2-cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of 2-cl-ib-meca. at administration of cpa and 2-cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a3 receptor may inhibit adenosine a1 receptor throughout a intracellular pathway in neurons. ps3a-c028 influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa 1 , j. yamada 2 , k. inoue 1 , y. yanagawa 3 , a. fukuda 1, 2 1 dept. physiol., hamamatsu univ. sch. med., japan; 2 dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; 3 dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad67-gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa 1 , hiroyuki hioki 1 , kouichi nakamura 1,2 , hisashi nakamura 1 , takeshi kaneko 1,2 1 dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter 3(vglut3)-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut3-positive cells showed immunoreactivity for 5-hydroxytryptamine (5ht), a significant proportion (12.3%) of vglut3-postive cells was 5ht-negative. in this study, triple immunofluorescence staining was performed for vglut3, 5ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad67) and tyrosine hydroxylase (th). our results showed that all of the vglut3-positive/5ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut3positive/5ht-negative neurons didn't show any immunoreactivities for gad67 nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut3-positive neurons in these nuclei. research funds: kakenhi 16200025, 17022020, 17650100 ps3a-c030 cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig 1 , mika ushimaru 1 , yoshiyuki kubota 1 , akiya watakabe 2 , tetsuo yamamori 2 , yuchio yanagawa 3 , yasuo kawaguchi 3 1 div. cerebral circuitry, nips, okazaki, japan; 2 div. brain biology, nibb, okazaki, japan; 3 dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin (5-ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, 60% of the cs were inhibited by 5-ht through 5-ht 1a r and 6% were excited through 5-ht 2a r. however, 44% of the fs cells were inhibited and 28% excited. these results show that cs cells are more inhibited by 5-ht than fs. the expression of 5-htr was confirmed by in situ hybridization. research funds: jsps pe04061 and 15300110 ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v4, teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer 3 but also in layer 5 and/or 6. in each of the 3 cases, about 50-100 gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad67, somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine (1) their other features, (2) possible species variability, (3) their functional significance. supported by riken bsi. withdrawn ps3a-c033 regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge 1,2 , susanna b. park 2 , greg j. stuart 2 , yasuo kawaguchi 1 1 national institute for physiological sciences, japan; 2 div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in 3 neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure (100 m) inhibited layer 5 pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers 2/3 were generally less responsive to ach, but ach inhibited layer 3 cells in visual cortex. prefrontal layer 5 pyramidal neurons were more responsive to ach than were layer 5 cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations (5 mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in 37% of non-fs interneurons (24 of 65 cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki 1,2 , kenzi saito 1,3 , yuchio yanagawa 1,2 1 department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; 2 sorst, jst, kawaguchi, japan; 3 sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad67-deficient mouse fetus. ps3a-c035 gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal 4 to 6 day in egl. its release, especially from premigratory zone, was inhibited by ni 2+ , but cd 2+ couldn't. we suggest that gaba excitation induces granule cell migration. ps3a-c036 gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi 1 , hiroaki wake 2 , junichi nabekura 2 , osamu takahashi 1 1 department of histology, kanagawa dental college, yokosuka, japan; 2 national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata 1 , kunihiko obata 2 , jonathan davies 3 , mark bellingham 3 , peter g. noakes 3 1 lab. for memory & learning, riken-bsi, wako, japan; 2 obata res. unit, riken-bsi, wako, japan; 3 sch. biomed. sci., univ. queensland, queensland, 4072, australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., 2005) . we investigated the involvement of gaba alone in the mn death using gad67-deficient mice, in which cerebral gaba is reduced to less than 10% of the wild-type. mn numbers at embryonic day (e) 18 were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e18 embryos and subjected to electrical recording from the c4 and c8 ventral roots and also gaba measurement. in gad67-deficient embryos, increase in number of brachial mns (139%) and decrease in both spontaneous discharges in the c4, c8 roots and gaba content (less than 20%) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek293 cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣1␤1␥2 and ␣2␤1␥2 combinations (75% reduction) but had no effect at ␣1␤1␥1 or ␣2␤1␥1. in slice recordings dmcm blocked ipscs by 70% in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥2 subunits whereas in the central amygdala some inputs contain ␥1 while others contain ␥2 subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ 1 -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of ␤-adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps3a-d040 hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane 1 , takashi iwai 2 , yasuo nagai 1 , junichiro oka 2 , hideo kimura 1 1 dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; 2 lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h 2 s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h 2 s. we examined the effect of h 2 s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na 2 s), a donor of h 2 s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na 2 s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na 2 s. these observations suggest that h 2 s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after 5 days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by (1 l) lidocaein (2%) 5 min before training. animals were allowed to self administer morphine (1 mg/kg per inf.) ten consecutive daily 2-h session. during all morphine self administration session lever pressing was measured. our results show that: (1) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. (2) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca 2+ wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of 17␤ estradiol from glucocorticoid activity yuki oishi 1 , suguru kawato 2 1 department of physics, graduate school of science, university of tokyo, tokyo, japan; 2 graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand, 17␤ estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as ␤-amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor. 17␤ estradiol rescued this reduction via estrogen receptor ␣ and ␤. so we found that 17␤ estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps3a-d044 the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex (0.2 mg/100 g b.w., sc) or sesame oil (vehicle control) everyday for 9-10 days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf-1 mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of ␤-estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf-1, estrogen receptors ␣ and ␤, and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf-1 and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and ␤ in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps3a-d046 mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of 6-hydroxydopamine. ps3a-d047 the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba 1 , kimi watanabe 1 , munekazu komada 2 1 institute for developmental research, aichi human service center, aichi, japan; 2 graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida 1,2 , asif k mustafa 2 , kenji hashimoto 3 , kumiko fujii 2,4 , kazuhisa maeda 2,5 , hiroshi ujike 6 , richard l. huganir 2 , solomon h. snyder 2 , akira sawa 2 1 dept. of systems biology, obi, suita, japan; 2 depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; 3 chiba univ. forensic mental health, chiba, japan; 4 dept. of psychiat, shiga univ. med. sci., shiga, japan; 5 div. of neuropsychiat, tottori univ., yonago, japan; 6 dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick1 as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick1 knockout mice were significantly lower than those of their wildtype littermates at age of p7, but not in adults, suggesting regulation of pick1 on sr at developing stage. in case-control association study, we observed an association of the pick1 gene with sz, which is more prominent in disorganized sz. our findings suggest that pick1 contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps3a-d050 epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe 1,2 , werner kilb 2 , ileana l. hanganu 2 , taizhe qian 2 , daiichiro nakahara 3 , atsuo fukuda 1 , heiko j. luhmann 2 1 dept. of physiol., hamamatsu, japan; 2 inst. of physiol., mainz, germany; 3 dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca3 region of an intact preparation. 5 mm taurine blocked epileptiform activity induced by mg 2+ free acsf and 20 m 4-ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps3a-d051 responses of pge 2 in undifferentiated and differentiated ng108-15 cells kayoko matsushima 1 , takashi imanishi 1 , akinori kawaguchi 1 , tetsuyuki wada 1 , shigeru yoshida 2 , seiji ichida 1 1 school of pharm. sci., kinki univ., osaka, japan; 2 school of pharm. sci., kinki univ., osaka, japan; 3 school of pharm. sci., kinki univ., osaka, japan; 4 school of pharm. sci., kinki univ., osaka, japan; 5 school of sci. & eng., kinki univ., osaka, japan; 6 school of pharm. sci., kinki univ., osaka, japan our previous findings showed that 5-ht-and bk-induced [ca 2+ ] i increases were enlarged in differentiated ng108-15 cells. for the next stage, we investigated the effect of pge 2 , an inflammatory mediator for 5-ht and bk, on the cells. ng108-15 cells were loaded with fura-2/am, and the change in [ca 2+ ] i was monitored by an image processor. the results showed: (1) pge 2 -induced response was decreased when ng108-15 cells were differentiated by bt 2 camp, (2) 10 −5 m ah6809 and sc19220 irreversibly inhibited pge 2 -induced response by about 50% and 26%, respectively, while 10 −5 m ah23848 and sulprostone had no effect, and (3) pge 2 -induced response was abolished under ca 2+free conditions in about 70% of both ng108-15 cells. these results indicate that the response to pge 2 , via ep1 and ep2 receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from 11-to 21-day-old rats. bath application of serotonin (5-ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during 5-ht application. under the current-clamp recording, 5-ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that 5-ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps3a-d053 searching for endogenous ligands of trace amine receptors in mammals (1) akira komatsu 1 , airi yamaguchi 2 , noriko makikusa 2 , osamu koizumi 2 1 dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; 2 neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by 2% dms, post-fixed by 4% formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps3a-d054 effects of mg 2+ on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa 1,2 , nahoko kasai 1 , akiyoshi shimada 1 , keiichi torimitsu 1,2 , kunihiko obata 3 , yuchio yanagawa 2,4 , tadaharu tsumoto 2,3 1 ntt basic research laboratories, kanagawa, japan; 2 sorst/jst, saitama, japan; 3 neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; 4 dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg 2+ plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca 2+ concentration are largely affected by mg 2+ removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg 2+ removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg 2+ concentration and their actions on neural activity using a mg 2+ indicator, kmg-20-am together with fluo4-am. we observed different effects of the mg 2+ removal on gabaergic and non-gabaergic neurons by using gad67-gfp knock-in mice. research funds: jst/sorst ps3a-e055 transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p7-p13. zn + cortical neurons were also labeled, intrinsic and extrinsic, from p5. unlike in the adult, these were in layer 5, instead of layers 2, 3, and 6. at p9, neurons occurred in layers 2, 3, and 5 and, in some areas, layer 4. at p15, zn + neurons first appeared in layer 6; and at p22, there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. 17024064 ps3a-e056 systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano 1,3 , ryohei kanzaki 2 , kei ito 1,3 1 center for bioinform., imcb, univ. of tokyo, tokyo, japan; 2 dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; 3 bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi 1,2 , taizo nakazato 2 , shigeru kitazawa 2 1 neurol, juntendo univ., tokyo, japan; 2 physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, 2005) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns2005). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse (100 to 250 mv for da; 300 to 450 mv for 5ht) was applied at 4 hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps3a-e059 time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui 1,4 , yuko sekino 2,3 , tomoaki sirao 1 1 dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; 2 div. of neural network, inst. med. sci. univ. of tokyo, tokyo; 3 crest, jst, kawaguchi, japan; 4 jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at 14 days in vitro (div), and analyzed the dynamic localization of gfp-da at 16 div. glutamate stimulation started gfp-da translocating within 10 s and completed in 2 min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in 10 min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps3a-e060 distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa 1 , jun shiota 1 , hiroyuki tsutsumishita 1 , hiroyuki sakagami 2 , masaaki tsuda 1 , akiko tabuchi 1 1 dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; 2 dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr-1, c-fos,␤-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi (17790055) to a.t. shoko shimizu 1 , shinsuke matsuzaki 1 , tsuyoshi hattori 1 , ko miyoshi 2 , masaya tohyama 1 1 department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; 2 department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia 1 (disc1) was identified as a novel gene disrupted by a (1;11) (q42.1;q14.3) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc1 at centrosome and residues 446-533 of disc1 (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc1 downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc1 to target to the centrosome. furthermore, we have shown that inhibition of the disc1-kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc1 is important to the disc1-kendrin interaction and that a truncated form of disc1 lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca 2+ or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca 2+ and cl − imaging. we found that the relatively low density a172 glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca 2+ oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps3a-e063 involvement of ca 2+ influx in the unpolarized non-vesicular release of fgf-1 hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf-1 lacking a conventional signal peptide sequence. we found that fgf-1 is co-released with s100a13, a ca 2+ binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf-1 is co-released with s100a13 from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf-1 and s100a13 required ca 2+ . the overexpression of s100a13 88-98 mutant lacking an ability of interaction with fgf-1 inhibited the their release, suggesting that s100a13 is a cargo molecule. the release of fgf-1 upon either stimulation was abolished by voltage-dependent n-type ca 2+ channel blocker. these findings suggest that ca 2+ influx may be involved in the unpolarized non-vesicular release of fgf-1. in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc12 cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that 5-ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca 2+ ] i ) in pc12 cells. however, it is unclear how [ca 2+ ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca 2+ ] i on actin dynamics in pc12 cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a23187. treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to 5-ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc12 cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp36 is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp36 is endogenously expressed in pc12 cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp36 is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp36 in actin dynamics in growth cones and found that knockdown of clp36 expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp36 deletion mutants that both of pdz and lim domains are necessary for the proper function of clp36. ps3a-e066 screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura 1 , tsuyoshi kobayashi 1 , yuchio yanagawa 2 , kunihiko obata 3 , fujio murakami 1,4 1 grad. sch. of frontier biosci., osaka univ., osaka, japan; 2 grad. sch. of medicine, gunma univ., maebashi, japan; 3 bsi, riken, wako, japan; 4 sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the 2208 clones tested, two genes, neuronatin and seizure related gene 6 (sez-6) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu 1,2 , tomoko matsumoto 1,2 , sakae mikami 3 , takashi nagasawa 3 , fujio murakami 1,2 1 grad. sch. of frontier biosci., osaka univ., japan; 2 sorst, jst, japan; 3 inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf-1 and its receptor cxcr4 in the precerebellar migration. we show that cxcr4 is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr4 and sdf-1 knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps3a-e068 involvement of an immunoglobulin superfamily molecule, neph2/mkirre in the migration of precerebellar neurons kazuhiko nishida 1,2 , kazuhide nakayama 1 , saori yoshimura 1,2 , fujio murakami 1,2 1 grad. sch. of frontier biosci., osaka univ., osaka, japan; 2 sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph1, neph2/mkirre, and neph3, only neph2/mkirre was strongly expressed in pcns. expression of neph2/mkirre was detected from e13.5 when pcns migrate tangentially. the expression level became weaker at p1, when pcns stop the radial migration, raising the possibility that neph2/mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph2/mkirre in the migration of pcns. hiroki umeshima 1,2 , toshio ohshima 3 , tomoo hirano 2 , mineko kengaku 1 1 lab. for neural cell polarity, riken bsi, wako, japan; 2 department of biophysics, kyoto university, kyoto, japan; 3 lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk5 (cdk5-dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin2-gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk5-dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito 1,2 , takahiko kawasaki 1,2 , tatsumi hirata 1,2 1 division of brain function, national institute of genetics, mishima, shizuoka, japan; 2 department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k252a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k252a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k252a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k252a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc51 functions with unc-76 to regulate axonal transport in drosophila hiroaki mochizuki 1 , hirofumi toda 1,3 , emiko suzuki 2 , joseph gindhart 4 , toshifumi tomoda 3 , katsuo furukubo-tokunaga 1 1 grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; 2 gene net. lab., natl. inst. genet. mishima; 3 beckman res. inst., city of hope, ca, usa; 4 dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc51, which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc51 is essential for neural development in drosophila. loss of function of drosophila unc51 results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc51 genetically interacts with unc-76, an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc51 mutants, unc-76 was separated from synaptotagmin vesicles. these results suggest that unc51 coordinates kinesin-cargo interaction via unc-76 to regulate dynamic axonal transport. ps3a-e072 change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi 1 , daisuke takahashi 2 1 life science inst. sophia university, tokyo, japan; 2 waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after 24 h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi (16500193) and kakenhi on priority areas (16027207) ps3a-e073 generation and analysis of region-specific rac1-deficient mice hidetoshi kassai 1 , masahiro fukaya 2 , eriko miura 2 , mizuho sakahara 1 , masahiko watanabe 1,2 , atsu aiba 1 1 div. cell biol., kobe univ. grad. sch. med., kobe, japan; 2 div. physiol. sci., hokkaido univ. grad. sch. med., japan rac1 is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac1 in the cns is poorly understood because of the embryonic lethality of rac1 knockout mice. in this study, we generated and analyzed region-specific rac1-deficient mice (emx1-rac1 ko mice) by the cre-loxp system, in which a promoter for emx1 homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx1-rac1 ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx1-rac1 ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac1 regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map1b containing nterminal 126 amino acids promoted neuronal death. to reveal the mechanism of map1b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map1b by two-hybrid system. alpha-tubulin was found to interact with map1b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map1b n-terminal has not yet been reported. beta-tubulin was also found to interact with map1b n-terminal. when ␤ tubulin was divided in 2 fragment at between amino acid 213 and 214, there was no interaction between ␤ tubulin fragments and map1b n-terminal. interaction needs the continuous region over aa 213 and 214. there were much proportion of round formed cos7 cells in n-terminal containing map1b transfected cells than in n-terminal lacking map1b transfected cells. there might be some interference in interaction between map1b and tubulin in cells express map1b containing n-terminal. otone endo 1,2 , masaaki mizuno 3 , yasukazu kajita 2 , jun yoshida 2 1 department of neurosurgery, ja kainan hospital, aichi, japan; 2 department of neurosurgery, nagoya university, nagoya, japan; 3 department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta 1 , masae yaguchi 1 , yumi matsuzaki 2 , yoshiaki toyama 3 , yutaka kawakami 4 , hideyuki okano 2 , masahiro toda 1,5 1 neuroimmunology research group, keio univ., tokyo, japan; 2 physiology, keio univ., tokyo, japan; 3 orthopaedic surgery, keio univ., tokyo, japan; 4 institute for advanced medical research, keio univ., tokyo, japan; 5 neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd11c + dcs, cd8␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd8␣ + dcs compared to cd11c + dcs. these results suggest that cd8␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps3a-e077 von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo 1 , hiroshi kanno 1 , takaakira yokoyama 1 , shuichi nakano 2 , naoki sugimoto 2 , nahoko kobayashi 3 , tetsuhiko yoshida 3 , isao yamamoto 1 1 dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; 2 fiber and dept. of chemistry, konan university, kobe, japan; 3 toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly 100%, and peptide with the elongin c binding site (residues 157-171) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps3a-e078 the effect of pueraria mirifica on erk1/2 and s-100 following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e2) on the expression of erk1/2 and s-100 following sciatic nerve crush and transection in rats. protein levels of perk1/2 and s-100 in distal segments of nerve at day 7 were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e2, caused a significant decrease in the expression of perk1/2 levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk1/2 phosphorylation. following treatments, levels of s-100 were significantly decreased both in crushed and transected nerves with respect to control group at p < 0.05. this estrogenic effect was blocked by ici182,780. because of their structural similarity to e2, pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day 7. this study suggested that pm as well as g could enhance nerve regeneration like e2 by interfering with the injury-induced erk signaling pathway. yasuhiro kato 1 , takafumi suzuki 2 , kunihiko mabuchi 2 1 department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; 2 department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed 5 untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf-3 and stat3, which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p6-10 wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y27632, a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level 7 days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin (5ht) positive cells increased in number progressively from 1 day to 6 weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and 5ht cells surrounded the axons and glia. thus, 5ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda 1 , yukio someya 2 , ryo kadota 2 , chikato mannoji 2 , tomohiro miyashita 2 , atsushi murata 3 , masashi yamazaki 2 1 department of orthopaedic surgery, togane hospital, 2 department of ortopaedic surgery, graduate school of medicine, chiba university, japan; 3 division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected 24 h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt (2.8 mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), 5-bromo-2 -deoxyuridine-5 -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days 1 after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days 2 after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day 2 after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps3a-f087 early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski 1 , hanna kozlowska 1 , marcin jurga 1 , aleksandra habich 1 , elzbieta wanacka 1 , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed 24 h, 48 h, 72 h and 7 days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue 1, 2, 3 and 7 days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext2, one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext2 mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican-1 and syndecan-1 were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe 1 , hiroe sagawa 2 , masahiro ichikawa 3 , yoshihito tokita 1 1 dept. perinatol., int. dev. res., kasugai, japan; 2 dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; 3 dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y39983, can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for 14d. after fixation, the neurites were stained with anti-tuj1 antibody to obtain number and length of tuj1 neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day 12, wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of 15-days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of 15-days-old embryonic mice by culturing in dmem/f12 medium for 9 days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of 0-9 div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for 3-9 div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for 7 days. dha increased the number of tuj1(+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first 24 h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi (17590205) ps3a-f092 interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j19 kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± 10 • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps3a-f094 effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating 3d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the 3d structure of surrounding stimuli defined by binocular disparity. ps3a-f095 rotational motion aftereffect in positive direction for 3-dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for 3-dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known 3dimensional mae. this finding suggests that the visual system has a mechanism that detect 3-dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j19 takanori uka, ryo sasaki department of physiology 1, juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch (3 deg) of a center/surround display presented 10 degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always 0 percent. for each of 11 subjects, we calculated direction discrimination thresholds (at 82% correct) at each surround size. consistent with crowding, thresholds increased when surround size was 4.5 and 6 degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was 9 and 12 degrees, relative to 6 degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps3a-f097 generation of receptive fields in higher visual areas based on v1 columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v2 and v4, respond to stimuli with complex shapes. how do these neurons integrate signals from v1? in particular, does the well-known columnar organization of v1 play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v1 neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about 15% of model neurons exhibit responses similar to those of v2 and v4 neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a 3-d surface slant is perceived (blakemore, 1980) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa 2006) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext(13041033), jsps(13308048), coe21 ps3a-f099 consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi 1 , kota sasaki 2 , izumi ohzawa 1,2 1 grad. school of frontier biosci., osaka univ., japan; 2 grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and 2-d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by 2-d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext(13041033), jsps(13308048), coe21 ps3a-f100 firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate 1,2 , hiroshi tamura 1,3 , ichiro fujita 1,3 1 graduate school of frontier biosciences, osaka university, osaka, japan; 2 jsps, japan; 3 crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi 16-07757 ps3a-f101 spatial-frequency dependency of receptive field size and surround suppression in lgn and v1 hironobu osaki 1 , tomoyuki naito 2 , osamu sadakane 2 , masahiro okamoto 3 , hiromichi sato 2,3 1 med. sch., osaka univ.; 2 grad. sch. med., osaka univ.; 3 grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v1), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v1 (n = 93) and lateral geniculate nucleus (lgn) (n = 63) in anesthetized cats. first, we found that v1 neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v1 has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura 1 , akichika mikami 2 , kazuo itoh 1 1 department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; 2 department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v3a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v3a, we injected a bi-directional tracer biotinylated dextran amine into the v3a. our results indicated that the v3a has connections with the occipital, parietal and temporal cortices. the v3a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v3a has commissural connections with the v3, the v3a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v2. these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps3a-g103 activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera (100 fps), and were also monitored with a 2d or 3d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity 10-40 ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps3a-g104 timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki 1,2 , arman abrahamyan 3 , catherine stevens 3 , andreas a. ioannides 1,2 1 brain science institute, riken, saitama, japan; 2 graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; 3 school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately 170 ms (n170) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within 100 ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about 100 ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto 1 , shoutaro akaho 1 , kenji fujikumi 2 , yasuko sugase-miyamoto 1 , masato okada 3 1 aist, ibaraki, japan; 2 ism, tokyo, japan; 3 university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of 45 neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of 45 neurons for 38 visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps3a-g106 relationship between color and shape selectivity in area teo of the monkey masaharu yasuda 1,2 , hidehiko komatsu 1,2 1 national institute for physiological science, okazaki, japan; 2 sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps3a-g107 neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a 3-choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was 0.58, a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across 2-3 simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant (17022025) ps3a-g108 behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki 1 , atsuko komori 1 , tomonori manabe 2 , toshihiko hosoya 2 , hiroshi sagara 3 , emiko suzuki 3 , hitoshi okamoto 2 , ichiro masai 1 1 masai initiative research unit, riken, wako, japan; 2 riken bsi, wako, japan; 3 ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde6c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde6c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde6c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until 9 day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde6c activity is important for the sensitivity of vision. ps3a-g109 localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: 50-94 years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from 50 to 80 years of age, and showed a decline and lack of ir in older retinas (85-94 years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from 50 to 91 years of age, and absent at 94 years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps3a-g110 temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa 1 , norio fujimaki 1 , toshihide imaruoka 2 1 nict, kobe, japan; 2 kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak (117, 215 ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity (125, 237 ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around 196 ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano 1,2 , derek arnold 3 , alan johnston 4 , tsunehiro takeda 1 1 univ. tokyo, chiba, japan; 2 ntt cs lab., kanagawa, japan; 3 univ. sydney, sydney, australia; 4 ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, 2003) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around 10 hz and matched with the alpha frequency of meg. as 10 hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical 10 hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps3a-g112 the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya 1,2 , kei ito 1,2,3 1 center for bioinform., imcb, univ. of tokyo, tokyo, japan; 2 dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; 3 bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified 12 discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal4 enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps3a-g113 dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi 1,2 , toshiki tani 2 , shigeru tanaka 1,2 1 graduate school of life science & systems engineering, kyushu institute of technology, japan; 2 laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of 5 weeks. we also found that when the goggles were removed after 1 or 2 weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, 2003) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than 0.3 hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps3a-g116 retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., 2003. neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada 1 , yasuhiro enami 1 , hiroshi jouhou 2 , takehiko saito 3 , kaj djupsund 4 1 tokyo metropol. univ., hino, tokyo; 2 astellas pharma. inc., osaka, japan; 3 suny upstate med. univ., center for vision and ophthal., ny, usa; 4 univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph 0 ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph 0 only 0.2 units from the baseline of 7.60, we found a remarkable reduction of the conduction velocity by 20-30%, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps3a-g118 analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi (17500195) ps3a-g119 stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of 30% (17 of 57) of neuron pairs (inter-neuronal distance >500 m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the 57 pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to 28 visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in 71% (12/17) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii 1,2 , toshihiko hosoya 1 1 bsi, riken, japan; 2 dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over 200 ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v1). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v1. this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v1 neurons. masayoshi tsuruoka 1 , masako maeda 1 , bunsho hayashi 1 , ikuko nagasawa 2 , tomio inoue 1 1 dept. physiol. showa univ. sch. dent. tokyo, japan; 2 dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t13-l2 dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention (80 mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil (5%, 1 ml). the increased emg activity significantly reduced following bilateral l6-s3 ventral rhizotomies. a baseline emg did not significantly alter when the l6-s3 ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps3a-g123 hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba40) was significantly greater (p < 0.001, uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < 0.001, uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm8 in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm8 agonist (l-menthol: 0.06-6 mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = 7) or boo rat (n = 8). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm8 on the urothelium cell of the urinary bladder. ps3a-g125 caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito 1 , a.d. craig 2 1 dept. physiol, shimane univ. sch. med., izumo, japan; 2 atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol 2005) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns40413 ps3a-g126 ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps3a-h128 fasting induced long-chain fatty acid receptor gpr120 expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor 120 (gpr120) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of 48 h fasting on gpr120 expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr120 mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. 48 h fasting induced gpr120 mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr120-like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr120 at least fasting period. ps3a-h129 ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that 3hydroxybutylate (3hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls (4v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of 4v respond to the change in 3hb. change in the intracellular calcium concentration ([ca 2+ ] i ) in vitro was measured in dispersed ependymocytes taken from the 4v in rats. the present results showed that the [ca 2+ ] i increased in response to 3hb, but the increase was blocked by ␣-cyano-4-hydroxycinnamic acid, which is a monocarboxylate transporter 1 (mct1) inhibitor. immunohistochemistry showed that mct1-immunoreactivities were located on the 4v ependymocytes. these results indicate that the ependymocytes may sense 3hb through a mct1-dependent mechanism. research funds: kakenhi 14360177 ps3a-h130 comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin (1.3 mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with 6-hydroxydopamine (6-ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at 4 weeks after 6-ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at 0 mm; anteroposterior, +3 mm; lateral and −5 and −7 mm; dorsoventral from bregma at 2 weeks after 6-ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better 6-ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b 17791323 most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: (1) retrieving the food with a rake (a motortool), (2) retrieving the hidden food with a mirror-attached rake, (3) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, (4) moving a mirror hung along the rail by hand to find the food, (5) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada 1,2 , shigehiro miyachi 2 , michiko imanishi 2 , masato taira 1 , masahiko takada 2 1 div. applied system neurosci., nihon univ. sch. med., tokyo, japan; 2 dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area 9 (9m), dorsal area 46 (46d), and ventral area 46 (46v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex 3 days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from 9m, 46d, and 46v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that 9m, 46v, and 46d receive multisynaptic projections from the entorhinal cortex, area 36, and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps3a-h137 new neural activities of reward anticipation and task errors h. ogawa 1 , h. ifuku 2 , t. nakamura 3 , s. hirata 4 1 kumamoto kinoh hosp, kumamoto, japan; 2 fac educ, kumamoto univ., kumamoto, japan; 3 nat kikuchi hosp, kumamoto, japan; 4 dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, 3 & 1-2) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca 2 s after led offset at correct trials. relations between cues and responses were reversed. of 169 reward-related neurons found, 88.7% showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = 96) only at correct trial, i-type (n = 5) at around possible reward onset only at incorrect trials, and c-i type (n = 49) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all 3 types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai 1 , takayuki hosokawa 2 , masato inoue 1 , akichika mikami 1 1 section of brain sciences, primate research institute, kyoto university, inuyama, japan; 2 department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps3a-h140 neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue1), and centrally allocated one of the objects presented in cue1 (cue2). the frequency of which object was presented as cue2, i.e., task context, was manipulated across blocks. we focused on cue2 period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps3a-h141 anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki 1 , barry j. richmond 3 , munetaka shidara 1,2 1 grad. sch. of tsukuba univ., ibaraki, japan; 2 neurosci. ri., aist, tsukuba, japan; 3 lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in 2 conditions: (1) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], (2) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition 84/120 neurons responded in all trials. in the certain condition 50/84 neurons responded in the rewarded trials only. 48 of these 50 showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas17022052), aist masamichi sakagami 1,3 , kosuke sawa 2 , xiaochuan pan 1,3 1 bsrc, tamagawa university, tokyo, japan; 2 senshu university, kanagawa, japan; 3 presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a1-b1-c1 and a2-b2-c2. after monkeys could acquire the sequences, new pairs of stimuli (i.e., d1 and d2, e1 and e2, etc) were introduced to associated with b1 or b2 (d1-b1, d2-b2, etc). the asymmetric reward rule was instructed by pairing c (c1 or c2) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a1 and a2, which were associated with c1 and c2 in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi (17022037), hsfp, presto, jst ps3a-h143 reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan 1,2 , kosuke sawa 1,3 , masamichi sakagami 1,2 1 bsrc, research institute, tamagawa university, japan; 2 presto, jst, japan; 3 department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a1-b1-c1 and a2-b2-c2. the asymmetric reward rule was instructed by pairing c (c1 or c2) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of 202 neurons from the lateral pfc, 31% showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about 10% of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated 3 times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every 4 pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than 3 consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps3a-h146 neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima 1 , hajime mushiake 1 , jun tanji 2 1 department of physiology, tohoku university school of medicine, sendai, japan; 2 brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the 2-sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps3a-h147 neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. 2005) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = 73) preferentially when the instruction was associated with small reward. the instructions activated 3 groups of putamen neurons preferring small reward-(n = 19), large reward-action (n = 3) and both types of action (n = 26). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by 30-50 and 100-150 ms, respectively. putamen neuron activation lead that of cm neurons by 50-70 ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi (17022032) ps3a-h148 encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t1), second (t2) and third (t3) choices with reward probabilities of about 30, 50 and 80%, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r1, 95%). most da neurons (66/85) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t1 to t2 and to t3 trials, then decreased in r1 trial. in another task with two repetition trials (r1 and r2, 96%), magnitude of start-cue responses decreased gradually from t3 to r1 and to r2 trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi (17022032) ps3a-h149 striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada 1,2 , hitoshi inokawa 1 , minoru kimura 1 1 dept. of physiol. kyoto prefect univ. med., kyoto, japan; 2 jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded 146 striatal projection neurons from a monkey. after depressing a start button, the monkey chose 1 of 3 target buttons with correct rates at 1st, 2nd, 3rd and repetition trials of 33, 50, 89 and 96%, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice (11/55) rather than to upcoming choice of target (2/55). during the target choice, another subset of neurons showed firings selective to choosing particular target (29/57) rather than to expected reward probability (12/57). after the target choices, another group of neurons fired related to expected reward (41/56) rather than to chosen action (19/56). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi (17022032), jsps fellows ps3a-h150 encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of 169 tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose 1 of 3 target buttons at average correct rates of 33 (first), 50 (second), 89 (third) and 96% (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans (80/169) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi (17022032) ps3a-i151 representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda 1 , k. samejima 2 , k. doya 3 , m. kimura 1 1 dept. physiol., kyoto pref. univ. med.; 2 brain sci. res. center, tamagawa univ.; 3 irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of 236 striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a 1 s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of 10, 50 or 90%. a linear regression of neuronal discharge rates showed: 39 neurons encoded reward values of either action during delay period before go signal, with most (64%) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = 33), while other 37 neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi (17022032) ps3a-i152 delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c1), first delay (d1), second cue (c2), second delay (d2), and response periods. activity during the d1 and d2 periods was analyzed in this study. firing rates during the d1 period linearly depended on c1 durations. on the other hand, d2 period activity depended on trial types (ls and sl), but not on the variety of c2 durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi (17021039) ps3a-i153 neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku 1 , ryoi tamura 1 , teruko uwano 1 , taketoshi ono 2 1 dept. integrative neurosci., univ. toyama, toyama, japan; 2 dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize 4 paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi (16500260 17021016) ps3a-i154 reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori 1 , mikio inagaki 1 , wu lisa 2 , taijiro doi 1 , eishi hirasaki 1 , hiroo kumakura 1 , ichiro fujita 1 1 osaka univ., japan; 2 massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from 17022025 and takeda science foundation ps3a-i155 response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and 7a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, (1) performance of visually guided sex discrimination task and (2) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) (16209006) (sa) and coe program in kit from the mext ps3a-i157 activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa 1 , satoshi katai 2 , masanori saruwatari 1 , masato inoue 1 , akichika mikami 1 1 section of brain sciences, primate research institute, kyoto university, inuyama, japan; 2 third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro 2004) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto 1 , tetsuya iidaka 2 1 department of psychology, nagoya university, nagoya, japan; 2 department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: 30-80 hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the 400-600 ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the 600-700 ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included 16 images (4 objects × 4 views). 4 novel objects were generated by deforming a prototype in four directions. for 30 deg-interval object sets, 4 views were obtained by rotating each object with the interval of 30 deg, 60 deg-interval set and 90 deg-interval set were with 60 deg and 90 deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across 60 deg separated views was significantly higher in the trials with 30 deg-interval sets than those with 60 deg-interval sets. similarly, the difference was also found in the performances across 90 deg separated views between those with 30 deg-interval sets and 90 deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps3a-i160 brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps3a-i161 obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, 2005 ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: 1682 middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps3a-i162 sirna-induced nr1 knockdown causes hypofunction of nmda-r and cognitive deficit m. saji 1,2 , t. utida 2 , a. ohnishi 2 , k. noda 1 , m. ogata 1 , h. akita 1 , n. suzuki 1,2 1 physiol, health sci. sch. kitasato univ., sagamihara, japan; 2 brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr1 using hvj-envelope vectors. we confirmed that marked down-regulation (50%) of nr1 expression occurred only in the hippocampus among various brain regions 4-8 days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr1 knockdown, the nr1 down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr1 knockdown, the nr1 down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka 1,2 , hiroshi ageta 2 , ikuko yao 2 , kaoru inokuchi 2 , yutaka kirino 1 , mitsutoshi setou 2,3 1 graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; 2 mitsubishi kagaku institute for life sciences, tokyo, japan; 3 okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl3 mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl3 is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl3 mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl3 wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps3a-i164 decreased interneurons in the pax6 mutant mouse limbic system hasumi haba 1 , tadashi nomura 1 , yoshinobu hara 1,2 , noriko osumi 1,2 1 div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; 2 crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax6 heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax6 heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at 8 weeks, but not at 4 weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp1) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine (0.2 mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: 16790712, 17025055 ps3a-i166 gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba 1,2 , satoko hattori 1 , hiroaki hori 1 , tetsuo nakabayashi 3 , hiroshi kunugi 1 , ryota hashimoto 1 1 department of mental disorder research, national institute of neuroscience; 2 tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; 3 musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp1) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of 38 patients with schizophrenia and 38 age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: 16790712, 17025055 ps3a-i167 genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto 1 , hiroko noguchi 1 , hiroaki hori 1 , tetsuo nakabayashi 2 , satoko hattori 1 , sachie chiba 1 , seiichi harada 2 , osamu saito 2 , hiroshi kunugi 1 1 department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; 2 musashi hospital, ncnp, kodaira, japan; 3 tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp1) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in 165 healthy volunteers and 72 patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: 16790712, 17025055 ps3a-i168 detection of 18f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura 1 , nicola bright 3 , toshio yanagida 1 , masatoshi takeda 2 , paul grasby 3 1 department of physiology, osaka university, japan; 2 department of psychiatry, osaka university, japan; 3 cyclotron unit, imperial college, hammersmith hospital, uk we used 18 f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and 18 f-dopa scans were obtained from a database of scans published in mcgowan et al. 2004 , archives general psychiatry. the 14 schizophrenic patients all met dsm-iv criteria on medication and 10 healthy volunteers were compared. we attempted to improve the quality of the 18 f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in 18 f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > 0.02). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p300 component of the visual eventrelated potential in 14 patients with schizophrenia and 14 healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p300 amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p300 amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p300 amplitude between the 2 faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area 10 between the 2 faces in controls, while in patients, there was no significant activity difference between the 2 faces. stimulation with crying face induced higher activities in the 10 and right 21 areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps3a-j170 inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura 1 , shoji tanaka 2 , koki yamashita 2 , motoichiro kato 1 , haruo kashima 1 1 department of neuropsychiatry, school of medicine, keio university; 2 department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps3a-j171 pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc1, a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as 5-ht6 are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in 7 of the 10 sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps3a-j173 alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta 1 , ryo yamamoto 1 , shigeki sugiura 2 , kaoru inokuchi 3 , nobuo kato 1 1 dept. integrat. brain sci., grad. sch. med., kyoto univ.; 2 nara med. univ.; 3 mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer1a/vesl-1s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer 1a protein by diffusion from patch pipettes. homer 1a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer 1a may be a biological mediator of ecs effects. masanori kasai 1 , nozomi miyagi 1 , norio kawashiro 2 , daisuke torizuka 2 1 dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; 2 sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant (1.0 mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the 11th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the 2nd, 7th, 14th, 21st, 28th and 35th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc (75.1 ± 12.1 g/dl, n = 7) on the 35the day was significantly lower than that in intact rats (139.4 ± 11.9 g/dl, n = 7). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps3a-j175 molecular cloning of a novel candidate for ethanol-responsive genes, yy1ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy1ap (yy1-associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy1-binding and transactivation domains previously demonstrated in yy1ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy1 genes was demonstrated in the same neurons. analogous to yy1ap as a co-activator of transcription factor yy1, it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps3a-j176 excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr2b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr2b-selective nmda antagonists ifenprodil (3 and 10 m) and ro25-6981 (0.1 and 1 m) markedly inhibited degeneration of orexin neurons induced by 24 h application of nmda (45 m) or qa (1.5 mm). we also show that stimulation of gaba a receptors by muscimol (10 and 30 m) or isoguvacine (30 and 100 m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba (100 m) plus a gaba uptake blocker nipecotic acid (1 mm) was abolished by a gaba a antagonist picrotoxin (100 m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr2b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh795). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of 1 ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps3a-j178 a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi 1 , osamu fukayama 2 , takashi sato 2 , takafumi suzuki 2 , kunihiko mabuchi 1,2 1 dept. biomed. eng., univ. tokyo, tokyo, japan; 2 dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps3a-j179 on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki 1 , yasuhiko jimbo 2 , kenji yasuda 1 1 department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; 2 department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip 8 um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are 100 khz in 64ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita 1 , tetsu okumura 1 , kazuo okanoya 2 , jun tani 1 1 lab. for behavior & dynamic cognition, riken-bsi, japan; 2 lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps3a-k181 local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura 1 , yuichi yamashita 1 , kazuo okanoya 2 , jun tani 1 1 behav & dynamic cognition, riken-bsi, saitama, japan; 2 biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata 1 , shinya ishii 1 , munetaka shidara 2 1 dept. of e&e engineering, oita univ., oita, japan; 2 grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [1] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, 2006 behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than 3 consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki 1 , shinya yamamoto 2 , sunao uchida 3 , shigeru kitazawa 4,5 1 faculty of hum sci., waseda univ., tokorozawa, japan; 2 neurosci. res. inst, aist, tsukuba, japan; 3 faculty of sport sci., waseda univ., tokorozawa, japan; 4 dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; 5 crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ±80 ms, s.d. = 80 ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about 50 ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi (15200031) ps3a-k185 single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama 1 , fumiko sato 2 , ken nakazawa 3 , kenichi ono 3 1 chiba university, japan; 2 field of home economics, faculty of education, chiba univ., japan; 3 department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm-3000, shi-madzu) over the frontal area (45 channels) for 7 volunteers (19-23 y). thirty tasks were presented visually every 30 s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between 2 tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was −0.4+/−0.45 between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi 1,2 , kosuke hamaguchi 3 , masato okada 1,2,3 1 department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; 2 jst, japan; 3 riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated 2 different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi (14084212) and (16500093) ps3a-k187 auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa 1,2 , sachiko koyama 1,2 , takashi omori 3 , takashi morotomi 4 1 research institue for electronic science, hokkaido university, sapporo, japan; 2 japan science and technology agency, saitama, japan; 3 graduate school of information science and technology, hokkaido university, sapporo, japan; 4 sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = 13). the erps were elicited by tones (500 hz) with different durations (8, 16, 32, 64, 128, 256 ms) and gaps (8, 16, 32, 64, 128 , 256 ms) embedded in a continuous pure tone (500 hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for 32 and 64 ms were high both for the tones (32 ms, 38%; 64 ms, 61%) and the gaps (32 ms, 46%; 64 ms, 46%). ps3a-k188 determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps3a-k189 theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to 50% till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, 1,6-diphenyl-1,3,5-hexatriene, and 1-[4 (trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc42 in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp5 expression correlates with wnts/bmps in the cortical hem, sp8 with fgfs in the cop, and sp9 with shh in the ventral midline and mge. by using in utero electroporation, we show that sp8 regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp8 and fgf8 exhibit reciprocal induction, indicating that sp8 is a positive feedback regulator of fgf8. surprisingly, though, ectopic expression of both sp8 and its dominant active form shift cortical areas in the opposite manner to fgf8, suggesting that sp8 activates additional targets that overcome fgf8 function. our results indicate that fgf10 is an additional target of sp8, showing effect on patterning similar to sp8. these findings indicate that sp8 balance the proper cortical arealization through fgf8 and fgf10. research funds: nihr37ns31558 ps3p-c003 fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira 1 , kotaro hattori 1 , takeshi yagi 2 , shigeki yuasa 1 1 dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; 2 kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi 1 , kazuko keino-masu 1 , tatsuyuki ohto 1 , satoshi kunita 2 , satoru takahashi 2 , masayuki masu 1 1 dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; 2 laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp1, has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp1 gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext1 gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp1-deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp1/sulfatase-1 and sulffp2/sulfatase-2, which have hs 6-o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp1 and sulffp2. here we report the isolation and the characterization of the third homologue, sulffp3. sulffp3 has about 56% and 71% overall amino acid homology with sulffp1 and sulffp2, respectively. at 24 h postfertilization, sulffp3 is expressed in the ventral region of spinal cord, whereas sulffp1 is expressed only in the floor plate and sulffp2 is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp3 will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp2 was shown to mediate the repulsive effect of sema3a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp2, 3, 4, and 5 are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class 3 semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps3p-c007 developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa 1 , n. yamamoto 2 , m. yoshimoto 2 , t. yasuda 1 , k. maruyama 1 , t. kage 1 , h. takeda 3 , h. ito 2 1 nat. inst. rad. sci., chiba, japan; 2 nippon med. sch., japan; 3 tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax6-or dlx2-positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er81 is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er81 homologue (zfer81), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer81 was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er81 expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is 70% identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta 1,2 , hiroo takahashi 1,2 , takafumi shintani 1,2 , kazuma etani 1 , masaharu noda 1,2 1 div. of mol. neurobiol., nibb, okazaki, japan; 2 crest, jst, japan in the developing chick retina, the expression of bmp4 is relieved by that of bmp2 at around e5 with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina2, are expressed in a double-gradient fashion from e6 on like ventroptin and bmp2. when bmp2 expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp2 knockdown and ephrina2-misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp4 to bmp2 thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en2, wnt7b and pcp2/l7 gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and 4di-10asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and 4di-10asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or 4di-10asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru 1,2 , takahisa taguchi 1 1 aist, ikeda, japan; 2 presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a 2-dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps3p-d014 laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps3p-d015 synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki 1 , toshio aoyagi 2 1 department of physics, kyoto university, kyoto, japan; 2 graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf-1 mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into 4 weekold wistar strain rats in several doses followed by successive injections of brdu for the next 4 days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as 1 mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps3p-d017 the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato 1 , james e. goldman 2 1 division of pharmacology, national institute of health sciences, tokyo, japan; 2 department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p0 rat svz, sagital sections of forebrain were made at p3 and cultured for 6 days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣3 integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of 2nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps3p-d020 localization of neurotrophin receptors trka in pc12 cells: 3d reconstruction analysis of membrane proteins tomoki nishida 1 , hiroshi jinnai 2 , tatuo arii 3 , akio takaoka 4 , ryoichi yoshimura 1 , yasuhisa endo 1 1 department of applied biology, kyoto institute of technology, kyoto, japan; 2 department of polymer science and engineering, kyoto institute of technology, kyoto, japan; 3 national institute for physiological sciences, myodaiji, okazaki, japan; 4 osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin-1 in pc12 cells, analyzed by high-voltage electron microscopy, and reconstructed 3d structure of their subcellular distribution by imod. our results indicated that localization of caveolin-1, known as an integral membrane protein of caveolae, was never found in the invagination structure in pc12 cells, but trka and caveolin-1 immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd1, a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/␤-catenin pathway. ccd1 mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd1 in the wnt-mediated neuronal development. there are three subtypes of mouse ccd1 gene products, ccd1a, ccd1b and ccd1c, which are generated by different promotor usage. mouse ccd1a as well as zebrafish ccd2a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi (15770137, 17300098) ps3p-d022 analysis of a role of r-spondin2 on proliferation of the cortical neuroepithelium yumiko hatanaka 1 , masahiro yamaguchi 2 , fujio murakami 3 , masayuki masu 1 1 grad. school of comprehensive human sci., univ. of tsukuba, japan; 2 grad. school of med., univ. of tokyo; 3 grad. school of frontier biosci., osaka univ r-spondin2 (rspo2) is a secreted activator of wnt/␤-catenin signaling (kazanskaya et al. 2004) . rspo2 is expressed in the developing medial cerebral wall and transgenic mice expressing rspo2 in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. 2005) . since wnt3a has a role for expansion of caudomedial cortical progenitor cells (lee et al. 2000) , these findings lead us to the idea that rspo2 may synergistically promote proliferation of cortical neuroepithlial cells together with wnt3a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a ␤-catenin/tcf reporter gene into these cells of embryonic day 11.5 mouse. an application of wnt3a on these cells increased level of the reporter expression, and an addition of rspo2 further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt3a and rspo2 directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il-1␤, il-11, lif, gdnf) or lowered o2 (3.5%), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells (1.86-fold) as compared to normal o2. cytokine cocktail significantly increased th (+) cells (2.11-fold) compared to nontreated control. treatment of lif and il-1␤ to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps3p-d024 structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui 1 , terukazu nogi 1 , mitsuharu hattori 2 , kenji iwasaki 1,3 , junichi takagi 1 1 research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; 2 dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; 3 core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer2 and vldlr), resulting in tyrosine phosphorylation of dab1. in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic 3d reconstruction of four-domain reelin fragment (i.e. r3-6) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r5-6) was capable of binding to the receptor (apoer2), and was also able to induce tyrosine phospholylation of dab1 in primary neuronal culture. ps3p-d025 effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax3 and pax7 compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps3p-d027 neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res 79:287, 2005) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi-3k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps3p-d028 fabp7 is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa 1 , miho matsumata 2,3 , yuji owada 2 , shigeki yuasa 1 , noriko osumi 2,3 1 natl. inst. of neurosci., ncnp, tokyo, japan; 2 tohoku univ. sch. of med., sendai, japan; 3 crest, jst pax6 transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax6 is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp7, a downstream of pax6, regulating maintenance of embryonic neural stem/progenitor cells (arai et al., 2005) . fabp7 was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). 56% of fabp7-expressing cells co-expressed gfap (a marker for early progenitors), and 33% of them co-expressed psa-ncam (a marker for late progenitors). fabp7 expression was also overlapped with pax6, and expression of fabp7 was down-regulated in the dg of pax6 deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp7 knockout mice. taking all together, it is concluded that fabp7 is required for maintenance of neural stem/progenitor cells in dg. ps3p-d029 involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day 4-7. sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani 1,6 , shiki okamoto 2 , masato imada 3 , kouichi itoh 4 , atsushi irie 5 , hitoshi sakuraba 6 , hideo kubo 7 1 department of molecular biologu, ohu univ., koriyama, japan; 2 dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; 3 dept. anatomy, nihon univ. shl. med., tokyo, japan; 4 dept. mol. pharma., univ. tokushima bunri, sanuki, japan; 5 dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; 6 department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; 7 dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam-2 shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam-2 is possible. in the present, we show that the isolated randam-2 high+ cells enrich nscs. the randam-2 high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam-2 low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam-2 is a usefule marker for the isolation of nscs by facs. yasuharu takamori 1 , yasuhisa tamura 1,3 , yosky kataoka 1,2 , yilong cui 1,3 , hisao yamada 1 1 department of anatomy and cell science, kansai medical university, osaka, japan; 2 department of physiology, osaka city university graduate school of medicine, osaka, japan; 3 morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b1 and b2 are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b1 (++), b2 (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b1 (+++), b2 (+), and mature neurons possess lamin a/c (++), b1 (+), b2 (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka 1 , yuichi tanaka 1 , tatsuhiro hisatsune 1 1 department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps3p-e033 new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba 1,2 , hideo namiki 2 , tatsunori seki 1 1 dept. of anat, juntendo univ. sch. of med., tokyo, japan; 2 integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p5. three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., 1987) . the dividing pattern is currently under investigation. akiya watakabe 1 , noritaka ichinohe 2 , sonoko ohsawa 1 , tsutomu hashikawa 3 , kathleen s. rockland 2 , tetsuo yamamori 1 1 div. of brain biol, nibb, okazaki, japan; 2 lab. for cortical organization and systematics, bsi, riken, wako, japan, 3 lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer 6 neurons in several areas of monkey neocortex. we previously reported that nurr1, ctgf and sema3e mrnas are specifically expressed in subsets of layer 6 neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer 6. by double ish, layer 6 neurons in monkeys are roughly divisible into cck(+) and sema3e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema3e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi 17024055 ps3p-e035 rbp-j regulates the cortical laminar formation kenji tanigaki 1 , kazue muraki 1 , norio yamamoto 2 , tasuku honjo 2 1 shiga medical center, research institute, shiga, japan; 2 department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps3p-e036 search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi 1 , tomoko ikawa 1 , yuya kasukawa 2 , hironori ueda 2,3 , kazuki kurimoto 4 , michinori saitou 4 , fumio matsuzaki 1,5 1 lab. for asymmetric cell division, cdb, riken, kobe, japan; 2 functional genomics subunit, cdb, riken, kobe, japan; 3 lab. for systems biology, cdb, riken, kobe, japan; 4 lab. for mammalian germ cell biology, cdb, riken, kobe, japan; 5 crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. 30 samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi (15700264) ryosuke tatsuno 1 , tomoaki sai 2,3 , masahiro otsu 2 , kuniko akama 1 , takashi nakayama 4 , tosifusa toda 5 1 grad. sch. of sci. and tech., chiba univ., chiba, japan; 2 lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; 3 dept. orthop. surg., jikei univ. sch. med., tokyo. japan; 4 dept. biochem., yokohama city univ. sch. med., yokohama, japan; 5 proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using 2-d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps3p-e038 identification of se90, a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified 150 genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se90 in developing and matured cns. teiichi furuichi 1 , akira sto 1,2 , yukiko sekine 1 , noriyuki morita 1 , tetsushi sadakata 1 , satoshi shoji 1 , jin-hong huang 1 , toshio kojima 2 1 laboratory for molecular neurogenesis, riken brain science institute, japan; 2 comparative systems biology team, riken genome sciences center, yokohama 230-0045, japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki 1 , makoto mizuno 2 , kazuhisa sakai 4 , yoshimoto kiyohara 4 , kazuhiko yamaguchi 3 , tsutomu hashikawa 4 , hiroyuki nawa 2 1 institute of biomedical engineering, waseda university, tokyo, japan; 2 department of molecular neurobiology, brain research institute, niigata university, niigata, japan; 3 laboratory for memory and learning, bsi, riken, saitama, japan; 4 laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg1 knock out mice (nrg1 type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps3p-e041 psd-95 protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama 1,2 , satoshi wakisaka 1 , shiho honma 1 , akira ito 1,2 , kei azuma 1,2 , mikihiko kogo 1 1 department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; 2 first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd-95 implicates in formation and maturation of excitatory synapses. psd-95 regulates the localization of the nmda receptor by means of binding with nr2. rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd-95 protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd-95 expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf97541. in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro-␤-erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps3p-e043 ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps3p-e044 comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi 1 , kenji mizutani 2 , hiroto ogawa 3 , kohji hotta 1 , kotaro oka 1 1 ctr. for biosci. and info, keio univ., yokohama, japan; 2 neuro, karolinska inst, stockholm, sweden; 3 bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to 50 cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion (25% or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps3p-e045 three dimensional (3d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in 3d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that 3d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether 3d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in 3 monkeys. of a total of 77 p-cells that were examined during both frontal and vergence-pursuit, 46% discharged for both, 40% only for vergence, and 14% only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had 3d pursuit signals. majority (70%) of these p-cells discharged before onset of vergence eye movements with the typical lead time of 50 ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi (17022001) ps3p-e046 information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey13308, rr00165 aya takemura 1 , yumi murata 1,3 , kenji kawano 1,2 1 neurosci. res. insti, aist, tsukuba, japan; 2 dept. integ brain sci., grad. sch. med., kyoto univ., japan; 3 grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid (15 mg/ml, 36-37 l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at 20 • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of 58 ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps3p-e049 the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara 1 , kenji kunita 2 , kaoru maeda 1 , takeo kiyota 1 1 department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; 2 institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were 17 undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for 30 ms with 800-ms intervals. the oscillation trial (0.5-hz frequency and 2.5-cm amplitude) (65-75 s) was repeated 5 times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the 5th trial. a significant correlation was shown between 5th-1st trial differences in mean speed and vep amplitude (r = 0.79). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps3p-e051 primary motor cortex contributes to generating manual following response toshitaka kimura 1 , naoki saijo 1 , hiroaki gomi 1,2 1 ntt cs labs, kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m1), that (1) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and (2) intracortical inhibition within m1 assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m1. such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo 1 , hiroaki gomi 1,2 1 ntt cs labs., kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in 8 radial directions applied during reaching. the data show that the direction of the initial phase (150-200 ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase (250-300 ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps3p-e053 spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa 1 , hiroaki gomi 1,2 1 ntt cs labs., kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps3p-f055 influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka 1 , takashi obu 2 , kazuyuki kanosue 3 1 asmew, waseda university, saitama, japan; 2 graduate school of human sciences, waseda university, saitama, japan; 3 faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = 6) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at 1.5, 1.75, and 2.0 hz. over 99% success rate was observed in all tasks except oppo (78-97%). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps3p-f056 simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu 1 , tetsuro muraoka 3 , kazuyuki kanosue 1,2,3 1 faculty of human sciences, waseda university, saitama, japan; 2 faculty of sport sciences, waseda university, saitama, japan; 3 asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at 1, 1.33, and 1.66 hz. they were asked to perform 4 tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps3p-f057 activities of erector spinae muscles during jaw clenching in man kayoko yasunaga 1,2 , tadachika yabushita 1 , kazuo toda 2 , kunimichi soma 1 1 orthodontic science, tokyo med. & dent. univ., tokyo, japan; 2 div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada 1 , sachiko motoyama 1 , futoshi mori 1 , shigemi mori 2 1 department of veterinary physiology, yamaguchi university, yamaguchi, japan; 2 national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for 12-20 weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps3p-f059 neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima 1 , futoshi mori 2 , akira murata 1 , masahiko inase 1 1 dept. of physiol., kinki univ. schl. of med., osakasayama, japan; 2 dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m1 of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m1 hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m1 modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m1 output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi (16500271) ps3p-f060 activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara 1,2 , nobuhiko hatanaka 1,2 , masahiko takada 3 , atsushi nambu 1,2 1 school of life science, the graduate university for advanced studies, japan; 2 division of system neurophysiology, national institute for physiological sciences, japan; 3 tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu 1 , sei-ichi sasaki 2 , yuichiro cho 1 , kenji sato 1 1 anatomy and physiological science, tokyo medical and dental university, tokyo, japan; 2 department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s1 and s2 spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl7 -s3 spinal segments which contain afferent fibers of eas muscles. after 3 weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for 4 consecutive days. we compared adaptation in day1 and that in day4. the gain decrease for the first 700 saccades in day 4 (0.140 ± 0.036) was larger than that in day 1 (0.080 ± 0.021) (p = 0.026, n = 5, paired-t test). the rate of adaptation in day 4 (1.739 ± 0.393 × 10 −4 /sac) was higher than that in day 1 (1.120 ± 0.240 × 10 −4 /sac) (p = 0.011). the overall gain change (1800 saccades) in day 4 (0.219 ± 0.030) was larger than that in day 1 (0.112 ± 0.022) (p = 0.002). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi (16300129) ps3p-f064 asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v1. the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = 7) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by 4 s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in 60% of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida 1 , xiaofeng lu 1,2 , shogo ohmae 1 , toshimitsu takahashi 1,2 , shigeru kitazawa 1,2 1 dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; 2 crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of 16 targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi 17022033 ps3p-f066 frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa 1 , soichi nagao 1,2 1 lab. for motor learning control, riken bsi, saitama, japan; 2 sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps3p-f068 projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki 1,2 , kei ito 1,2,3 1 dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; 2 center for bioinform., imcb, univ. of tokyo, japan; 3 bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal4 enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening 4,000 strains, we identified about 180 candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps3p-f069 immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura 1 , yoshitaka ohtubo 1 , takashi kumazawa 2 , kiyonori yoshii 1 1 graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; 2 department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi (16300094) and the 21st coe program (center #19) granted by mext of japan ps3p-f070 inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa 1 , atsushi kuhara 1 , hitoshi inada 1 , eiji kodama 1 , takafumi mizuno 1 , ikue mori 1,2 1 lab. of mol. neurobiol., nagoya univ., japan; 2 institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx-7 gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx-7 mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx-7 mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps3p-f071 postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut1 and -2) during rat ob development from postnatal day 1 (p1) to p10 by in situ hybridization and immunohistochemistry. at p1 vglut1 immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut1 mrna signals were detectable in the mitral cell layer from p1 to p10. in contrast, vglut2 ir was prominent in the glomerulus at all days examined, and only at p1 and p3 in mitral cells. despite mitral vglut2 ir disappeared at p10, the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps3p-f072 v1r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito 1,2 , masumi ichikawa 3 , yuji mori 2 , kimiko hagino-yamagishi 1 1 tokyo metrop. inst. med. sci., tokyo, japan; 2 the univ. of tokyo, tokyo, japan, 3 tokyo metrop. inst. neurosci., tokyo, japan in rodent, v1r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v1r gene, which is expressed in the olfactory epithelium (oe). to examine when the v1rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified 23 v1r sequences. these v1rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v1rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps3p-f073 analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t1r2/t1r3 heterodimer plays a role as a sweet taste receptor. but, mice lacking t1r3 showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t1r3-independent sweetener binding site also exist in mice. in this study, to predict binding sites on t1r2/t1r3 and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [(1) sucrose, glcose, fructose, (2) saccharin, aspartame, acesulfame-k, glycine, (3) d-phenylalanine, (4) d-tryptophan, (5) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t1r2 and t1r3, respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu 1 , sachiko saito 2 , yuko murata 3 , ding ming 4 , tatsu kobayakawa 5 , robert f. margolskee 4 , yuzo ninomiya 1 1 sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; 2 saito sachiko taste and smell research institution, ibaraki, japan; 3 national res. institute of fisheries sci., kanagawa, japan; 4 dept. of physiol. & biophys., mount sinai sch. med., new york, usa; 5 national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t2r receptors which coupled with ggustducin. ps3p-f075 newborn infant body odor attenuates their mother's postpartum moods shota nishitani 1 , mayumi kokuryo 1 , tsunetake miyamura 2 , kazuyuki shinohara 1 1 div. neurobiol. & behav., nagasaki university, japan; 2 obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps3p-f076 human prefrontal activity in taste encoding: an fnirs study masako okamoto 1 , mari matsunami 2 , haruka dan 1 , tomoko kohata 2 , kaoru kohyama 1 , ippeita dan 1 1 national food research institute, tsukuba, japan; 2 nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = 10) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes (42 subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes (36 subfamilies) were expressed in the dorso-medial zone (zone 1) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone 1. these results indicate that there is a clear boundary between zone 1 and non-zone 1 areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone 1 tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi (16500196) ps3p-g079 taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to (1) best-taste profiles and (2) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi (11671860) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼100 members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi (16300105) ps3p-g081 identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka 1 , kazuhisa sakai 2 , tsutomu hashikawa 2 , kensaku mori 1 , masahiro yamaguchi 1 1 dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; 2 laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps3p-g083 nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi (15209061), kakenhi (17791325) ps3p-g084 integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than 25% of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi (17023012) ps3p-g085 odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to 8 classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than 2 classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi (13gs0007) ps3p-g086 odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa 1 , takaaki sato 2 , akira shimizu 1 , ken-ichiro tsutsui 1 , toshio iijima 1 1 div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; 2 res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range (20 hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps3p-g087 chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when 3 m lysine was spotted on ammonium chloride background, 0.003-0.01 m and 0.05 m background did not influence lysine chemotaxis, while 0.03 m background augmented and 0.07-0.1 m background suppressed the chemotaxis. in contrast, when 0.1 m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps3p-g088 glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu 1,2,3 , takeshi ishihara 1 , eisuke nishida 3 , yuichi iino 2 1 dept. biol., fac. sci., kyushu univ., japan; 2 mol. genet. res. lab., univ. of tokyo, japan; 3 grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for 5 min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr-1, a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr-1 appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr-1. in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr-1. these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr-1 localization in aiy neurons. kohei ueno 1 , yoshiaki kidokoro 2 1 dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; 2 inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼1%), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the 26th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal4/uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps3p-g090 spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki 1,2 1 graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; 2 department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps3p-g091 medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka 1 , yoshinori sahara 2 , shinichiro horie 2 , masumi ichikawa 3 , shun nakamura 2 1 st. marianna univ. schl. med., kawasaki, japan; 2 ntl. inst. neurosci., ncnp, tokyo, japan; 3 tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than 10 s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps3p-g093 synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about 1 hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi (17023012) ps3p-g094 rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii 1,2 , ken yoshikawa 2 , yuki takatori 1 , koichiro kawashima 2 1 dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; 2 dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca 2+ signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca 2+ signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m 1 , m 3 , m 4 and m 5 machr subtypes, and ␣3, ␣5, ␣6, ␣7, ␣9, ␣10 and ␤4 nachr subunits. transfection of anti-m 3 , anti-m 5 and anti-␣7 small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca 2+ signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m 3 or anti-m 5 sirna. ca 2+ signals evoked by nicotine were reduced by anti-␣7 sirna. these findings indicate that m 3 , m 5 machr and ␣7 nachr subtypes play major roles in ca 2+ signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi (16590060) ps3p-g101 is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto 1 , sachiho kubo 2 , seiji matsuda 3 , naoki maruyama 2 1 dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; 2 dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; 3 dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, 2006) research funds: kakenhi (16590831) ps3p-g102 dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day 50. electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day 45. analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around 25 hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c1 from kansai medical university ps3p-h103 unusually folded sod1 species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno 1 , yumiko simazaki 1 , fuminori saitoh 1 , ryosuke takahashi 2 , toshiyuki araki 1 1 national institute of neuroscience (ncnp), tokyo, japan; 2 dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod1 protein is thought to be responsible for the selective loss of motoneurons in sod1-related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod1 species are not yet clarified. since we have detected accumulation of unusual sod1 species in motoneuronal axons from g93a sod1-tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod1 species. immunoprecipitation analyses revealed specific interaction of unusual sod1 species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod1-tg compared with wt sod1-tg mice. these data strongly suggest that the toxic property of unusual sod1 proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c (17590907) ps3p-h104 relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately 30% of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day 24. the mice started to show ncl symptoms on p25, and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from 30% to 50% of that in the wild-type mice. research funds: kakenhi (10343253) ps3p-h105 neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro2a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura 1 , katsumi higaki 1 , junichiro matsuda 2 , yoshiyuki suzuki 3 , eiji nanba 1 1 division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; 2 national institute of biomedical innovation, osaka, japan; 3 clinical research center, international university of health and welfare, tochigi, japan g m1 -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal ␤-galactosidase, excessive lysosomal accumulation of gm1 is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm1 is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm1 accumulation of lysosome and pm and the membrane fluidity was also reduced. gm1-bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m1 -gangliosidosis. katsuya inoue 1,2 , katsuaki endo 1 , takamitsu fujikawa 1 , seijyun fukuda 2 , tatsuo nakamura 2 1 department of physical therapy, university of aino, osaka, japan; 2 institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after 40 min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after 40 min occulusion. ps3p-h108 development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando 1 , yuya kawanaka 1 , minoru saito 2 , hiroaki mochizuki 1 , ken honjo 1 , hirofumi toda 1,3 , toshifumi tomoda 3 , akira sawa 4 , katsuo furukubo-tokunaga 1 1 grad. school of life & envir. sci., univ. tsukuba, japan; 2 molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; 3 beckman res. inst., city of hope. california, usa; 4 dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia-1 (disc1) gene, originally identified at the breakpoint of a chromosome (1;11) (q42.1; q14.3) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc1 functions in vivo, we expressed disc1 in drosophila and examined developmental and behavioral phenotypes. overexpression of disc1 resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps3p-h110 effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake 1 , ayumi kamo 1 , kenji minato 1 , hitoshi haruna 1 , hiritsugu fukuda 2 , yuji murata 2 , takayoshi hosono 1 1 department of bomedical engineering, osaka electro-communication university, japan; 2 graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of 22 of 25 7-day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing 8% oxygen at 40 • c for 15 min. after the insult, 11 of the 22 rats were put in a box at 35 • c for 12 h (hypothermia, h-group). the other 11 rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps3p-h112 tau hyperphosphorylation in ts1cje, a partial trisomy 16 mouse model for down syndrome ebrahim abdul 1 , a. shimohata 1 , w. yu 1 , m. yamaguchi 1 , m. murayama 3 , d. chui 3 , t. akagi 2 , t. takeuchi 1 , k. amano 1 , h.s. karthik 1 , t. hashikawa 2 , h. sago 4 , c.j. epstein 5 , a. takashima 3 , k. yamakawa 1 1 research scientist; 2 lab. for neural arch.; 3 lab. for alzheimers disease; 4 div. of fetal med. ncchd; 5 ucsf, usa although down syndrome (ds) or trisomy 21 is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts1cje, a ds mouse model partially trisomic for chromosome 16, shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome 21q22, has about 97 genes. importantly, sod1 and app, which may contribute to the ds phenotype, are excluded from the ts1cje trisomic segment. here we report that ts1cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk3␤ and jnk/sapk activities without alterations in a␤pp metabolism. our results suggest that genes on the trisomic ts1cje segment other than app and sod1 can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: 17790737 ps3p-h113 increased oxidative stress and mitochondrial dysfunction in ts1cje, a down syndrome mouse model atsushi shimohata 1 , ebrahim a. s. 1 , m. yamaguchi 1 , w. yu 1 , h. sago 2 , c.j. epstein 3 , k. yamakawa 1 1 lab. for neurogenetics, riken-bsi, japan; 2 div. of fetal med. ncchd, japan; 3 dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome 21(hsa21) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts1cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome 16 extending from sod1 to znf295, containing 97 genes syntenic with hsa21. since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts1cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts1cje brains. collectively, our results implicate dosage imbalanced genes other than sod1 and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts1cje ds mental retardation-like phenotype. ps3p-h114 polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan 1,2 , kouko tatsumi 2 , takayuki manabe 2 , takahira yamauchi 1,2 , eri makinodan 2 , juro shimoda 1 , toshifumi kishimoto 1 , akio wanaka 2 1 department of psychiatry, nara medical university, kashihara, japan; 2 department of 2nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded (62q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. 7200 genes, encompassing around 50% of the drosophila genome, were screened, resulting in the identification of 370 candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps3p-h117 analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. (2005) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the 2 , 3 ,-cyclic nucleotide 3 -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps3p-h118 dysregulation of sodium channel ␤4 subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel ␤4 (␤4) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified ␤4 as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in ␤4 started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of ␤4 expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of ␤4 gene in pc12 cells. forskolin, an activator of the camp/pka pathway, did not affect b4 promoter activity, indicating that ␤4 is not camp-responsive gene. these findings strongly suggest that sodium channel ␤4 subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps3p-h119 repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki 1 , yuji takahashi 1 , jun goto 1 , mutsuo oyake 2 , toshiya sato 3 , shoji tsuji 1 1 department of neurology, the university of tokyo, tokyo, japan; 2 department of neurology, brain research institute, niigata university, niigata, japan; 3 center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of 76 (q76), 96 (q96), 113 (q113), or 129 (q129). in the accelerating rotarod (9w), the latencies of q76, q96, q113 and q129 were 113%, 81%, 34% and 24%, respectively. in the open field, moving distances of q96, q113, and q129 were decreased to 98%, 50%, and 20%, respectively, while that of q76 was increased to 134%. home cage activity was decreased depending on the repeat length. the q113 mice, however, showed increased ratios of the activity during the light time to that during the total day at 8 weeks (115%) and 24 weeks (208%), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps3p-h120 the mice lacking schnurri-2 show multiple behavioral abnormalities related to psychiatric disorders keizo takao 1 , nobuyuki yamasaki 1 , keiko toyama 1 , tsuyoshi takagi 2 , shunsuke ishii 2 , tsuyoshi miyakawa 1 1 hmro, kyoto university graduate school of medicine, kyoto, japan; 2 riken, tsukuba, japan schnurri-2 (shn-2) is a zinc finger transcription factor, a mouse homologue of human hiv-ep2, that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn-2 is known to play important roles in the mammalian immune systems. however, the role of shn-2 in the central nervous system (cns) is still unknown. to investigate the functional significance of shn-2 in mammalian brain, we analyzed the shn-2 knockout (ko) mice using a comprehensive behavioral test battery. shn-2 ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn-2 mice. our results demonstrate a critical role of shn-2 in cns and suggest that shn-2 ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h121 comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h122 effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c57bl/6j, c57bl/6n, c57bl/6c, 129svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than 1200 mice (, including wild type and mutant mice from 25 strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h124 comprehensive behaivoral analysis of ryanodine receptor type3 knockout mouse suzuko ohsako 1 , koichi tanda 1,2 , nobuyuki yamasaki 1 , keiko toyama 1 , hiroshi takeshima 3 , tsuyoshi miyakawa 1 1 kyoto university graduate school of medicine, kyoto, japan; 2 dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; 3 dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca 2+ signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr3 is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr3 deficiency, we subjected ryr3 knockoout mice to a battery of behavioral tests. ryr3 knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr3 in regulating general locomotor activity and social behavior. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h125 comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama 1 , koichi tanda 1,2 , nobuyuki yamasaki 1 , tsuyoshi miyakawa 1 1 hmro, kyoto university graduate school of medicine, kyoto, japan; 2 dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h126 primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi 1 , keiko shimizu 2 1 grad. sch. human and environmental std, kyoto univ., kyoto, japan; 2 primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected 6-ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. 6-ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in 6-ohda injected monkeys. these results suggest that monkeys injected 6-ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi (17021022) ps3p-i127 training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata 1,2,3 , noriyuki higo 1,3 , takao oishi 1,3,4 , akiko yamashita 5 , keiji matsuda 1 , motoharu hayashi 4 1 neurosci. res. inst, aist, tsukuba, japan; 2 grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; 3 crest, jst, kawaguchi, japan; 4 dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; 5 div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext (17021055) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about 2,500 g1 animals (dba/2j × enu-treated c57bl/6j) for home-cage activity, open-field activity, and passive avoidance response, and obtained 13 lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in 5 of 13 mutant lines. hyperactivity was predominant phenotype, and 7 of 13 mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps3p-i129 ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., 2005) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg7-knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., 2005) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc3, and found that in our ncl model mice, positive signals for ubiquitin and lc3 were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)(17790141) and creative scientific research (16gs0315) ps3p-i130 activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama 1 , eiichi jodo 1 , yoshiaki suzuki 2 , ken-yo hoshino 2 , yukihiko kayama 1 1 dept. of physiology, fukushima medical university, fukushima, japan; 2 dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch23390, mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps3p-i131 increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara 1 , hiroyuki sakagami 2 , hisatake kondoh 2 , kohji fukunaga 1 1 department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps3p-i132 severity of audiogenic seizures is influenced by multiple factors in vlgr1-mutated mice hideshi yagi 1,2 , makoto sato 1,2 1 division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; 2 research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr1-mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c57bl/6 backcrossed vlgr1-mutated mice and 129/svs4 backcrossed vlgr1-mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr1-mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps3p-i133 reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota 1 , ken kadota 1,2 1 department of bioenvironmental medicine, university of chiba, chiba, japan; 2 chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of 5 mg/kg of map i.p. for 12 days (d 1 d 12) and then withdrawn from the drug for 7 28 days (wd 7 wd 28). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg 1) and mfc. the decrease in the density of pac was first observed in cg 1 and then in mfc. the reduction began on d 6 and advanced to higher levels on d 12 and subsequently wd 7. these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg 1 and mfc, particularly in layers ii and iii. ps3p-i134 up-regulation of ␤ 2 -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of ␤ 2 -adrenergic receptor (␤ 2 -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of ␤ 2 -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons. ␤ 2 -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of ␤ 2 -ar-immunoreactive cells were labeled with ki-67, a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of ␤ 2 -ar-immunoreactive cells also exhibited fgf-2-ir. these findings suggest that ␤ 2 -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf-2. ps3p-i135 effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita 1 , norihiro muraoka 2 , hiroshi hasuo 1 , takashi akasu 1 , minoru shigemori 2 1 dept. of physiology, kurume univ. sch. of med., kurume, japan; 2 dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh482). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity (10-50 v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was 30-40 v. the amplitude of slow peak in fpi group was about 50% larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps3p-i136 comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca 2+ imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca 2+ imaging. the slices (300 m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for 1 h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of 1 ml/min. green autofluorescence (>520 nm) of the slices illuminated by blue light (460-490 nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca 2+ imaging was also performed in the slices. slices were incubated in acsf medium containing 10 m of fluo3/4 am for 1 h at 37 • c. the ca 2+ image was recorded with an excitation wavelength of 460-490 nmand an emission wave length of >520 nm. the autofluorescence and ca 2+ responses wereobserved in slices perfused with l-glutamate (10 mm). takuya hayashi 1 , hiroshi sato 1 , shinichi abe 2 , takashi hanakawa 2 , hiroshi watabe 1 , hidenao fukuyama 2 , babak aldekani 3 , hidehiro iida 1 1 department of investigative radiology, national cardiovascular center research institute, osaka, japan; 2 human brain research center, kyoto university, kyoto, japan; 3 nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area 9 corresponded to that revealed by the tracer (mncl 2 ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h17-kokoro-025 ps3p-i138 optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata 1,2 , yasutake mori 1,2 , tsuya taneda 1,2 , hiroaki okuda 1,2 , masaya tohyama 1,2 1 department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; 2 21st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps3p-i140 a model selection of glm applied to fmri data using aic jobu watanabe 1 , fumikazu miwakeichi 2 , andreas galka 3,4 , ryuta kawashima 4 , tohru ozaki 3 , sunao uchida 1,5 1 institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; 2 department of medical system engineering, faculty of engineering, chiba university, chiba, japan; 3 institute for statistical mathematics, tokyo, japan; 4 institute of experimental and applied physics, university of kiel, keil, germany; 5 new industry creation hatchery center, tohoku university, sendai, japan; 6 faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for 20-30 min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps3p-j148 flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata 951-8585, japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps3p-j149 effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune 1,2 , takashi tsukiura 1 , hiroko mochizuki-kawai 1 , chisato suzuki 1,2 , toshio iijima 2 1 neurosci. res. inst., aist, tsukuba, japan; 2 div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps3p-j150 different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno 1 , kenji kansaku 2 , yu aramaki 2 , mitsuo kawato 1 1 atr cns, kyoto, japan; 2 institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = 14) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, 50 or 10 yen is provided with a probability of 0.8, but only with 0.2 if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < 0.01) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps3p-j151 brain networks for communicative speech production: feeling inference and speech content production yuko sassa 1,2 , motoaki sugiura 3 , hyeonjeong jeong 1,4 , keisuke wakusawa 1,5 , kaoru horie 6 , shigeru sato 6 , ryuta kawashima 1 1 niche, tohoku university, sendai, japan; 2 ristex, jst, tokyo, japan; 3 miyagi university of education, sendai, japan; 4 gsics, tohoku university, sendai, japan; 5 department of pediatrics, tohoku university, sendai, japan; 6 the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the 21st coe program ps3p-j152 the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi 1 , yasuhisa fujibayashi 2 , yoshiharu yonekura 2 , tatsuya asai 2 1 department of human and intelligence systems, university of fukui, fukui, japan; 2 biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps3p-j153 imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai 1 , kuniyoshi shinya 1 , tetsuo araki 2 , masahiro kusakabe 2 , yasuhisa fujibayashi 3 , yoshiharu yonekura 3 1 department of nuclear power and energy safety engineering, university of fukui, fukui, japan; 2 department of human and intelligence systems, university of fukui, fukui, japan; 3 biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura 1,2 , winfried denk 3 , michael hausser 2 1 department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; 2 university college london, london, uk; 3 max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps3p-k161 analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka 1 , yasuhiro tanaka 1 , takeshi kaneko 1,2 1 dept. of morphological brain science, kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad67-gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after 3 h incubation in nmdg-based solution than in nacl-based solution. cutting at 0 • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at 20 • c. the viability after 3 h incubation was better kept at 20 • c than at 0 or 37 • c. we thus recommend that slices be cut at 20 • c in na-free solution, and incubated at 20 • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi (16200025,17022020,17650100) ps3p-k162 contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh 1 , yoshichika yoshioka 2 1 faculty of engineering, iwate university, morioka, japan; 2 iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal (2.95 ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals (3.03, 3.23, and 3.34 ppm). a phantom solution mimicking the normal condition (gsh:gssg = 100:1) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = 7:3:1:1), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was 1.9 mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area 9) and the right superior and middle frontal gyri (brodmann area 10) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps3p-k164 cortical commissural connection in macaque and human callosum using diffusion mri rishu piao 1 , takuya hayashi 1 , hiroshi sato 1 , shinichi abe 1,2 , takashi hanakawa 2 , hidenao fukuyama 2 , hidehiro iida 1 1 national cardiovascular center, osaka, japan; 2 human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in 9 subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps3p-k165 optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong 1,2 1 niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, 2 niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the 3-d neural cortical activity using the 2-d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps3a-c192 sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp8) nobuyuki karasawa 1 , kazuko watanabe 2 , keiki yamada 3 1 seijoh universitry, tokai, japan, 2 dept. physio., sch. med., gifu univ., gifu, japan, 3 dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, 2,4-diamino-6-hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp8) mice. time course of immunoreactive intensity for serotonergic (5-ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that 5-ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and 5-ht neurons. katsuya yamada 1,2,5 , yoshihiro matsumura 2 , takashi miki 3 , makoto wakui 1 ␣-smooth muscle actin + arterioles were fewer in kir6.1(−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir6.1(−/−) barrel cortex than in wild-type one for short (2 s, 5 hz) but not long (30 s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps3a-h135 learning to use sensory-tools by japanese monkeys yumiko yamazaki 1 , hiromi namba 1 support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps2p-h147 mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h1 receptor (h1r) in cognition in physiological and pathological conditions by using h1r mutant (h1−/−) mice. in normal condition, several behavioral studies indicated h1−/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h1−/− mice. these results indicate h1 receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h1−/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h1−/− mice. mutation of h1 receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h1r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h1r impairs cognition in normal conditions, whereas h1r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi (17390156; 14370027) ps3a-h133 5-ht2a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura 1,2 , hirohito-m. kondo 2 , makio kashino 2,3 1 department of psychology, tokai women's university, kakamigahara, japan; 2 ntt communication science laboratories, ntt corporation, atsugi, japan; 3 shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of 5-ht neurotransmission. we examined whether this 5-ht2a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the 5-ht2a receptor gene polymorphism in impulsive behavior (nomura et al., 2005) , together with the present findings, this observation indicates the involvement of 5-ht2a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps3a-h134 role of cortical thin arterioles in neurovascular coupling; analyses of kir6.1-containing atpsensitive potassium channel-deficient mice ps3p-g095 rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal 1 , chinmay k. mukhopadhyay 2 , birendra n. mallick 1 1 school of life sciences, jawaharlal nehru university, new delhi, india; 2 special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv-1 and the direct infection of hsv-1 are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv-1 jrfl-infected macrophages or with hsv-1. the generation of primary neurospheres did not suppressed by hiv-1-infection or by hsv-1 infection at no more that moi 10. in the secondary neurospheres, cd133 + neural stem cells were intact in these infections, although beta-3-tubulin + cells were decreased in hiv-1 infection and intact in hsv-1-infection. in the differentiation assay, neun + nfp + neurons in hiv-1-infection and gfap + s100 + astrocytes in hsv-1 infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv-1 infection and in hsv-1 infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv-1 and hsv-1 infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi 17790579 grant-in-aid for young scientists(b) ps3p-g097 effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga 1,2 , taeko yamauchi 2 , toshihiro konagaya 2 , hideki ohira 1 1 department of psychology, nagoya university, japan; 2 department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka 1 , shen jin-song 2 , saburo saito 1 , shigeki yuasa 3 1 dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; 2 dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; 3 dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori 1 , tomoko yamamoto 1 , kiyoshi matsumura 2 , hiroshi hosokawa 1 , shigeo kobayashi 1 1 dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; 2 dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e 2 (pge 2 ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge 2 but also induce an enzyme cyclooxygenase-2 (cox-2) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox-2, and whether cox-2 is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox-2 was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox-2 specific inhibitor, ns-398. these results indicate that aa itself or its metabolites induces cox-2 that accelerates the formation of pge 2 from aa, and, hence, enhances fever. mitsunari abe 1 , tatsuya mima 1 , shinichi urayama 1 , toshihiko aso 1 , nobukatsu sawamoto 2 , hidenao fukuyama 1 1 human brain research center, kyoto university graduate school of medicine, japan; 2 nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at 0.9 hz (10 min; 90% of the rest motor threshold) applied over the left primary motor area (m1). we scanned 4 sets of dwi (before, and 0 min, 10 min and 20 min after rtms) using 3-t mr scanner, and calculated apparent diffusion coefficient (adc). in 3 out of 4 subjects, the adc decreased (mean 1.72 × 10 −5 /mm 2 s −1 ) in the left m1 just after the rtms, which recovered at 20 min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v6 and v6a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v6a located more dorsal has a larger receptive field size then v6. both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v6 and v6a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v1 in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v6 and v6a.ps3p-i144 measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro 9-channel magnetometer consisting of a 3 × 3 matrix of superconducting quantum interference device (squid) with its integrated base of 2.5 × 2.5 mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with 60-80 ms peak by the white led flashing stimulation and the aef wave with 10-20 ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of 19 bits was used, along with a "blue dye" (rh 1691) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were 0.1-0.05% of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto 1 , yoko hoshi 1 , chie sato 1 , susumu terakawa 2 1 tokyo institute of psychiatry, tokyo, japan; 2 photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals (586 and 610 nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around 20-ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps3p-j147 multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh414, a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a 4t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a 4-s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba18), superior occipital gyrus around the parieto-occipital sulcus (ba19) and superior parietal lobule (ba7). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps3p-j155 influence of task difficulty during meter inspection: an fmri study naoki miura 1,2 , makoto takahashi 3 , jobu watanabe 2,4 , shinya uchida 2,5 , shigeru sato 6 , kaoru horie 6 , masaharu kitamura 2 , toshio wakabayashi 2 , katsuki nakamura 1,7 , ryuta kawashima 2 1 crest, jst, kawaguchi, japan; 2 niche, tohoku univ. sendai, japan; 3 graduate school of engineering, tohoku univ. sendai, japan; 4 bme institute, waseda univ., tokyo, japan; 5 idac, tohoku univ., sendai, japan; 6 lbc research center, tohoku univ., sendai, japan; 7 department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps3p-j156 neural connectivity among brain areas related to language function shinya uchida 1,2,3 , naoki miura 2,4 , jobu watanabe 5 , shigeo kinomura 1 , kazunori sato 1 , yasuyuki taki 1 , kentaro inoue 1 , ryoi goto 1 , ai fukushima 2 , kaoru horie 6 , shigeru sato 6 , katsuki nakamura 3 , hiroshi fukuda 1 , ryuta kawashima 2 1 department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; 2 niche, tohoku university, sendai, japan; 3 national institute of neuroscience, ncnp, kodaira, japan; 4 japan science and technology agency, kawaguchi, japan; 5 bme institute, asmew, waseda university, tokyo, japan; 6 graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than 80% of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa 1,2 , motoaki sugiura 3 , yuko sassa 1,4 , hyeonjeong jeong 1,5 , kaoru horie 5 , shigeru sato 5 , hiroyuki yokoyama 2 , kazuie inuma 2 , ryuta kawashima 1 1 niche, tohoku univ., sendai, japan; 2 department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; 3 miyagi univ. of education, sendai, japan; 4 ristex, jst, tohoku univ., sendai, japan; 5 gsics, tohoku univ., sendai, japan; 6 lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda 1 , yasumasa okamoto 1 , kazuhiro shishida 1 , akiko kinoshita 1 , shigeru toki 1 , kazutaka ueda 2 , hidehisa yamashita 1 , shigeto yamawaki 1 1 department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; 2 training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada 1,3,4 , kenji yoshimi 1,2 , noriyuki higo 4 , yong-ri ren 2 , hideki mochizuki 2 , yoshikuni mizuno 2 , shigeru kitazawa 1,3,4 1 dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; 2 dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; 3 crest, jst, tokyo, japan; 4 neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba-1 positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma −5.9 mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections.